WO2014045621A1 - 肺炎球菌表面タンパク質aを含む肺炎球菌ワクチン - Google Patents
肺炎球菌表面タンパク質aを含む肺炎球菌ワクチン Download PDFInfo
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- WO2014045621A1 WO2014045621A1 PCT/JP2013/058401 JP2013058401W WO2014045621A1 WO 2014045621 A1 WO2014045621 A1 WO 2014045621A1 JP 2013058401 W JP2013058401 W JP 2013058401W WO 2014045621 A1 WO2014045621 A1 WO 2014045621A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion
Definitions
- the present invention relates to a pneumococcal vaccine containing pneumococcal surface protein A.
- Streptococcus pneumoniae is a major respiratory pathogenic bacterium that causes pediatric and adult diseases such as meningitis and sepsis (Invasive pneumococcal disease: IPD) and community-acquired pneumonia.
- the current pneumococcal vaccine antigen is a capsular polysaccharide that determines the serotype of the bacterium, and at least 93 serotypes are known to date.
- PCV7 7-valent pneumococcal conjugate vaccine
- CCM 197 detoxified diphtheria toxin
- PCV13 13-valent pneumococcal conjugate vaccine
- Non-patent Document 1 Croney et al. Collected a large-scale survey of pneumococcal strains derived from pediatric invasive infection in Alabama in the United States that developed between 2002 and 2010 before vaccination with PCV13. The type contained only 60%, and the remaining 40% of strains contained 17 serotypes not included in PCV13 (Non-patent Document 1). In Japan, public subsidies for PCV7 vaccination for children started in 2011, but in 2012, an increase in IPD due to non-vaccine-containing serotypes was reported (Non-patent Document 2). It is unrealistic to continue adding non-vaccine serotype capsular polysaccharides as antigens to current vaccines, suggesting the limitations of current pneumococcal vaccines based on capsular polysaccharides I can say that.
- PspA pneumococcal surface protein A
- PspA is a novel pneumococcal vaccine. It is attracting attention as an antigen.
- PspA has a structure consisting of each domain shown in FIG. 1, and an antigenic epitope recognized by an antibody that has a protective effect against pneumococci exists in the ⁇ -helix and proline-rich regions of PspA. It is known (Non-Patent Documents 3 and 4).
- PspA is roughly classified into three families according to the gene sequence of its antigenic epitope region, and further classified into six subgroups called clades.
- the PspA family of Streptococcus pneumoniae isolated from the clinic accounts for 98% or more in Family 1 and 2 (Non-patent Document 5). It is known that PspA also acts as a pathogenic factor and inhibits the binding of complement C3 to the surface of Streptococcus pneumoniae (Non-Patent Document 6).
- anti-PspA-specific antibodies are complements of PspA. It is known that it acts antagonistically to the inhibitory activity and exhibits an infection-protecting activity against this bacterium (Non-patent Documents 7 and 8).
- Non-patent Document 9 Since the cross-reactivity of different PspA is diverse (Non-patent Documents 10 and 11), it is based on PspA to select combinations of clades belonging to family 1 and family 2 that show wider cross-reactivity. It is considered important for the development of vaccines.
- Patent Documents 1 and 2 describe that PspA protein is useful as an immunologically active component of a vaccine against pneumococcal infection.
- Non-patent document 12 examines the vaccine effect of a fusion protein of PspA of family 1 clade 1 and PspA of family 2 clade 4, and a fusion protein of PspA of family 1 clade 1 and PspA of family 2 clade 3. .
- Non-Patent Document 13 the vaccine effect of a fusion protein of PspA of family 1 clade 2 and PspA of family 2 clade 4 is examined.
- the present invention finds a clade combination or strain combination that exhibits a broad cross-reaction among PspA combinations of pneumococcal surface protein antigens and can induce an immune response against a wide range of clinically isolated pneumococcal strains.
- An object of the present invention is to provide a novel pneumococcal vaccine based on PspA.
- a fusion protein combining multiple PspAs a single protein antigen showing a broad cross-reaction and capable of inducing an immune response against a wide range of clinically isolated pneumococcal strains is provided, and a novel pneumococcal vaccine is provided The task is to do.
- the present invention includes the following inventions in order to solve the above problems.
