RS virus vaccine and its use

Abstract

To provide a RS virus vaccine having high therapeutic and / or preventive effects against RS virus.SOLUTION: The present invention relates to a vaccine for preventing or treating RS virus infection, comprising a combination of (a) an antigen protein derived from the F protein of RS virus, and (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds, and (ii) an adjuvant comprising a complex consisting of lentinan, the vaccine being administered to a human, the antigen protein being administered in an amount of 100 to 400 μg per dose, and the adjuvant being administered in an amount of 100 to 400 μg per dose.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a vaccine for preventing or treating respiratory syncytial virus infection, which uses an antigen protein derived from respiratory syncytial virus in combination with an adjuvant containing a complex of an oligodeoxynucleotide and lentinan, and also relates to the use of the vaccine in preventing and / or treating respiratory syncytial virus infection. [Background technology]

[0002] Respiratory syncytial virus (hereinafter referred to as RSV or RS virus) is a major cause of lower respiratory tract disease (LRI) in infants. RSV infection remains a significant cause of death, particularly in infants, young children, and the elderly. Therefore, RSV is recognized worldwide as a priority health issue (Non-Patent Document 5).

[0003] Therefore, RSV vaccines are being developed in various countries. For example, development of a stable pre-fusion RSV F protein (or a fragment thereof), a composition comprising the protein, and its use for preventing and / or treating RSV infection (Patent Documents 3 and 6-8), development of an RSV F protein mutant, a nucleic acid or vector encoding an RSV F protein mutant, a composition comprising an RSV F protein mutant or a nucleic acid, and their use (Patent Document 4), and development of a vaccine comprising a recombinant RSV antigen for treating and / or preventing RSV infection (Patent Document 5) are known. In addition, development of an immunogenic composition comprising at least two paramyxovirus F protein antigens selected from human metapneumovirus (hMPV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV) (the antigen is a recombinant F protein polypeptide, which is modified to stabilize the trimer pre-fusion conformation) is also known (Patent Document 10).

[0004] It is also known that it is effective for a vaccine to contain an adjuvant in addition to the active ingredient, a recombinant antigen protein, etc. For example, a vaccine composition containing at least about 1 μg and up to about 200 μg of RSV soluble F protein and at least about 1 μg and up to about 20 μg of an adjuvant containing a lipid Toll-like receptor (TLR) agonist has been developed (Patent Document 9). CpG oligonucleotides (CpG ODN) are short (approximately 20 bases), single-stranded synthetic DNA fragments containing immunostimulatory CpG motifs. They are known to be potent agonists of Toll-like receptor 9 (TLR9), activating dendritic cells (DCs) and B cells to produce type I interferon (IFN) and inflammatory cytokines (Non-Patent Documents 1 and 2), and acting as adjuvants for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) responses (Non-Patent Documents 3 and 4).

[0005] There are at least four types of CpG ODNs, K type (also called B type), D type (also called A type), C type, and P type, which differ in their backbone sequences and immunostimulatory properties (Non-Patent Document 6). Among them, K type CpG ODNs contain multiple non-palindromic CpG motifs and are known to strongly activate B cells to produce interleukin 6 (IL-6) and activate and mature plasmacytoid DCs (pDCs), but hardly produce interferon α (IFN-α) (Non-Patent Documents 7 and 8).

[0006] In addition, mouse and humanized CpG ODNs with a poly dA PO backbone linked to the 5' end, complexed with schizophyllan (SPG), a soluble β-glucan derived from Schizophyllum commune, have been shown to enhance cytokine production and act as influenza vaccine adjuvants and preventive or therapeutic agents for Th2 cell-related diseases (Non-Patent Documents 9, 10, Patent Document 2). Furthermore, it is also known that a complex containing a K-type CpG ODN having a poly(dA) tail at the 3' end and an SPG has vaccine adjuvant activity (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 041318 [Patent Document 2] JP 2008-100919 A [Patent Document 3] International Publication No. 2017 / 174568 [Patent Document 4] International Publication No. 2017 / 109629 [Patent Document 5] International Publication No. 2009 / 079796 [Patent Document 6] International Publication No. 2017 / 005848 [Patent Document 7] International Publication No. 2020 / 099383 [Patent Document 8] International Publication No. 2014 / 174018 [Patent Document 9] International Publication No. 2014 / 168821 [Patent Document 10] International Publication No. 2010 / 149743 [Non-patent literature]

[0008] [Non-Patent Document 1] Hemmi, H., et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740-745 (2000). [Non-Patent Document 2] Krieg, AM Therapeutic potential of Toll-like receptor 9 activation. Nature reviews. Drug discovery 5, 471–484 (2006).

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[0009] An object of the present invention is to provide an RS virus vaccine that has a high therapeutic and / or prophylactic effect against RS virus. [Means for solving the problem]

[0010] To achieve the above objectives, the inventors have investigated the short-term and long-term immunogenicity and potential efficacy of vaccine components, vaccination doses and regimens, in order to rapidly achieve a reduction in antigen dosage, an increase in immune response, an improvement in the quality of immune response, fewer administrations, a longer-lasting immune response, prevention of RSV infection, prevention of aggravation of the infection, and prevention of infection. They then discovered that when a complex consisting of a polydeoxynucleotide containing a specific humanized K-type CpG oligodeoxynucleotide and lentinan is immunized together with a specific RS virus antigen in a specific ratio, it induces both humoral and cellular immune responses against the RS virus antigen, thereby completing a new RS virus vaccine.

[0011] The present invention includes the following inventions. [1] A vaccine for preventing or treating respiratory syncytial virus infection, comprising: (a) an antigenic protein derived from the F protein of respiratory syncytial virus, and (b)(i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of Including a combination of Administered to a human, and The above vaccine, wherein the antigen protein is administered in a single dose of 100 to 400 μg, and the adjuvant is administered in a single dose of 100 to 400 μg. [2] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg. [3] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg. [4] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg. [5] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg. [6] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg. [7] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg. [8] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg. [9] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

[10] The vaccine described in [1], wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 400 μg.

[11] The vaccine according to any one of [1] to

[10] , which is administered once to ten times every one to five years, and the number of administrations per year is no more than two.

[12] A vaccine described in any of [1] to

[11] , which is administered once or twice a year.

[13] The vaccine described in

[11] or

[12] , which is administered twice a year, with the second administration being administered four weeks after the first administration.

[14] The vaccine described in any of [1] to

[13] , wherein the administration is intramuscular administration.

[15] A vaccine described in any of [1] to

[14] , wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

[16] The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The vaccine according to any one of [1] to

[15] , comprising a complex consisting of:

[17] The antigen protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 4). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO: 4. The vaccine according to any one of [1] to

[16] , which is a protein.

[18] The vaccine described in any of [1] to

[17] , wherein the antigen protein and the adjuvant are held in the same container.

[19] The vaccine described in any of [1] to

[17] , wherein the antigen protein and the adjuvant are held in separate containers and are mixed prior to administration.

[20] The vaccine described in any of [1] to

[19] , wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.1.

[21] The vaccine described in any of [1] to

[20] , wherein the complex has a triple helix structure.

[22] A method for preventing or treating respiratory syncytial virus infection in humans, comprising: (a) an antigenic protein derived from the F protein of respiratory syncytial virus, and (b)(i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of administering to a human a vaccine comprising a combination of The above method, wherein the antigen protein is administered in a single dose of 100 to 400 μg, and the adjuvant is administered in a single dose of 100 to 400 μg.

[23] The method according to

[22] , wherein the antigen protein is administered in a single dose of 100 μg, and the adjuvant is administered in a single dose of 100 μg.

[24] The method according to

[22] , wherein the antigen protein is administered in a single dose of 100 μg, and the adjuvant is administered in a single dose of 200 μg.

[25] The method according to

[22] , wherein the antigen protein is administered in a single dose of 100 μg, and the adjuvant is administered in a single dose of 400 μg.

[26] The method according to

[22] , wherein the antigen protein is administered in a single dose of 200 μg, and the adjuvant is administered in a single dose of 100 μg.

[27] The method according to

[22] , wherein the antigen protein is administered in a single dose of 200 μg, and the adjuvant is administered in a single dose of 200 μg.

[28] The method according to

[22] , wherein the antigen protein is administered in a single dose of 200 μg, and the adjuvant is administered in a single dose of 400 μg.

[29] The method according to

[22] , wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 100 μg.

[30] The method according to

[22] , wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 200 μg.

[31] The method according to

[22] , wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 400 μg.

[32] The method according to any of

[22] to

[31] , wherein the compound is administered once to ten times every one to five years, and the number of administrations per year is no more than two.

[33] The method according to any of

[22] to

[32] , wherein the method is administered once or twice a year.

[34] The method according to

[32] or

[33] , wherein the compound is administered twice a year, with the second administration being administered four weeks after the first administration.

[35] The method according to any one of

[22] to

[34] , wherein the administration is intramuscular administration.

[36] The method described in any of

[22] to

[35] , wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

[37] The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The method according to any one of

[22] to

[36] , comprising a complex consisting of

[38] The antigenic protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 4). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO: 4. The method according to any one of

[22] to

[37] , wherein the compound is a protein.

[39] The method according to any of

[22] to

[38] , wherein the antigen protein and the adjuvant are held in the same container.

[40] The method according to any of

[22] to

[38] , wherein the antigen protein and the adjuvant are held in separate containers and mixed prior to administration.

[41] The method according to any of

[22] to

[40] , wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.1.

[42] The method according to any one of

[22] to

[41] , wherein the complex has a triple helix structure.

[43] Use of an antigen protein in combination with an adjuvant in the manufacture of a vaccine for preventing or treating respiratory syncytial virus infection in humans, comprising: the antigenic protein is (a) an antigenic protein derived from an F protein of RS virus, The adjuvant comprises: (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3'-terminus of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are substituted with phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of The above use, wherein the antigen protein is administered in a single dose of 100 to 400 μg, and the adjuvant is administered in a single dose of 100 to 400 μg.

[44] The use according to

[43] , wherein the antigen protein is administered in a single dose of 100 μg, and the adjuvant is administered in a single dose of 100 μg.

[45] The use according to

[43] , wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

[46] The use according to

[43] , wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

[47] The use according to

[43] , wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

[48] ​​The use according to

[43] , wherein the antigen protein is administered in a single dose of 200 μg, and the adjuvant is administered in a single dose of 200 μg.

[49] The use according to

[43] , wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

[50] The use according to

[43] , wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 100 μg.

[51] The use according to

[43] , wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

[52] The use according to

[43] , wherein the antigen protein is administered in a single dose of 400 μg, and the adjuvant is administered in a single dose of 400 μg.

[53] The use of any of

[43] to

[52] , wherein the compound is administered once to ten times every one to five years, and the number of administrations per year is no more than two.

[54] The use described in any of

[43] to

[53] , wherein the compound is administered once or twice a year.

[55] The use of

[53] or

[54] , wherein the compound is administered twice a year, with the second administration being administered four weeks after the first administration.

[56] The use described in any of

[43] to

[55] , wherein the administration is intramuscular administration.

[57] The use described in any of

[43] to

[55] , wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

[58] The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The use according to any one of

[43] to

[57] , comprising a complex consisting of:

[59] The antigenic protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 4). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO: 4. The use according to any one of

[43] to

[58] , wherein the compound is a protein.

[60] The use described in any of

[43] to

[58] , wherein the antigen protein and the adjuvant are held in the same container.

[61] The use described in any of

[43] to

[58] , wherein the antigen protein and the adjuvant are held in separate containers and mixed prior to administration.

[62] The use described in any of

[43] to

[61] , wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.1.

[63] The use described in any of

[43] to

[62] , wherein the complex has a triple helix structure.

[64] An antigenic protein in combination with an adjuvant for the prevention or treatment of respiratory syncytial virus infection in humans, comprising: the antigenic protein is (a) an antigenic protein derived from an F protein of RS virus, The adjuvant comprises: (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3'-terminus of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are substituted with phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of The antigen protein and the adjuvant are administered in combination to a human; The antigen protein combined with an adjuvant, wherein the antigen protein is administered in a single dose of 100 to 400 μg, and the adjuvant is administered in a single dose of 100 to 400 μg.

[65] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 100 μg, and the adjuvant is administered in a single dose of 100 μg.

[66] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

[67] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

[68] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

[69] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg.

[70] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

[71] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg.

[72] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

[73] An antigen protein combined with an adjuvant according to

[64] , wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 400 μg.

[74] An antigen protein combined with an adjuvant according to any one of

[64] to

[73] , which is administered once to ten times every one to five years, and the number of administrations per year is no more than two.

[75] An antigen protein combined with an adjuvant according to any one of

[64] to

[74] , which is administered once or twice a year.

[76] An antigen protein combined with an adjuvant according to

[74] or

[75] , administered twice a year, with the second administration being given four weeks after the first.

[77] The antigen protein combined with an adjuvant according to any one of

[64] to

[76] , wherein the administration is intramuscular administration.

[78] An antigen protein combined with an adjuvant described in any of

[64] to

[77] , wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

[79] The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An antigen protein combined with an adjuvant according to any one of

[64] to

[78] , comprising a complex consisting of:

[80] The antigenic protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 4). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO: 4. An antigen protein combined with an adjuvant according to any one of

[64] to

[79] , wherein the adjuvant is a protein.

[81] An antigen protein combined with an adjuvant according to any one of

[64] to

[80] , wherein the antigen protein and the adjuvant are held in the same container.

[82] An antigen protein combined with an adjuvant according to any one of

[64] to

[80] , wherein the antigen protein and the adjuvant are held in separate containers and mixed prior to administration.

[83] An antigen protein combined with an adjuvant described in any of

[64] to

[82] , wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.1.

[84] An antigen protein combined with an adjuvant according to any one of

[64] to

[83] , wherein the complex has a triple helix structure. Effect of the Invention

[0012] The present invention provides respiratory syncytial virus vaccines and methods of use that induce both humoral and cellular immune responses against respiratory syncytial virus antigens. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly by this.

[0014] 1. Respiratory syncytial virus Respiratory syncytial virus (RSV) is classified as a member of the Orthopneumovirus genus of the Pneumoviridae family, and is known to cause acute respiratory infections and acute lower respiratory tract infections such as bronchiolitis and pneumonia in children. RS viruses are classified into subgroups A and B (RSV A strain and RSV B strain), and the virus surface contains a G protein that contributes to adhesion to the host and an F protein that causes membrane fusion, and these two proteins, the F protein and the G protein, are thought to be involved in infection. In this specification, when describing RS virus, it includes all RS viruses, and includes all common RS viruses that cause infectious diseases.In this specification, RSV A strain specifically includes RSV A2 and the like, and RSV B strain specifically includes RSV B WV / 14617 / 85 and the like, but the RS virus of the present invention is not limited thereto.

[0015] 2. Antigenic proteins derived from respiratory syncytial virus The G protein of RS virus is highly glycosylated and antigenically heterogeneous. In contrast, the F protein is relatively conserved, and some anti-F protein neutralizing mAbs such as palivizumab can protect against strains A and B. Therefore, the antigen protein used in the present invention is preferably the F protein of RS virus (also referred to as the antigen protein of the present invention). The antigen protein of the present invention includes a protein having the entire amino acid sequence of the F protein encoded in the genome of RS virus, but also includes a protein having an amino acid sequence obtained by editing the entire amino acid sequence of the F protein by a conventional genetic recombination technique for the purpose of improving productivity during production and improving immunogenicity during vaccination, such as a glycoprotein in which a part of the sequence involved in cell membrane fusion function and the C-terminal sequence are deleted.

[0016] The amino acid sequence of the antigenic protein of the present invention is not particularly limited as long as it can generate immunity against RS virus. The antigenic protein of the present invention includes, for example, (A)(i) peptide I, which contains an amino acid sequence having 95% or more (96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3) or is a peptide consisting of said amino acid sequence, and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; (ii) Peptide II, which contains an amino acid sequence having 95% or more (96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO: 4 (provided that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 4) or consists of the amino acid sequence. Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO: 4. It may be a protein (CAS RN: 2769988-12-5). Alternatively, as an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (or the amino acid sequence shown in SEQ ID NO: 4), an amino acid sequence in which one to several amino acid residues have been substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 3 (or the amino acid sequence shown in SEQ ID NO: 4) can also be selected as the amino acid sequence of the antigen protein of the present invention. The "several" means, for example, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0017] In this specification, sequence identity between two types of amino acid sequences can be determined by aligning the sequences using default parameters of ClustalW version 2 (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG (2007), "Clustal W and Clustal X version 2.0", Bioinformatics. 23 (21): 2947-2948).

[0018] The antigen protein can be prepared according to a conventional method. For example, the antigen protein can be produced by expressing a vector having a nucleic acid molecule encoding the antigen protein of the present invention in a host cell. The host cell can be selected from, for example, mammalian cells, such as Chinese hamster ovary (CHO) cells, human cell lines (e.g., HEK293 cells and their derivatives), yeast, fungi, insect cells, and the like. Furthermore, when producing the antigen protein of the present invention in a host cell, it is necessary to introduce a nucleic acid molecule encoding the protein in an expressible state into the host cell, and to culture the host cell. The nucleic acid molecule encoding the antigen protein of the present invention may be codon-optimized by a conventional method for expression in a host cell such as a mammalian cell. In one embodiment, the antigen protein of the present invention may be produced by a genetically modified CHO cell. The nucleic acid sequences used for protein expression may be produced and / or synthesized and / or cloned by methods commonly used in the art.