- [1] Full length or a fragment thereof of at least family 1 pneumococcal surface protein A (excluding pneumococcal surface protein A from pneumococcal Rx1 strain and pneumococcal surface protein A from pneumococcal St435 / 96 strain);
- a pneumococcal vaccine comprising a fusion protein comprising the full length of a family 2 pneumococcal surface protein A or a fragment thereof.
- the pneumococcal vaccine according to [2], wherein the fusion protein is any of the following (1) to (3).
- Fusion protein [4] fusion protein comprising at least pneumococcal surface protein A of family 1 clade 2 and pneumococcal surface protein A of family 2 clade 5 The pneumococcal vaccine according to [3] above, which is any one of 4) to (6).
- a fragment of pneumococcal surface protein A is at least a proline-rich region
- the pneumococcal vaccine according to any one of [1] to [4] above, comprising all or part of [6]
- the pneumococcus according to [5] wherein the pneumococcal surface protein A fragment comprises all or part of a proline-rich region and all or part of an ⁇ -helix region adjacent thereto.
- the pneumococcal surface protein A of family 1 clade 2 is derived from a pneumococcal strain selected from the group consisting of D39, WU2, E134, EF10197, EF6796, BG9163 and DBL5 [2] The pneumococcal vaccine according to 1.
- Streptococcus pneumoniae expressing pneumococcal surface protein A of family 2 clade 3 is derived from TIGR4, BG8090 or AC122, and pneumococcal surface protein A of family 2 clade 4 is derived from EF5668, BG7561, BG7817 or BG11703
- the pneumococcal vaccine according to [3] wherein the family 2 clade 5 pneumococcal surface protein A is derived from ATCC 6303 or KK910.
- a pneumococcal vaccine characterized by comprising: [11] The pneumococcal vaccine according to [10], wherein the pneumococcal surface protein A is any one of the following (i) to (iii): (I) Family 1 Clade 2 and Family 2 Clade 3 only (ii) Family 1 Clade 2 and Family 2 Clade 4 only (iii) Family 1 Clade 2 and Family 2 Clade 5 only [12] The pneumococcal vaccine according to any one of [1] to [11]. [13] The pneumococcal vaccine according to any one of [1] to [12] above, which comprises a vaccine component against pathogens other than pneumococci.
- pneumococcal vaccine that exhibits a wide range of cross-reactions and can induce an immune response against a wide range of clinically isolated pneumococcal strains. Inoculation with the pneumococcal vaccine of the present invention can induce protective immunity against pneumococcal infections in children and adults.
- FIG. 1 It is a figure which shows the structure of PspA protein. It is a figure which shows the structure of three types of fusion PspA protein produced in Example 1, (A) is PspA2 + 4, (B) is a figure which shows the structure of PspA2 + 5, (C) is PspA3 + 2. (A) is a figure which shows the result of SDS-PAGE of each fusion PspA protein, (B) is a figure which shows the result of the western blotting of each fusion PspA protein.
- FIG. It is a figure which shows the result of having measured the anti- PspA antibody titer in the immune serum obtained from the mouse
- the present invention provides a pneumococcal vaccine comprising a fusion protein comprising at least the full length of Family 1 PspA or a fragment thereof and the full length of Family 2 PspA or a fragment thereof.
- a pneumococcal vaccine comprising a fusion protein comprising at least the full length of Family 1 PspA or a fragment thereof and the full length of Family 2 PspA or a fragment thereof.
- the fusion protein containing PspA (family 1 clade 2) derived from Streptococcus pneumoniae Rx1 strain used for the fusion protein of PspA of family 1 clade 2 and PspA of family 2 clade 4 described in Non-Patent Document 13 is Excluded from the invention.
- Non-Patent Document 12 Streptococcus pneumoniae St435 / 96 strain used as PspA of Family 1 clade 1
- Fusion protein comprising PspA (family 1 clade 1) derived from, for example, Table 1 of, 2002. is also excluded from the present invention.
- a part of the gene encoding PspA of Streptococcus pneumoniae St435 / 96 strain and its amino acid sequence are registered in a database such as GenBank with accession number AY082387.
- “family 1 PspA” does not include PspA derived from the Rx1 strain and the St435 / 96 strain.
- the fusion protein (hereinafter sometimes referred to as “the fusion protein of the present invention”) contained in the pneumococcal vaccine of the present invention comprises at least family 1 PspA and family 2 PspA. It only has to be included.
- the fusion protein of the present invention may contain 3 or more types of PspA, and may contain proteins other than PspA and capsular polysaccharides (for example, carrier protein and capsular vaccine antigen).
- it is a fusion protein composed of two types of PspA, that is, a fusion protein composed of family 1 PspA and family 2 PspA.
- the fusion protein may contain an amino acid sequence other than PspA such as a tag sequence, a vector-derived sequence, or a restriction enzyme-derived sequence.
- the order of each protein to be fused is not limited.
- the N-terminal side is Family 1 and the C-terminal side is Family 2
- the N-terminal side may be family 2 and the C-terminal side may be family 1 in this order.
- the order is not limited.
- PspA derived from Streptococcus pneumoniae whose family and clade have already been identified can be suitably used for the fusion protein of the present invention.
- PspA derived from pneumococci whose family and clade have not been identified it is preferable to use them after identifying the family and clade.
- the PspA family and clade are obtained by PCR-amplifying a portion expected to contain the ⁇ -helix region and the proline-rich region of the PspA gene, determining the base sequence, and about 400 bp upstream of the proline-rich region in the obtained base sequence.
- PspA identified in clade 1 or 2 is judged as family 1
- PspA identified in clade 3 or 4 or 5 is judged as family 2
- PspA identified in clade 6 is judged as family 3 (references: non-patent literature 5, Swiatlo E, Brooks-Walter A, Briles DE, McDaniel LS.
- Oligonucleotides identity conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumonia. Gene 1997, 188: 279-284 Further, the PspA gene is amplified using primers specific to family 1 and family 2, and if the PCR product size is about 1000 bp, it is judged as family 1, and if the PCR product size is about 1200 bp, the family (Reference: Vela Coral MC, Fonseca N, Castaneda E, Di Fabio JL, Hollingshead SK, Briles DE. Pneumococcal surface protein A of invasive Streptococcus pneumonia iso s ren child : 832-6.).
- pneumococci whose families and clades have been identified include pneumococci described in Tables 1 and 2, and any of these pspA derived from pneumococci (excluding Rx1) is suitable for the fusion protein of the present invention. Can be used.
- the pneumococcal surface protein A of family 1 is preferably clade 2. More preferred are the following fusion proteins (1) to (3). (1) Fusion protein comprising at least PspA of Family 1 clade 2 and PspA of Family 2 clade 3 (2) Fusion protein comprising at least PspA of family 1 clade 2 and PspA of family 2 clade 4 (3) At least family 1 Fusion protein comprising PspA of clade 2 and PspA of family 2 clade 5 More preferably, the following fusion proteins (4) to (6) are used.
- the vaccine of the present invention preferably contains the fusion protein (1) or (4).
- PspA used for the fusion protein may be full length or a fragment thereof. All of two or more types of PspA constituting the fusion protein may be full length, may be a mixture of the full length and a fragment, or all may be a fragment.
- PspA has a signal sequence, ⁇ -Helix, Proline-rich, Choline binding, and C-terminal tail. It is expressed as a protein (precursor) consisting of each of the above regions, from which the signal sequence is cleaved to become mature PspA. Therefore, the full length PspA means a portion obtained by removing the signal sequence region from the PspA shown in FIG.
- the fragment of PspA used for the fusion protein of the present invention is not particularly limited as long as it consists of a part of full-length PspA and can induce protective immunity against pneumococcal infection against the living body. It is preferable to include all or part of the region.
- the fragment of PspA may contain all or part of the ⁇ -helix region adjacent to the proline-rich region. Furthermore, the fragment of PspA preferably does not contain the C-terminal tail region, and more preferably does not contain the choline-binding region and the C-terminal tail region.
- the fragment of PspA used for the fusion protein of the present invention consists of only a part of the proline-rich region, only a part of the proline-rich region, or an ⁇ -helix region adjacent to a part of the proline-rich region.
- a part, a part of the ⁇ -helix region adjacent to a part of the proline rich region, a part of the ⁇ -helix region adjacent to the whole proline rich region, and a proline rich region And all of the ⁇ -helix region. More preferably, it is a fragment consisting of the entire proline-rich region and the entire ⁇ -helix region.