[0019] 3. Polydeoxynucleotides The polydeoxynucleotide (PDN) used in the present invention is a polydeoxynucleotide (also referred to as the polydeoxynucleotide of the present invention) that includes a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid. In this specification, polydeoxynucleotide and PDN have the same meaning. In this specification, polydeoxyadenylic acid may also be referred to as "poly(dA)" or "poly(dA)".

[0020] In the polydeoxynucleotide of the present invention, poly dA is located on the 3' side of K-type CpG ODN, so that the complex of the present invention (details will be described below) has an immunostimulatory activity specific to D-type CpG ODN in addition to the immunostimulatory activity specific to K-type CpG ODN. Therefore, more specifically, the polydeoxynucleotide of the present invention is a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid bound to the 3' side of the humanized K-type CpG oligodeoxynucleotide.

[0021] K-type CpG ODN contains one or more non-palindromic unmethylated CpG motifs and has the structural and functional properties of activating B cells to produce IL-6 but hardly inducing IFN-α production in plasmacytoid dendritic cells (pDCs). The unmethylated CpG motif refers to a short nucleotide sequence containing at least one cytosine (C)-guanine (G) sequence, in which the 5th position of cytosine in the cytosine-guanine sequence is not methylated. In the following description, CpG means unmethylated CpG unless otherwise specified. The K-type CpG ODN used in the present invention contains a non-palindromic structure containing one or more CpG motifs, and the K-type CpG ODN may be composed of a non-palindromic structure containing one or more CpG motifs. The polydeoxynucleotide of the present invention, including the humanized K-type CpG ODN, has an immunostimulatory activity specific to K-type CpG ODN in humans (for example, activity of activating human B cells to produce IL-6). Here, in the present specification, "humanized" means having agonistic activity against human TLR9. The humanized K-type CpG ODN may have a four-base CpG motif consisting of TCGA or TCGT. In addition, one humanized K-type CpG ODN may contain two or three four-base CpG motifs. The humanized K-type CpG ODN of the present invention may contain at least one, at least two, or two or three four-base CpG motifs consisting of TCGA or TCGT. When the K-type CpG ODN has two or three four-base CpG motifs, these four-base CpG motifs may be the same or different.

[0022] The humanized K-type CpG oligodeoxynucleotide is not particularly limited as long as it has the above-mentioned CpG motif, but may, for example, comprise a nucleotide sequence having 95% or more (96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the nucleotide sequence shown in SEQ ID NO:1, or may consist of said nucleotide sequence.

[0023] The polydeoxyadenylic acid bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide may be poly dA having a length of 20 to 60 nucleotides (e.g., 30 to 50 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides) or, for example, 30 to 45 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides)). When the polydeoxyadenylic acid attached to the 3' end of the humanized K-type CpG oligodeoxynucleotide is 40 nucleotides in length, the polydeoxynucleotide of the present invention may be a polydeoxynucleotide that includes or consists of the nucleotide sequence represented by SEQ ID NO:2.

[0024] The humanized K-type CpG ODN and poly dA may be linked directly by a covalent bond, or may be linked via a spacer sequence. The spacer sequence refers to a nucleotide sequence containing one or more nucleotides inserted between two adjacent components. The length of the spacer sequence is not particularly limited as long as the complex of the present invention has immunostimulatory activity, but may usually be 1 to 10 nucleotides, 1 to 5 nucleotides, or 1 to 3 nucleotides. The humanized K-type CpG ODN and poly dA may be linked directly by a covalent bond.

[0025] The polydeoxynucleotide of the present invention may have an additional nucleotide sequence at its 5'-end and / or 3'-end in addition to the humanized K-type CpG ODN, poly dA, and an optional spacer sequence. The length of the additional nucleotide sequence is not particularly limited as long as the complex of the present invention has immunostimulatory activity, but is usually 1 to 10 nucleotides long, preferably 1 to 5 nucleotides long, and more preferably 1 to 3 nucleotides long.

[0026] In the polydeoxynucleotide of the present invention, in the humanized K-type CpG ODN and polydeoxyadenylic acid, some or all of the phosphodiester bonds are appropriately replaced with phosphorothioate bonds (i.e., one of the non-bridging oxygen atoms is replaced with a sulfur atom as described in WO 95 / 26204) or phosphorodithioate bonds so as to be resistant to in vivo degradation (e.g., degradation by exonucleases or endonucleases), and preferably, some or all of the phosphodiester bonds in the polydeoxynucleotide of the present invention are replaced with phosphorothioate bonds. In the polydeoxynucleotide of the present invention, phosphorothioate bonds are expected to not only provide resistance to degradation, but also enhance immunostimulatory activity (e.g., activity to activate B cells to produce IL-6), and to provide a high yield of CpG-β-1,3-glucan complex. In this specification, a phosphorothioate bond is synonymous with a phosphorothioate backbone, and a phosphodiester bond is synonymous with a phosphate backbone.

[0027] Therefore, more specifically, the polydeoxynucleotide of the present invention may be a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid of 20 to 60 nucleotides in length bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and in which some or all of the phosphodiester bonds of the polydeoxynucleotide are substituted with phosphorothioate bonds or phosphorodithioate bonds. Alternatively, the polydeoxynucleotide of the present invention may be a polydeoxynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

[0028] The polydeoxynucleotides of the present invention include any pharma- ceutically acceptable salts, esters, or salts of such esters of polydeoxynucleotides. Pharmaceutically acceptable salts of the polydeoxynucleotide of the present invention preferably include metal salts such as alkali metal salts, such as sodium salt, potassium salt, and lithium salt, alkaline earth metal salts, such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts, such as ammonium salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, Examples of the salt include amine salts, such as organic salts like piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; inorganic salts like hydrohalogen salts like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrates, perchlorates, sulfates, and phosphates; organic salts like lower alkane sulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, arylsulfonates like benzenesulfonate and p-toluenesulfonate, acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts like glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, and aspartate salts.

[0029] The polydeoxynucleotide of the present invention may be in the form of a single strand, a double strand, or a triple strand, but is preferably a single strand.

[0030] The polydeoxynucleotide of the present invention is preferably isolated. "Isolated" means that the polydeoxynucleotide has been subjected to an operation for removing factors other than the target component and is free from the naturally occurring state. The purity of the (isolated) polydeoxynucleotide (the percentage of the mass of the target polydeoxynucleotide in the total mass of the evaluation target) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more.

[0031] The polydeoxynucleotide of the present invention has excellent immunostimulatory activity by itself, and also has the property of forming a triple helix structure together with lentinan. The method for producing the polydeoxynucleotide of the present invention is not particularly limited, and a conventional method can be used, for example, synthesis can be performed using the solid phase phosphoramidite method.

[0032] 4. Lentinan The lentinan used in the present invention (also referred to as the lentinan of the present invention) is a β-1,3-glucan that contains a large amount of 1,6-glucopyranoside branches (for example, side chain ratio of 33 to 40%). Lentinan (LNT) is a known β-1,3-1,6-glucan derived from shiitake mushrooms, and has the molecular formula (CH 10O5)n, and its molecular weight is about 300,000 to 700,000. Lentinan is practically insoluble in water, methanol, ethanol (95), or acetone, but is soluble in polar organic solvents such as DMSO and aqueous sodium hydroxide. Lentinan enhances the activity of activated macrophages, killer T cells, natural killer cells, and antibody-dependent macrophage-mediated cytotoxicity (ADMC) (Hamuro, J., et al.: Immunology, 39, 551-559, 1980; Hamuro, J., et al.: Int. J. Immunopharmacol., 2, 171, 1980; Herlyn, D., et al.: Gann, 76, 37-42, 1985). In animal experiments, it has been shown that combined administration of lentinan with chemotherapy drugs has a tumor growth inhibitory effect and a life-prolonging effect against syngeneic and autologous tumors. In addition, administration of lentinan alone has also been shown to have a tumor growth inhibitory effect and a life-prolonging effect. In clinical trials, Lentinan has been shown to extend the survival time of patients with inoperable or recurrent gastric cancer when used in combination with oral tegafur (Drug interview form "Lentinan 1 mg for intravenous injection "Ajinomoto""). Furthermore, the lentinan of the present invention may be substantially free of chloroform.