- the ⁇ -helix region is a signal sequence from the region predicted from the Rx1 strain-derived PspA that has an ⁇ -helix structure using the secondary structure prediction program (positions 1 to 288). It is a region excluding (1st to 31st positions) and can be defined as a region showing high homology with this amino acid sequence.
- the proline-rich region is located between the ⁇ -helix region and the choline binding region, and can be defined as a region where proline residues frequently appear.
- the choline-binding region is a region in which a repetitive sequence consisting of 20 amino acids (based on TGWLQVNGSWYYLNANGAMA (SEQ ID NO: 24)) that is relatively highly conserved exists 10 times, and this amino acid sequence is high. It can be defined as a region showing homology.
- the C-terminal tail region can be defined as a region from immediately after the final repetitive sequence present in the choline-binding region to the C-terminal stop codon.
- the length of the PspA fragment is not particularly limited as long as it can induce an immune response against a living body. It is preferably at least 27 residues, more preferably 108 residues or more, and even more preferably 300 residues or more (references: DanielsDCC, Coan P, King J, Hale J, Benton KA). , Briles DE, Hollingshead SK.
- the Proline-Rich Region of Pneumococcal Surface Proteins A and C Contains Surface-Accessible Epitopes Common to All Pneumococci and Elicits Antibodyin MedistInfect. .
- PspA of Family 1 clade 2 for example, PspA derived from pneumococcal strains D39, WU2, E134, EF10197, EF6796, BG9163, DBL5 and the like is preferable. More preferably, it is PspA derived from D39 or WU2.
- PspA of family 2 clade 3 PspA derived from pneumococcal strains TIGR4, BG8090, AC122 and the like is preferable. More preferably, it is PspA derived from TIGR4.
- PspA of family 2 clade 4 PspA derived from pneumococcal strains EF5668, BG7561, BG7817, BG11703 and the like is preferable. More preferably, it is PspA derived from EF5668.
- PspA of family 2 clade 5 PspA derived from pneumococcal strains ATCC 6303, KK910 and the like is preferable. More preferred is PspA derived from ATCC 6303.
- the fusion protein of the present invention is preferably a combination of D39-derived PspA and EF5668-derived PspA, a combination of D39-derived PspA and ATCC 6303-derived PspA, or a combination of WU2-derived PspA and TIGR4-derived PspA.
- a combination of WU2-derived PspA and TIGR4-derived PspA is more preferable, and a combination of TIGR4-derived PspA on the N-terminal side and WU2-derived PspA on the C-terminal side is more preferable.
- the fusion protein of the present invention preferably comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3 or 5.
- a fusion protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 is more preferred.
- the amino acid sequence represented by SEQ ID NO: 1 is a fusion protein of D39-derived PspA and EF5668-derived PspA, a vector-derived sequence containing a polyhistidine tag from the N-terminal side, D39 PspA amino acid sequence (Accession No. ABJ54172, 619aa) from position 32 to position 401, an EcoRI-recognized nucleotide sequence-derived sequence, and the amino acid sequence of PspA of EF5668 (Accession No. AAC62252, 653aa) positions 32 to 454 are linked in order.
- the amino acid sequence represented by SEQ ID NO: 3 is a fusion protein of D39-derived PspA and ATCC 6303-derived PspA, a vector-derived sequence containing a polyhistidine tag from the N-terminal side, the amino acid sequence of P39 of D39 (Accession No. ABJ54172, 619aa) from position 32 to position 401, an EcoRI-recognized nucleotide sequence-derived sequence, and an amino acid sequence in which positions 32 to 461 of PspA partial amino acid sequence (Accession No. AF071820, 461aa) of ATCC 6303 are linked in order.
- the amino acid sequence represented by SEQ ID NO: 5 is a fusion protein of TIGR4-derived PspA and WU2-derived PspA, a vector-derived sequence containing a polyhistidine tag from the N-terminal side, the amino acid sequence of PspA of TIGR4 (Accession No. AAK74303, 744aa) from position 32 to position 524, the sequence derived from the EcoRI recognition base sequence, and the amino acid sequence from position 32 to position 409 of the partial amino acid sequence of PspA of WU2 (Accession No. AAF27710, 415aa).
- the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1.
- One to several amino acids have been deleted, substituted or added means that the number can be deleted, substituted or added by a known mutant peptide production method such as site-directed mutagenesis (preferably 10 Or less, more preferably 7 or less, and even more preferably 5 or less) amino acids are deleted, substituted or added.
- a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a protein obtained by isolating and purifying a naturally occurring protein.
- the substantially identical amino acid sequence is at least 80% identical to the amino acid sequence represented by SEQ ID NO: 1, more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% Or an amino acid sequence that is 99% identical.
- fusion protein comprising the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1
- a fusion protein having substantially the same activity as the fusion protein comprising the amino acid sequence represented by SEQ ID NO: 1 is preferable.
- the substantially homogeneous activity includes the activity of inducing an immune response against a wide range of pneumococcal strains, and this activity is equivalent to the fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (for example, about 0.5 to 20 times, preferably about 0.5 to 2 times).
- the fusion protein of the present invention is constructed by constructing a recombinant expression vector into which a gene encoding the fusion protein of the present invention has been inserted by known genetic engineering techniques, and introducing this into a suitable host cell as a recombinant protein. It can be produced by expressing and purifying.
- the fusion protein of the present invention uses a gene encoding the fusion protein of the present invention and a known in vitro transcription / translation system (for example, a cell-free protein synthesis system derived from rabbit reticulocytes, wheat germ or E. coli). Can be manufactured.
- the vaccine of the present invention is highly useful in that an immune response can be induced against pneumococci even without an adjuvant.
- the vaccine of the present invention may contain one or more adjuvants.
- the vaccine of the present invention contains an adjuvant, it can be appropriately selected from known adjuvants.
- aluminum adjuvant for example, aluminum salt such as aluminum hydroxide, aluminum phosphate, aluminum sulfate or a combination thereof), Freund's adjuvant (complete or incomplete), TLR ligand (for example, CpG, Poly (I : C), Pam3CSK4, etc.), BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin, formylmethionyl peptide and the like.
- it is an aluminum adjuvant, a TLR ligand, or a combination thereof.
- the amount of the adjuvant blended is not particularly limited as long as it is an amount that non-specifically enhances the immune response to the fusion protein of the present invention, and may be appropriately selected depending on the type of adjuvant.
- an aluminum adjuvant aluminum hydroxide
- CpG aluminum hydroxide
- about 1 to 100 times the amount of aluminum adjuvant and about 1 to 50 times the amount of CpG may be added to the fusion protein of the present invention in a mass ratio. preferable.
- the vaccine of the present invention may further contain a vaccine component against pathogens other than pneumococci. That is, the present invention provides a mixed vaccine comprising the fusion protein of the present invention, which is a vaccine component against pneumococci, and a vaccine component against pathogens other than pneumococci.
- the vaccine component with respect to pathogens other than a pneumococcus is not specifically limited, For example, the vaccine component already used as a mixed vaccine is mentioned.
- diphtheria toxoid diphtheria toxoid, pertussis toxoid, pertussis antigen, tetanus toxoid, inactivated poliovirus, attenuated measles virus, attenuated rubella virus, attenuated mumps virus, Haemophilus influenzae type b polysaccharide antigen, hepatitis B virus HBs
- examples include antigens, inactivated hepatitis A virus antigens, and the like.
- the combined vaccines currently used include diphtheria / pertussis / tetanus mixed vaccine (DPT vaccine), diphtheria / pertussis / tetanus / inactivated polio vaccine (DPT-IPV vaccine), measles / rubella mixed vaccine (MR vaccine), Measles, rubella, mumps cold vaccine (MMR), Haemophilus influenzae type b / hepatitis B virus vaccine, hepatitis A virus / hepatitis B virus vaccine, diphtheria / pertussis / tetanus / hepatitis B virus / inactivated polio vaccine, etc. It is preferable to add a fusion protein that is a component of the pneumococcal vaccine of the present invention to the components of these mixed vaccines.
- the vaccine of the present invention can be administered by oral administration or parenteral administration.
- parenteral administration include intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, intranasal administration, transdermal administration, and transmucosal administration.
- Preferred is parenteral administration, and more preferred is intradermal administration, subcutaneous administration or intramuscular administration.
- the vaccine of the present invention can be formulated by appropriately blending the fusion protein of the present invention, a pharmaceutically acceptable carrier, and further additives.
- preparations for oral administration such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches It can be set as the formulation for administration. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ
- Carriers or additives that can be blended are not particularly limited.