[0033] The method for producing the lentinan of the present invention is not particularly limited, and for example, the method described in Patent Document 1 may be used. In addition, as a method for producing lentinan of the present invention, as a means for obtaining lentinan of higher purity and in higher yield, it is effective to isolate and use the floating fraction produced by adding an organic solvent such as ethanol to the hot water extract obtained from the fruiting body of Lentinus edodes or a hot aqueous solution containing lentinan, and to perform a high-concentration alkali treatment at a low temperature. According to this production method, the purification step using chloroform, which is required in the commonly used production method of lentinan, is not necessary. In addition, the lentinan obtained by this production method can improve the efficiency of forming the complex of the present invention. Specifically, the lentinan of the present invention may be obtained by a process including the following steps: (1) a step of separating and obtaining a floating fraction, the step comprising the steps of: (a) performing a step of separating and obtaining a floating fraction; (a) Extraction of lentinan from shiitake mushrooms with hot water (b) Add alcohol to the hot water extract of (a) and mix. (c) Leave it at 18℃ to 25℃ for 15 to 24 hours. (d) collecting the floating fraction produced by (c); (2) dissolving the separated floating fraction in a 2.5M aqueous sodium hydroxide solution to a concentration of 25 mg / mL, treating at 2°C to 8°C for 15 to 30 hours, then adding purified water to a floating fraction concentration of 5 mg / mL and a sodium hydroxide concentration of 0.5 M, and treating at 2°C to 8°C for 15 to 30 hours to destroy the higher-order structure of lentinan; (3) A process for controlling the molecular weight of lentinan to 150 kD to 270 kD by reacting 5 mg / mL of lentinan obtained in step (2) with 0.5 M sodium hydroxide solution at 40°C to 50°C for a predetermined time.

[0034] 5. Adjuvants (complexes) The adjuvant used in the present invention is an adjuvant consisting of a complex of the polydeoxynucleotide of the present invention and lentinan (also referred to as the complex of the present invention), or an adjuvant containing the complex.

[0035] As used herein, the term "complex" refers to a product obtained by association of multiple molecules via non-covalent or covalent bonds such as electrostatic bonds, van der Waals bonds, hydrogen bonds, and hydrophobic interactions.

[0036] The complex of the present invention may be in the form of a triple helix structure. In a preferred embodiment, two of the three strands forming the triple helix structure are lentinan strands, and one is a polydeoxyadenylic acid strand in the polydeoxynucleotide of the present invention. The complex may also include a portion that does not form a triple helix structure.

[0037] The composition ratio of polydeoxynucleotide and lentinan in the complex of the present invention can vary depending on the chain length of polydeoxyadenylic acid in polydeoxynucleotide and the length of lentinan. For example, when the length of the β-1,3-glucan chain constituting lentinan and the chain length of polydeoxyadenylic acid are equivalent, two β-1,3-glucan chains and one polydeoxynucleotide of the present invention can be associated to form a triple helix structure. Generally, the chain length of polydeoxyadenylic acid is short compared to the β-1,3-glucan chain, so multiple polydeoxynucleotides of the present invention are associated with two β-1,3-glucan chains via polydeoxyadenylic acid to form a triple helix structure.

[0038] The polydeoxynucleotide of the present invention, by forming a complex with lentinan, acquires the immunostimulatory activity specific to D-type CpG ODN (e.g., activity of activating plasmacytoid dendritic cells to produce IFN-α) without requiring the sequence of D-type CpG ODN. That is, the complex of the present invention has both the immunostimulatory activity specific to K-type CpG ODN (e.g., activity of activating human B cells to produce IL-6) and the immunostimulatory activity specific to D-type CpG ODN (e.g., activity of activating human plasmacytoid dendritic cells to produce IFN-α).

[0039] The method for preparing the complex of the present invention is not particularly limited, but can be carried out under the same conditions as those described in Patent Document 1, for example. That is, lentinan, a type of β-1,3-glucan that naturally exists as a triple helix structure, is dissolved in an aprotic organic polar solvent or an alkaline aqueous solution and unwound into a single strand. The single-stranded lentinan solution thus obtained is mixed with a solution of the polydeoxynucleotide of the present invention (a buffer aqueous solution with a pH close to neutral) and kept for an appropriate time, for example, kept overnight at 5°C. As a result, the two lentinan strands and the poly dA strand in the polydeoxynucleotide form a triple helix structure, thereby forming the complex of the present invention.

[0040] The mixing ratio of the polydeoxynucleotide of the present invention to lentinan can be appropriately set taking into consideration the length of the poly dA chain, etc., and the molar ratio (LNT / PDN) may usually be 0.02 to 2.0, or 0.1 to 0.5.

[0041] In one embodiment, the complex of the present invention has a rod-shaped particle shape. The particle size is equivalent to the particle size formed by the lentinan used as a material naturally exhibiting a triple helical structure, and the average particle size may be 10 to 100 nm, or 20 to 50 nm, or may be 100 to 180 nm, or 130 to 180 nm. The particle size can be measured by dissolving the complex in water and measuring it by dynamic light scattering at 80°C using a Malvern Instruments Zeta Sizer.

[0042] The complex of the present invention is preferably isolated. The purity of the "isolated complex" (the percentage of the mass of the target complex in the total mass of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more.

[0043] 6. Vaccines The vaccine used in the present invention is a medicine (also referred to as the vaccine of the present invention) containing a combination of the antigen protein of the present invention and the adjuvant of the present invention. The vaccine of the present invention has a high therapeutic effect and / or preventive effect against either or both of the RSV A strain and the RSV B strain of RS virus. In humans, the ratio of the antigen protein antibody titer one year after the previous administration of the vaccine of the present invention to the antigen protein antibody titer four weeks after the previous administration of the vaccine of the present invention is at least 0.1, and may be 0.2 or more, or may be 0.5 or more. The antibody titer can be examined, for example, by the same method as in this embodiment. Furthermore, the vaccine of the present invention can induce both humoral and cellular immune responses. For example, as shown in the Examples, the humoral immune response can be confirmed by anti-RSV neutralizing activity in the blood, and the cellular immune response can be confirmed by antigen-specific IFN-γ production response.

[0044] The vaccine of the present invention may be administered once to ten times every year to five years, in which case the number of administrations per year may be two or less. For example, the vaccine of the present invention may be administered once every two years or once every five years, for example, twice in the first year and once or twice in the fifth year. The vaccine of the present invention may also be administered once or twice per year. When administered twice per year, the second administration may be administered four weeks after the first administration.

[0045] The vaccine of the present invention can be obtained by formulating the antigen protein of the present invention and an adjuvant according to a conventional method. The vaccine of the present invention may further contain a pharmacologically acceptable carrier. The vaccine of the present invention may further contain an antigen other than the antigen derived from RS virus. The vaccine of the present invention may contain additives such as pharmacologic acceptable excipients, solvents, stabilizers, buffers, dispersants, pH adjusters, and preservatives in addition to the antigen protein derived from RS virus F protein and the adjuvant of the present invention. Examples of the carrier and additive include polysaccharides, polylactic acid, polyglycolic acid, amino acids (such as L-histidine), amino acid salts (such as L-histidine hydrochloride hydrate), polymerized amino acids, amino acid copolymers, sucrose, trehalose, lactose, sugar alcohols (such as D-sorbitol), emulsifiers (such as polysorbate 20), acetate buffer salts, phosphate buffer salts, citrate buffer salts, borate buffer salts, tartrate buffer salts, Tris buffer salts, glycine buffer salts, and arginine buffer salts, as well as combinations thereof.

[0046] The vaccine of the present invention is provided in a dosage form suitable for oral or parenteral administration. Vaccines for parenteral administration include, for example, injections, suppositories, etc., and the injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. The vaccine of the present invention may also be an intramuscular injection for intramuscular administration. In addition, the vaccine of the present invention, for example, an injection, may contain an antigen protein and an adjuvant in the same container. In addition, the vaccine of the present invention, particularly an injection, may contain an antigen protein and an adjuvant in separate containers, and the antigen protein and the adjuvant may be administered simultaneously or mixed before administration. Such an injection can be prepared according to a known method. For example, the injection may be prepared by dissolving or suspending the polydeoxynucleotide of the present invention and / or the adjuvant in a sterile aqueous solvent that is usually used for injections. Examples of the aqueous solvent for injection include distilled water; physiological saline; and buffers such as phosphate buffer, carbonate buffer, Tris buffer, and acetate buffer. The pH of such an aqueous solvent is 5 to 10, and preferably 6 to 8. The prepared injection may be filled into an appropriate ampule or vial.

[0047] For example, a powder formulation of the vaccine of the present invention may be prepared by subjecting a suspension of the antigen protein and / or adjuvant of the present invention to vacuum drying, freeze-drying, etc. The vaccine of the present invention may be stored in a powder state, and when used, the powder may be dispersed in an aqueous solvent for injection, for use.