- various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption
- Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
- additives used in solid preparations for oral administration include excipients such as lactose, mannitol, glucose, microcrystalline cellulose, and corn starch; binders such as hydroxypropylcellulose, polyvinylpyrrolidone, and magnesium aluminate metasilicate Dispersing agents such as corn starch; disintegrating agents such as calcium calcium glycolate; lubricants such as magnesium stearate; solubilizing agents such as glutamic acid and aspartic acid; stabilizers; hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, etc.
- excipients such as lactose, mannitol, glucose, microcrystalline cellulose, and corn starch
- binders such as hydroxypropylcellulose, polyvinylpyrrolidone, and magnesium aluminate metasilicate Dispersing agents such as corn starch
- disintegrating agents such as calcium calcium glycolate
- lubricants such as magnesium stearate
- solubilizing agents such as
- Water-soluble polymers such as cellulose, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, etc .; sucrose, powdered sugar, sucrose, fructose, glucose, lactose, reduced maltose water candy, powder returned Maltose water syrup, glucose fructose liquid sugar, fructose glucose liquid sugar, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin, sodium saccharin, etc., white sugar, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc. Coating agents and the like.
- Liquid preparations for oral administration are prepared by dissolving, suspending or emulsifying in commonly used diluents.
- the diluent include purified water, ethanol, a mixed solution thereof and the like.
- this liquid agent may contain a wetting agent, a suspending agent, an emulsifier, a sweetening agent, a flavoring agent, a fragrance, a preservative, a buffering agent and the like.
- additives used for injections for oral administration include isotonic agents such as sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, glucose, propylene glycol; phosphate buffer, Buffers such as acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon aminocaproate buffer; methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate , Preservatives such as butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax; hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol Thickeners such as sodium hydrogen sulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic
- solubilizers for example, alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; nonionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysolecithin, and pluronic polyol You may mix
- Liquid preparations such as injections can be stored after removing moisture by freezing or lyophilization. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.
- the vaccine of the present invention can be administered to any animal (human, non-human) having an immune system.
- mammals such as humans, monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats and mice; and birds such as chickens, ducks and geese.
- the vaccine of the present invention is preferably intended for human children and adults.
- the number of administrations and the administration interval of the vaccine of the present invention are not particularly limited. For example, a single dose may be administered, or multiple doses may be administered at intervals of about 2 days to about 8 weeks.
- the dose of the vaccine varies depending on the administration subject, administration method, etc., but the single dose is preferably about 0.01 ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg, more preferably about 1 ⁇ g to More preferably, it is about 0.1 mg.
- the present invention includes a method for preventing or treating pneumococcal infection comprising administering to an animal an effective amount of the vaccine of the present invention.
- the vaccine of the present invention is superior in the following respects as compared with a conjugated pneumococcal vaccine using a capsular polysaccharide as an antigen.
- a single fusion protein antigen is effective against a wide variety of pneumococcal strains.
- a protein antigen Since it is a protein antigen, a fusion step with a carrier protein is unnecessary, and the production cost is low.
- a single fusion protein is used, and there is no need to mix multiple antigens. 5) It is only necessary to purify one type of fusion protein (vaccine antigen), the manufacturing process is easy, and the manufacturing cost can be suppressed.
- the present invention comprises at least the full length of PspA of family 1 clade 2 or a fragment thereof and the full length of PspA of family 2 selected from the group consisting of clade 3, clade 4 and clade 5 or a fragment thereof.
- the pneumococcal vaccine is included. That is, a pneumococcal vaccine comprising at least clade 2 PspA and clade 3 PspA, at least clade 2 pspA and clade 4 pspA, and at least clade 2 pspA and clade 5 pspA pneumococcal vaccine is there.
- the pneumococcal vaccine of this form may contain the above three combinations of PspA as separate proteins.
- the pneumococcal vaccine of this embodiment can be carried out in the same manner as the pneumococcal vaccine containing the fusion protein of the present invention, except that the above-mentioned three combinations of PspA may be contained as
- the PspA contained in the pneumococcal vaccine is preferably any one of the following (i) to (iii).
- PspA of clade 2 is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 26 Proteins consisting of the same amino acid sequence are preferred.
- the PspA of clade 3 is preferably a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27.
- the PspA of clade 4 is preferably a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 28.
- the PspA of clade 5 is preferably a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 29.
- the amino acid sequence represented by SEQ ID NO: 25 consists of the full length of the ⁇ -helix region and the proline-rich region of D39-derived PspA (the amino acid sequence is shown in SEQ ID NO: 30 and the base sequence of the encoding gene is shown in SEQ ID NO: 31, respectively). This is a full-length protein and corresponds to positions 32 to 401 in the amino acid sequence of P39A of D39 (SEQ ID NO: 30).
- the amino acid sequence represented by SEQ ID NO: 26 consists of the full-length ⁇ -helix region and the proline-rich region of PspA derived from WU2 (the partial amino acid sequence is shown in SEQ ID NO: 32, and the base sequence of the encoded gene is shown in SEQ ID NO: 33).
- the amino acid sequence represented by SEQ ID NO: 27 consists of the full length of the ⁇ -helix region and the proline-rich region of TIGR4-derived PspA (the amino acid sequence is shown in SEQ ID NO: 34, and the base sequence of the encoding gene is shown in SEQ ID NO: 35, respectively). It is a protein consisting of the full length and corresponds to positions 32 to 524 of the amino acid sequence of PspA of TIGR4 (SEQ ID NO: 34).
- the amino acid sequence represented by SEQ ID NO: 28 consists of the full length of the ⁇ -helix region and the proline-rich region of PspA derived from EF5668 (the amino acid sequence is shown in SEQ ID NO: 36, and the base sequence of the encoding gene is shown in SEQ ID NO: 37). It is a protein consisting of the full length and corresponds to positions 32 to 454 of the amino acid sequence of PspA (SEQ ID NO: 36) of EF5668.
- the amino acid sequence represented by SEQ ID NO: 29 consists of the full length of the ⁇ -helix region and the proline-rich region of PspA derived from ATCC 6303 (the partial amino acid sequence is shown in SEQ ID NO: 38 and the base sequence of the encoding gene is shown in SEQ ID NO: 39). And corresponds to positions 32 to 461 of the amino acid sequence of PspA of ATCC 6303 (SEQ ID NO: 38).
- a protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by any of SEQ ID NOs: 25 to 29 is the same as the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 described above. .
- the present invention provides a polynucleotide encoding the fusion protein of the present invention.
- the polynucleotide can be present in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA).
- the polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be any of double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. In the case of a single strand, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand).
- the polynucleotide of the present invention may be fused to a polynucleotide encoding a tag label (tag sequence or marker sequence) on the 5 ′ side or 3 ′ side thereof. Furthermore, it may contain sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences).
- UTR untranslated region
- the polynucleotide of the present invention can be prepared by obtaining polynucleotides encoding two or more types of PspA constituting the fusion protein and combining them.
- Polynucleotides encoding each PspA constituting the fusion protein can be separately obtained by a known DNA synthesis method, PCR method or the like.
- the base sequence is designed by appropriately selecting the codon of each amino acid, and chemically synthesized using a commercially available DNA synthesizer. be able to.
- the base sequence information of the gene encoding the target PspA is obtained from a known database (GenBank, etc.) (see the accession numbers in Tables 1 and 2), and a primer for amplifying the region encoding the desired PspA
- the desired DNA fragment can be obtained in large quantities by designing PCR and performing PCR or the like using the genomic DNA of pneumococci expressing the PspA as a template.
- the polynucleotide of the present invention can be prepared by genetically binding it.
- Examples of the polynucleotide encoding the fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 1 include, but are not limited to, the polynucleotide consisting of the base sequence represented by SEQ ID NO: 2.
- Examples of the polynucleotide encoding the fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 3 include, but are not limited to, a polynucleotide comprising the base sequence represented by SEQ ID NO: 4.
- Examples of the polynucleotide encoding the fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 5 include, but are not limited to, the polynucleotide consisting of the base sequence represented by SEQ ID NO: 6.
- the present invention provides an expression vector used for producing the fusion protein of the present invention.
- the expression vector of the present invention is not particularly limited as long as it contains a polynucleotide encoding the fusion protein of the present invention, but a plasmid vector (pSP64, pBluescript, etc.) having an RNA polymerase recognition sequence is preferred.
- Examples of the method for producing an expression vector include, but are not limited to, a method using a plasmid, phage, or cosmid.