[0048] The vaccine of the present invention is for human use, and the antigen protein is administered in an amount of 100 to 400 μg per dose, and the adjuvant is administered in an amount of 100 to 400 μg per dose. The vaccine of the present invention may be administered in an amount of 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, or 400 μg per dose of the antigen protein. The vaccine of the present invention may be administered in an amount of 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, or 400 μg per dose of the adjuvant. Furthermore, the vaccine of the present invention may be administered in such a manner that the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg, or the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg, or the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg, or the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 1 .... The dose of the antigen protein may be 200 μg and the dose of the adjuvant may be 200 μg, the dose of the antigen protein may be 200 μg and the dose of the adjuvant may be 400 μg, the dose of the antigen protein may be 400 μg and the dose of the adjuvant may be 100 μg, the dose of the antigen protein may be 400 μg and the dose of the adjuvant may be 200 μg, or the dose of the antigen protein may be 400 μg and the dose of the adjuvant may be 400 μg and the dose of the adjuvant may be 400 μg. In this specification, the dose of the adjuvant is synonymous with the dose of the complex of the present invention.

[0049] 7. Vaccine mechanism The complex of the present invention contained in the vaccine of the present invention has excellent immunostimulating activity, and is therefore used as an adjuvant, i.e., an immunostimulant. By administering the adjuvant of the present invention to a human, an immune response can be induced in the human. In particular, the complex of the present invention has the activity characteristics of K-type CpG ODN, and stimulates peripheral blood mononuclear cells to produce a large amount of both type I interferon (Pan-IFN-α, IFN-α2, etc.) and type II interferon (IFN-γ), and is therefore useful as a type I interferon production inducer, a type II interferon production inducer, or a type I and type II interferon production inducer. Since it induces the production of both type I and type II interferon, the complex of the present invention or a vaccine containing the same is useful for preventing or treating diseases for which either or both type I and type II interferon are effective. Examples of diseases for which type I interferon is effective include RS virus infection according to the present invention, other viral infections (e.g., hepatitis C virus (HCV), herpes virus, papilloma virus, influenza virus, etc.), and cancer. Diseases for which type II interferon is effective include allergies, infections caused by intracellular parasitic protozoa (e.g., Leishmania) and bacteria (e.g., Listeria, Mycobacterium tuberculosis), etc. For acute viral infections including RS virus and influenza virus, both type I interferon and type II interferon enhance immune responses related to viral elimination, and therefore the complex of the present invention or a vaccine containing the same is expected to be effective against acute viral infections.

[0050] Furthermore, the polydeoxynucleotide or complex of the present invention, particularly the complex of the present invention, has a strong adjuvant activity, and when the polydeoxynucleotide or complex of the present invention is administered to a mammal, for example, a human, together with an antigen, it can strongly induce an immune response against the antigen. In particular, the complex of the present invention strongly induces both humoral and cellular immune responses against the antigen.

[0051] By including an antigen derived from RS virus in the vaccine of the present invention, an immune response against the antigen is induced, and a mechanism for immunologically eliminating RS virus containing the antigen from the body is established. Therefore, the vaccine of the present invention for inducing an immune response against the antigen protein is useful for preventing or treating RS virus infection.

[0052] The complex of the present invention potently induces both humoral and cellular immune responses to antigens, and is therefore used in combination with antigens derived from intracellular respiratory syncytial virus (RSV), which are known to be recognized by cytotoxic T lymphocytes.

[0053] Since the complex of the present invention potently induces both type I and type II interferons, in one embodiment, respiratory syncytial virus that causes acute viral infection against which both type I interferon and type II interferon are effective is selected as the virus.

[0054] The contents of all publications, including patents and patent applications, cited in this specification are hereby incorporated by reference in their entirety to the same extent as if fully set forth herein.

[0055] The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. Furthermore, the examples should not be construed as being limiting in any sense. EXAMPLES

[0056] [Manufacturing example: Preparation of antigen protein] The antigen protein was a glycoprotein composed of F1 and F2 chains, in which a part of the sequence involved in the cell membrane fusion function and the C-terminal sequence were deleted from the entire amino acid sequence of the F protein of the RS virus. Specifically, a dimeric antigen protein drug substance was prepared by intermolecular cross-linking between the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4, in which a part of the F protein of the RS virus is modified, between C293 of the amino acid sequence shown in SEQ ID NO: 3 and C12 of the amino acid sequence shown in SEQ ID NO: 4, and between C66 of the amino acid sequence shown in SEQ ID NO: 3 and C44 of the amino acid sequence shown in SEQ ID NO: 4, by disulfide bonds. The antigen protein was produced by genetically recombinant CHO cells according to a conventional method. Hereinafter, in this example, the antigen protein is also referred to as RSV-rec-F.

[0057] F1 chain 1 AIASGVAVSK VLHLEGEVNK IKSALLSTNK AVVSLSNGVS VLTSKVLDLK 51 NYIDKQLLPI VNKQSCSISN IETVIEFQQK NNRLLEITRE FSVNAGVTTP 101 VSTYMLTNSE LLSLINDMPI TNDQKKLMSN NVQIVRQQSY SIMSIIKEEV 151 LAYVVQLPLY GVIDTPCWKL HTSPLCTTNT KEGSNICLTR TDRGWYCDNA 201 GSVSFFPQAE TCKVQSNRVF CDTMNSLTLP SEVNLCNVDI FNPKYDCKIM 251 TSKTDVSSSV ITSLGAIVSC YGKTKCTASN KNRGIIKTFS NGCDYVSNKG 301 VDTVSVGNTL YYVNKQEGKS LYVKGEPIIN FYDPLVFPSD EFDASISQVN 351 EKINQSLAFI RKSDELLHNV NAGKSTTNIM ITT (SEQ ID NO: 3) (In the amino acid sequence shown in SEQ ID NO:3, intramolecular cross-links are formed between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 via disulfide bonds.)

[0058] F2 chain 1 QNITEEFYQS TCSAVSKGYL SALRTGWYTS VITIELSNIK KNKCNGTDAK 51 VKLIKQELDK YKNAVTELQL LMQSTQATNN RARR (Sequence number 4)

[0059] [Manufacturing Example: Preparation of Adjuvant] The adjuvant, which is a complex used in the present invention, was produced by referring to the production method described in Patent Document 1 (WO2015 / 041318) except for the production method of lentinan. The lentinan of the present invention was prepared by the following method. Lentinan was extracted from shiitake mushrooms with hot water, and alcohol was added to the obtained hot water extract and mixed, and the mixture was left at 18°C ​​to 25°C for 15 to 24 hours, and the resulting floating fraction was collected to separate and obtain the floating fraction. The floating fraction was then dissolved in 2.5M aqueous sodium hydroxide solution so that the concentration of the separated and obtained floating fraction was 25mg / mL, and treated at 2°C to 8°C for 15 to 30 hours, and then purified water was added to make the floating fraction concentration 5mg / mL and the sodium hydroxide concentration 0.5M, and treated at 2°C to 8°C for 15 to 30 hours to destroy the higher-order structure of lentinan. The obtained 5mg / mL lentinan was reacted with 0.5M sodium hydroxide solution at 40°C to 50°C for a predetermined time, thereby controlling the molecular weight of lentinan to 150kD to 270kD, and purified to obtain the lentinan used in the present invention.

[0060] The polydeoxynucleotide of sequence number 2, which is composed of a humanized K-type CpG oligodeoxynucleotide having the nucleotide sequence of sequence number 1 and a 40-nucleotide polydeoxyadenylic acid linked to the 3' end of the humanized K-type CpG oligodeoxynucleotide, was synthesized using the conventional solid-phase phosphoramidite method. The adjuvant drug substance of the present invention, which is a complex of lentinan and polydeoxynucleotide, was prepared as follows. Lentinan was dissolved in 0.25 M NaOH aqueous solution at 20°C to a concentration of approximately 40 mg / mL, and left overnight at 5°C. The polydeoxynucleotide of the present invention was separately dissolved in water for injection to a concentration of 0.56 mM, and cooled to 5°C. The prepared lentinan solution and polydeoxynucleotide solution were mixed so that the weight ratio of lentinan to polydeoxynucleotide was approximately 0.85:1, and then 0.3 M NaH2PO4 aqueous solution was added to neutralize, forming a complex to produce an adjuvant drug substance. The formation of the complex was confirmed by monitoring the absorption at 260 nm using size exclusion chromatography to shift the polydeoxynucleotide to a higher molecular weight side.

[0061] Humanized K-type CpG oligodeoxynucleotides AsTsCsGsAsCsTsCsTsCsGsAsGsCsGsTsTsCsTsCsC (SEQ ID NO: 1) (The s in the above sequence indicates that the internucleoside phosphodiester bond has been replaced with a phosphorothioate bond.)

[0062] Humanized K-type CpG oligodeoxynucleotide + 40 nucleotide long polydeoxyadenylic acid AsTsCsGsAsCsTsCsTsCsGsAsGsCsGsTsTsCsTsCsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsAsA (sequence number 2) (The s in the above sequence indicates that the internucleoside phosphodiester bond has been replaced with a phosphorothioate bond.)

[0063] [Manufacturing example: Vaccine (active drug)] The antigen protein (RSV-rec-F) drug substance was sterile filtered and filled into a washed and sterilized colorless glass vial. After filling, the vial was stoppered with a washed and sterilized butyl rubber stopper and then sealed with an aluminum cap to produce an RSV-rec-F formulation. The adjuvant formulation was also produced from the adjuvant drug substance in the same manner. In addition to RSV-rec-F and the adjuvant, the vaccine (active drug) contained pharma- ceutical acceptable additives such as stabilizers and buffers. The RSV-rec-F formulation and the adjuvant formulation were mixed before administration and administered to a subject or a test animal. The vaccine of the present invention may be a formulation in which an antigen protein and an adjuvant are mixed from the beginning. In this example, the amount of adjuvant is synonymous with the amount of complex.

[0064] [Preliminary experiment] Non-clinical trial For immunogenicity tests, RSV-rec-F alone, adjuvant alone, or the vaccine of the present invention (RSV-rec-F and adjuvant) was administered intramuscularly to BALB / c mice, and the anti-RSV-rec-F antibody response in the blood, anti-RSV neutralizing activity in the blood, and RSV-rec-F-specific cellular immune responses were evaluated. In the antibody response test, a positive correlation was observed between the RSV-rec-F dose and the anti-RSV-rec-F immunoglobulin G (IgG) titer in the blood, and a positive correlation was also observed between the adjuvant dose and the anti-RSV-rec-F IgG titer in the blood. In the neutralizing activity test, RSV-rec-F dose-dependent anti-RSV neutralizing activity in the blood was observed, and the addition of adjuvant resulted in significantly higher anti-RSV neutralizing activity in the blood. In cellular immune response tests, the addition of adjuvant significantly increased RSV-rec-F-specific interferon (IFN)-γ production levels, but significantly decreased RSV-rec-F-specific interleukin (IL)-13 production levels compared to the RSV-rec-F alone group.

[0065] These results suggest the usefulness of administering RSV-rec-F in combination with an adjuvant in terms of vaccine antibody responses, neutralizing activity, and specific cellular immune responses. In addition, in an infection protection test, BALB / c mice were intramuscularly administered RSV-rec-F alone, adjuvant alone, or the vaccine of the present invention (RSV-rec-F and adjuvant), and then infected with RSV and the virus titer in the lungs was measured. As a result, a significant reduction in the virus titer in the lungs was observed by administering a combination of RSV-rec-F (1 or 5 μg / body) and adjuvant (10 μg / body) compared to the group administered RSV-rec-F alone. This result suggests the usefulness of administering RSV-rec-F in combination with an adjuvant in terms of the infection protection effect of RSV-rec-F.

[0066] [Example 1] Phase 1 study A single-center, randomized, placebo-controlled, double-blind, dose-escalation study in which some personnel (those who manage and prepare the investigational drug) may be unblinded. the purpose: To evaluate the safety and tolerability of the vaccine according to the present invention when administered intramuscularly to healthy Japanese adults (aged 20 to 50 years) and healthy elderly people (aged 65 to 80 years). To evaluate the immunogenicity of the vaccine when administered intramuscularly to healthy adult and healthy elderly Japanese subjects.

[0067] Step 1 Cohorts 1 to 3 (including a placebo group) were composed of healthy adult subjects. Eight subjects in each cohort were randomly assigned to either the active drug group or the placebo group in a 3:1 ratio. At each step, treatment began in cohort 1 and progressed to the next cohort. Step 2 After completing step 1, cohorts 1 to 3 (including a placebo group) were formed from healthy elderly subjects. Eight subjects in each cohort were randomly assigned to either the active drug group or the placebo group in a 3:1 ratio. In each step, treatment began in cohort 1 and progressed to the next cohort.

[0068] Dosage and administration method The drug was administered intramuscularly into the deltoid muscle of the upper arm twice at four-week intervals. The number of subjects in each cohort receiving the active drug was 6, and the doses of antigen protein (RSV-rec-F) and adjuvant were as shown in the table below. The amounts of antigen protein and adjuvant were set based on the results of non-clinical pharmacological studies.

[0069] [Table 1]

[0070] In the table below, cohorts 1 to 3 refer to the active drug administration groups of each cohort unless otherwise specified.

[0071] [Evaluation Example 1] Measurement of anti-RSV neutralizing activity in blood Venous blood was collected from the subject's forearm vein to prepare serum, which was then frozen and transported to the measurement facility. The serum was inactivated and used for the measurement. Serially diluted sample serum and WHO standard serum were mixed with RSV (A2 strain, 50 PFU) to perform a neutralization reaction, and the reaction mixture was added to HEp-2 cells and cultured for 3 days. The RSV F protein expressed in RSV-infected HEp-2 cells was detected with an anti-RSV F protein antibody labeled with horse radish peroxidase. The serum dilution ratio (ED50) at which the RSV proliferation rate was 50% was determined for each of the sample serum and standard serum, and the anti-RSV neutralizing activity (IU / mL) was calculated using the following formula: anti-RSV neutralizing activity of each sample (IU / mL) = ED50 of each sample / (ED50 of standard serum / 2000).

[0072] [Table 2]

[0073] [Table 3]

[0074] In the elderly, neutralizing activity increased relative to baseline for all doses and time points in the active drug groups (cohorts 1 to 3), and the lower limit of the 95% confidence interval for GMFR exceeded 1. In adults, neutralizing activity also increased relative to baseline for all doses and time points in the active drug groups, and the lower limit of the 95% confidence interval for GMFR exceeded 1 on Day 29 and Day 57 in Cohort 1, and on Day 29 in Cohort 2. Thus, induction of humoral immunity was demonstrated in both adults and elderly people. There was no significant difference in values ​​between Day 29 and Day 57 in either the adult or elderly cohort. There was no clear positive dose-response relationship in the GMFR of neutralizing activity in either adults or elderly subjects.

[0075] [Evaluation Example 2] Measurement of anti-RSV-rec-F antibody titer in blood Venous blood was collected from the subject's forearm vein, and serum was prepared, frozen, and transported to the measurement facility. Serum was diluted and anti-RSV-rec-F antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) using RSV-rec-F immobilized on a solid phase. Anti-RSV-rec-F antibody titers in samples were expressed in U / mL using a calibration curve prepared by simultaneously measuring serially diluted WHO standard serum (defined as 2,000 U / mL).

[0076] [Table 4]

[0077] [Table 5]

[0078] In both adults and elderly subjects, GMT increased from baseline for all doses and time points, and the lower limit of the 95% confidence interval for GMFR exceeded 1, indicating induction of humoral immunity. There were no significant differences between the values ​​on Day 29 and Day 57 in either cohort. In elderly subjects, GMT and GMFR tended to be higher in cohort 3 than in cohorts 1 and 2, but there was no clear positive dose-response in adults.

[0079] [Evaluation Example 3] Measurement of RSV-rec-F specific IFN-γ production response Venous blood was collected from the subject's forearm vein in an anticoagulant-containing blood collection tube and transported to the measurement facility. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. Using thawed PBMCs, RSV-rec-F-specific IFN-γ production responses were measured by enzyme-linked immunospot (ELISpot) using a partial peptide of RSV-rec-F as antigen stimulation. The stimulation time was 20 hours. The results were calculated by subtracting the number of spots without stimulation and dividing them into 10 6 The numbers were expressed as the number of spots per PBMC.

[0080] [Table 6]

[0081] [Table 7]

[0082] On day 57, the number of INF-γ-producing cells increased in the active drug treatment group compared to baseline, suggesting the induction of cellular immunity. In adults, the increase from baseline in the number of INF-γ-producing cells on Day 57 was greater in the active drug groups (cohorts 1 to 3) than in the placebo group, and there was a tendency for the number to increase dose-dependently between the active drug groups. In addition, the values ​​on Day 57 tended to be higher than on Day 29. In elderly subjects, the N number on Day 57 in the placebo group was very small, making it difficult to compare with the placebo, but in Cohort 2 and Cohort 3, where measurements were obtained on Day 57, the number of INF-γ-producing cells increased 8-fold and 12-17-fold, respectively, compared to baseline. In both adult and elderly subjects, the number of INF-γ-producing cells increased after administration.

[0083] From the above results, it was shown that humoral immunity was induced in adults and elderly people by administration of the vaccine according to the present invention. On the other hand, no clear positive dose-response was observed in the humoral immune response between the dose groups. In addition, there was no significant difference in the humoral immune response between Day 29 and Day 57. In addition, in adults and elderly people, the number of INF-γ-producing cells was increased in the active drug administration group compared to baseline on Day 57, suggesting that cellular immunity was induced by vaccine administration. Although the number of data is limited, the number of INF-γ-producing cells tended to increase in a dose-dependent manner in adults and tended to be higher on Day 57 than on Day 29.

[0084] [Evaluation Example 4] Safety and tolerability evaluation No serious adverse events occurred in either adult or elderly dose cohort. In this evaluation example, specific adverse events were defined as injection site adverse events (injection site erythema, injection site swelling, injection site induration, injection site pain, injection site heat, injection site pruritus) and systemic adverse events (fever, fatigue, headache, rash [systemic / local], myalgia) that occurred between the first and second administration and 14 days after administration. The incidence of specific adverse events in step 1 (healthy adults) was 0% in the placebo group and 83.3% (15 / 18) in the overall active treatment group. Specific adverse events that were relatively common in the active treatment group and their incidence were injection site pain (0% in the placebo group, 72.2% [13 / 18] in the overall active treatment group, same order below), fatigue (0%, 22.2% [4 / 18]), and injection site heat (0%, 16.7% [3 / 18]). The incidence of specific adverse events in step 2 (healthy elderly subjects) was 16.7% (1 / 6) in the placebo group and 61.1% (11 / 18) in the active treatment group overall. The incidences of specific adverse events that were relatively common in the active treatment group were injection site pain (16.7% [1 / 6], 38.9% [7 / 18]), injection site erythema (0%, 33.3% [6 / 18]), injection site induration (0%, 33.3% [6 / 18]), injection site swelling (0%, 27.8% [5 / 18]), and injection site heat (0%, 22.2% [4 / 18]).

[0085] In adults, the unspecified adverse events (events other than local and systemic adverse events) that occurred in two or more subjects across all cohorts receiving active treatment were injection site tenderness* (27.8%; 5 / 18 subjects), all of which were determined to be causally related to administration of the active treatment. In elderly subjects, unspecified adverse events occurring in two or more subjects across all active drug cohorts were injection site redness** 16.7% (3 / 18 subjects) and injection site induration** 11.1% (2 / 18 subjects), both of which were determined to be causally related to administration of the active drug. Furthermore, in adult and elderly subjects who received the vaccine according to the present invention, the severity of specific adverse events was mild or moderate, and most of them recovered within 5 days.

[0086] The severity of all unspecified adverse events was mild or moderate, and most events disappeared within a few days. (*: If there was no pain when moving the vaccinated arm, and pain was felt only when pressing firmly on the injection site, this was collected as "injection site tenderness." (Pain was considered to be spontaneous.) **: Redness and induration measuring less than 2.5 cm in longest diameter was collected as an unspecified adverse event, not an injection site adverse event.) From the above, the safety and tolerability of the vaccine of the present invention when administered intramuscularly to adults and elderly people was confirmed in the dose range of 100 to 400 μg of antigen and 100 to 400 μg of adjuvant.

[0087] [Example 2] Phase 2 study Multicenter, randomized, double-blind, dose-finding study in which some personnel (those managing and preparing clinical trials drugs) may be unblinded the purpose: To confirm the immunogenicity of each vaccine administration group and to explore the optimal dose in healthy Japanese subjects aged 60 to 80 years

[0088] In Example 1, the GMT (Day 29 and Day 57) of blood anti-RSV neutralizing activity and anti-RSV-rec-F antibody titer after inoculation of healthy elderly people with an antigen amount of 100 to 400 μg was increased from the baseline point because the lower limit of the 95% confidence interval of GMFR exceeded 1 in all groups and time points (see the results of blood anti-RSV neutralizing activity and blood anti-RSV-rec-F antibody titer). The GMFR of blood anti-RSV neutralizing activity (Day 29 and Day 57) after inoculation of healthy elderly people with an antigen amount of 100 to 400 μg was about 2.8 to 4.5 times the baseline, and was highest (about 4.1 to 4.5 times) when the antigen amount was 100 μg, and no clear positive dose-response relationship was observed in the dose range (see the results of blood anti-RSV neutralizing activity). On the other hand, the GMFR (Day 29 and Day 57) of the anti-RSV-rec-F antibody titer relative to the baseline was higher with an antigen dose of 400 μg (approximately 33-39 times) than with an antigen dose of 100-200 μg (approximately 14-20 times) (see results for blood anti-RSV-rec-F antibody titers). Based on the above, the antigen amount in the dose finding test of this Example 2 was set in the dose range of 100 to 400 μg, with the dose being set around 100 μg and 400 μg, which are the lower and upper limits of the dose range.

[0089] In the test of Example 1, the average value of the RSV-rec-F specific IFN-γ production response (Day 29 and Day 57) after inoculation of 100 to 400 μg of adjuvant to healthy elderly people showed a numerically increasing tendency from the baseline point at any dose, and the baseline ratio of the average value was higher at 400 μg (about 12 to 16 times) than at 100 to 200 μg of adjuvant (about 8 to 12 times) (see the results of RSV-rec-F specific IFN-γ production response). In healthy adults, the baseline ratio of the average value of the RSV-rec-F specific IFN-γ production response at Day 57 after inoculation of 100 to 400 μg of adjuvant was higher at 400 μg (about 4.2 times) than at 100 to 200 μg of adjuvant (about 3.1 times) (see the results of RSV-rec-F specific IFN-γ production response). From the above, the amount of adjuvant in the dose-finding test of this Example 2 was set to a dose range of 100 to 400 μg.

[0090] (Basis for setting combination of antigen and adjuvant amounts) In Example 2, the dose of the antigen (RSV-rec-F) was set at three levels (100, 200 and 400 μg), including the lower and upper limits of the dose range of 100 and 400 μg, and the dose of the adjuvant was set at four levels (0, 100, 200 and 400 μg) covering the dose range including the use of the antigen alone (0 μg), considering that it is a new adjuvant, and a factorial design was created by combining them. With this test design, it was thought that information on the dose-response relationship of immunogenicity and safety when the antigen is used alone and in combination with each dose of the adjuvant could be obtained, and based on that, the optimal dose combination of the antigen and the adjuvant could be determined. Therefore, one of the 10 dose patterns marked with an O in the table below will be administered intramuscularly to the deltoid muscle of the upper arm twice at four-week intervals. Note that "n" in the table below indicates the number of people to be administered the drug.

[0091] [Table 8]

[0092] Each of the following items will be evaluated. Geometric mean titer (GMT) and geometric mean fold rise (GMFR) of serum anti-RSV (RSV / A, RSV / B) neutralizing activity on Day 29 and Day 57 GMT and GMFR of anti-RSV-rec-F antibody on Day 29 and Day 57 Summary statistics of RSV-rec-F-specific IFN-γ production responses on Day 29 and Day 57

[0093] In addition, the following items will also be evaluated: Geometric mean titer (GMT) and geometric mean fold rise (GMFR) of serum anti-RSV (RSV / A, RSV / B) neutralizing activity in Month 6 and Month 12 GMT and GMFR of anti-RSV-rec-F antibodies in Month 6 and Month 12 Summary statistics of RSV-rec-F-specific IFN-γ production responses in Month 6 and Month 12 [Industrial Applicability]

[0094] The present invention provides respiratory syncytial virus vaccines and methods of use that induce both humoral and cellular immune responses against respiratory syncytial virus antigens. [Sequence List Free Text]

[0095] SEQ ID NO: 1: Nucleotide sequence of humanized K-type CpG oligodeoxynucleotide SEQ ID NO: 2: Nucleotide sequence of a polydeoxynucleotide consisting of a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide SEQ ID NO: 3: Amino acid sequence of the F1 chain protein of RS virus SEQ ID NO: 4: Amino acid sequence of the F2 chain protein of RS virus

Claims

1. A vaccine for preventing or treating RS virus infection, (a) an antigenic protein derived from the F protein of respiratory syncytial virus, and (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid of 20 to 60 nucleotides in length bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of Including a combination of Administered to a human, and The above vaccine, wherein the antigen protein is administered in an amount of 100 to 400 μg per dose, and the adjuvant is administered in an amount of 100 to 400 μg per dose.

2. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg.

3. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

4. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

5. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

6. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg.

7. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

8. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg.

9. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

10. 2. The vaccine of claim 1, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 400 μg.

11. The vaccine according to any one of claims 1 to 10, which is administered 1 to 10 times every 1 to 5 years, and the number of administrations per year is not more than 2.

12. The vaccine of any one of claims 1 to 11, which is administered once or twice a year.

13. 13. The vaccine of claim 11 or 12, which is administered twice a year, with the second administration being administered four weeks after the first.

14. The vaccine of any one of claims 1 to 13, wherein the administration is intramuscular.

15. The vaccine according to any one of claims 1 to 14, wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

16. The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The vaccine according to any one of claims 1 to 15, comprising a complex consisting of:

17. The antigen protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:4 (provided that the cysteine ​​residue in the amino acid sequence shown in SEQ ID NO:4 is maintained). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO:

4. The vaccine of any one of claims 1 to 16, which is a protein.

18. The vaccine according to any one of claims 1 to 17, wherein the antigen protein and the adjuvant are held in the same container.

19. The vaccine according to any one of claims 1 to 17, wherein the antigen protein and the adjuvant are held in separate containers and are mixed before administration.

20. The vaccine according to any one of claims 1 to 19, wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.

1.

21. The vaccine according to any one of claims 1 to 20, wherein the complex is in the form of a triple helix structure.

22. A method for preventing or treating respiratory syncytial virus infection in humans, comprising: (a) an antigenic protein derived from the F protein of respiratory syncytial virus, and (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid of 20 to 60 nucleotides in length bound to the 3' end of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of administering to a human a vaccine comprising a combination of The above method, wherein the antigen protein is administered in an amount of 100 to 400 μg per administration, and the adjuvant is administered in an amount of 100 to 400 μg per administration.

23. The method according to claim 22, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg.

24. The method according to claim 22, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

25. The method according to claim 22, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

26. The method according to claim 22, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

27. The method according to claim 22, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg.

28. The method according to claim 22, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

29. The method according to claim 22, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg.

30. The method according to claim 22, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

31. The method according to claim 22, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 400 μg.

32. The method according to any one of claims 22 to 31, which is administered from 1 to 10 times every 1 to 5 years, and the number of administrations per year is not more than 2.

33. 33. The method of any one of claims 22 to 32, administered once or twice a year.

34. 34. The method of claim 32 or 33, wherein the administration is given twice a year, and the second administration is given four weeks after the first administration.

35. The method of any one of claims 22 to 34, wherein the administration is intramuscular.

36. The method according to any one of claims 22 to 35, wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

37. The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The method of any one of claims 22 to 36, comprising a complex consisting of:

38. The antigen protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:4 (provided that the cysteine ​​residue in the amino acid sequence shown in SEQ ID NO:4 is maintained). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO:

4. The method according to any one of claims 22 to 37, wherein the compound is a protein.

39. The method according to any one of claims 22 to 38, wherein the antigen protein and the adjuvant are held in the same container.

40. The method according to any one of claims 22 to 38, wherein the antigen protein and the adjuvant are held in separate containers and are mixed prior to administration.

41. The method according to any one of claims 22 to 40, wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.

1.

42. The method according to any one of claims 22 to 41, wherein the complex is in the form of a triple helix structure.

43. 2. Use of an antigenic protein in combination with an adjuvant in the manufacture of a vaccine for preventing or treating respiratory syncytial virus infection in humans, comprising: the antigenic protein is (a) an antigenic protein derived from an F protein of RS virus, The adjuvant comprises: (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3'-terminus of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of The above use, wherein the antigen protein is administered in a single dose of 100 to 400 μg, and the adjuvant is administered in a single dose of 100 to 400 μg.

44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg.

45. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

46. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

47. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

48. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg.

49. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

50. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg.

51. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

52. 44. The use according to claim 43, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 400 μg.

53. The use according to any one of claims 43 to 52, which is administered from 1 to 10 times every 1 to 5 years, and the number of administrations per year is not more than 2.

54. 54. The use according to any one of claims 43 to 53, administered once or twice a year.

55. 55. The use of claim 53 or 54, wherein the administration is given twice a year, the second administration being given four weeks after the first administration.

56. The use according to any one of claims 43 to 55, wherein the administration is intramuscular administration.

57. The use according to any one of claims 43 to 55, wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate or phosphorodithioate bonds.

58. The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan The use according to any one of claims 43 to 57, comprising a complex consisting of

59. The antigen protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:4 (provided that the cysteine ​​residue in the amino acid sequence shown in SEQ ID NO:4 is maintained). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO:

4. The use according to any one of claims 43 to 58, which is a protein.

60. The use according to any one of claims 43 to 58, wherein the antigen protein and the adjuvant are held in the same container.

61. The use according to any one of claims 43 to 58, wherein the antigen protein and the adjuvant are held in separate containers and are mixed prior to administration.

62. The use according to any one of claims 43 to 61, wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the immediately preceding administration of the vaccine is at least 0.

1.

63. The use according to any one of claims 43 to 62, wherein the complex is in the form of a triple helix structure.

64. An antigenic protein in combination with an adjuvant for the prevention or treatment of respiratory syncytial virus infection in humans, comprising: the antigenic protein is (a) an antigenic protein derived from an F protein of RS virus, The adjuvant comprises: (b) (i) a polydeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylic acid having a length of 20 to 60 nucleotides bound to the 3'-terminus of the humanized K-type CpG oligodeoxynucleotide, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An adjuvant comprising a complex consisting of The antigen protein and the adjuvant are administered in combination to a human; The antigen protein combined with an adjuvant, wherein the antigen protein is administered in a single dose of 100-400 μg, and the adjuvant is administered in a single dose of 100-400 μg.

65. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 100 μg.

66. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 200 μg.

67. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 100 μg and the adjuvant is administered in a single dose of 400 μg.

68. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 100 μg.

69. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 200 μg.

70. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 200 μg and the adjuvant is administered in a single dose of 400 μg.

71. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 100 μg.

72. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 200 μg.

73. 65. The antigen protein combined with an adjuvant according to claim 64, wherein the antigen protein is administered in a single dose of 400 μg and the adjuvant is administered in a single dose of 400 μg.

74. An antigen protein combined with an adjuvant according to any one of claims 64 to 73, which is administered 1 to 10 times every 1 to 5 years, and the number of administrations per year is not more than 2.

75. An antigenic protein in combination with an adjuvant according to any one of claims 64 to 74, administered once or twice a year.

76. 76. An antigenic protein in combination with an adjuvant according to claim 74 or 75, administered twice a year, the second administration being given four weeks after the first administration.

77. The antigen protein in combination with an adjuvant according to any one of claims 64 to 76, wherein said administration is intramuscular administration.

78. 78. An antigen protein combined with an adjuvant according to any one of claims 64 to 77, wherein the humanized K-type CpG oligodeoxynucleotide comprises a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and some or all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds.

79. The adjuvant (i) a polydeoxynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:2, in which some or all of the phosphodiester bonds of the polydeoxynucleotide are replaced by phosphorothioate bonds or phosphorodithioate bonds; and (ii) Lentinan An antigen protein in combination with an adjuvant according to any one of claims 64 to 78, comprising a complex consisting of:

80. The antigen protein is (A)(i) Peptide I, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 (with the proviso that cysteine ​​is maintained in the amino acid sequence shown in SEQ ID NO: 3), and which is intramolecularly cross-linked by disulfide bonds between C167 and C197, C176 and C187, C212 and C221, C236 and C247, and C270 and C276 in the amino acid sequence shown in SEQ ID NO: 3; and (ii) Peptide II comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:4 (provided that the cysteine ​​residue in the amino acid sequence shown in SEQ ID NO:4 is maintained). Including, (B) Peptide I and Peptide II are intermolecularly cross-linked by disulfide bonds between C293 of the amino acid sequence represented by SEQ ID NO: 3 and C12 of the amino acid sequence represented by SEQ ID NO: 4, and between C66 of the amino acid sequence represented by SEQ ID NO: 3 and C44 of the amino acid sequence represented by SEQ ID NO:

4. An antigen protein in combination with an adjuvant according to any one of claims 64 to 79, which is a protein.

81. The antigen protein combined with the adjuvant according to any one of claims 64 to 80, wherein the antigen protein and the adjuvant are held in the same container.

82. The antigen protein combined with an adjuvant according to any one of claims 64 to 80, wherein the antigen protein and the adjuvant are held in separate containers and are mixed prior to administration.

83. The antigen protein combined with the adjuvant according to any one of claims 64 to 82, wherein the ratio of the antigen protein antibody titer one year after administration of the vaccine to the antigen protein antibody titer four weeks after the previous administration of the vaccine is at least 0.

1.

84. An antigen protein in combination with an adjuvant according to any one of claims 64 to 83, wherein the complex is in the form of a triple helix structure.

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