- the specific type of vector is not limited, and a vector that can be expressed in a host cell can be appropriately selected.
- a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector.
- a host transformed with the expression vector of the present invention is cultured, cultivated or raised, and then subjected to conventional techniques (eg, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange) from the culture. Chromatography, affinity chromatography, etc.), the fusion protein of the present invention can be recovered and purified.
- the expression vector preferably contains at least one selectable marker.
- selectable markers include dihydrofolate reductase, neomycin resistance gene, etc. for eukaryotic cell culture, and tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene for culture in Escherichia coli and other bacteria. Etc.
- the host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, bacteria such as E. coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, animal cells (eg, CHO cells) , COS cells, and Bowes melanoma cells).
- bacteria such as E. coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, animal cells (eg, CHO cells) , COS cells, and Bowes melanoma cells).
- a method for introducing the expression vector into a host cell that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. .
- the present invention provides a transformant introduced with the expression vector of the present invention.
- the transformant of the present invention includes not only cells, tissues or organs but also organisms. Further, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms, plants and animals exemplified as the host cell.
- the transformant of the present invention can be suitably used for production of the fusion protein of the present invention. These transformants are preferably those that stably express the fusion protein of the present invention, but may be transiently expressed.
- the present invention further includes the following inventions.
- a fusion protein comprising at least the full length of a family 1 pneumococcal surface protein A or a fragment thereof and the full length of a family 2 pneumococcal surface protein A or a fragment thereof.
- the fusion protein according to [1] which is any of the following (1) to (3).
- Fusion protein consisting of pneumococcal surface protein A of family 1 clade 2 and pneumococcal surface protein A of family 2 clade 3 (2) pneumococcal surface protein A of family 1 clade 2 and pneumococcal surface of family 2 clade 4 Fusion protein consisting of protein A (3) Fusion protein consisting of pneumococcal surface protein A of family 1 clade 2 and pneumococcal surface protein A of family 2 clade 5 [3] Pneumococcal surface protein A of family 2 is clade 3 The fusion protein according to [1] or [2] above, wherein [4] The fusion protein according to [1], wherein the pneumococcal surface protein A fragment contains at least all or part of a proline-rich region.
- the fusion protein according to [1] which consists of the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3 or 5.
- [7] A polynucleotide encoding the fusion protein according to any one of [1] to [6].
- a pneumococcal vaccine comprising the fusion protein according to any one of [1] to [6].
- the pneumococcal vaccine according to [11] further comprising an adjuvant.
- a method for preventing or treating pneumococcal infection comprising administering an effective amount of the fusion protein according to any one of [1] to [6] to an animal.
- the fusion protein according to any one of [1] to [6] for use in the prevention or treatment of pneumococcal infection.
- Example 1 Production of fusion PspA protein
- three types of fusion PspA proteins shown in FIGS. 2 (A), (B) and (C) were prepared using P39A derived from D39, TIGR4, EF5668, ATCC 6303 and WU2. did. All gene cloning procedures are described in E. This was performed using E. coli DH5 ⁇ . E. E. coli DH5 ⁇ was cultured in LB medium (1% bactoptryptone, 0.5% yeast extract, 0.5% NaCl), and kanamycin was added to LB medium to a final concentration of 30 ⁇ g / ml when necessary.
- DNA fragments encoding the N-terminal ⁇ -helix and proline-rich region of PspA of each strain were obtained by PCR amplification using the primers shown in Table 4 and the genomic DNA of each strain as a template.
- the PCR product was inserted into the pET28a (+) vector to create a fusion PspA protein expression vector. Specifically, it is as follows.
- a PspA PCR product derived from pneumococcus D39 (primers P1 and P2) or TIGR4 (primers P7 and P8) having a restriction enzyme NdeI recognition sequence added to the 5 ′ end and an EcoRI recognition sequence added to the 3 ′ end was pET28a (+ )
- the restriction enzyme cleavage sites of the vector were inserted into NdeI and EcoRI recognition sites.
- pneumococci in which a restriction enzyme EcoRI recognition sequence is added to the 5 ′ end side and a XhoI recognition sequence is added to the 3 ′ end side of the restriction enzyme EcoRI and XhoI recognition site of the pET28a (+) vector into which D39-derived PspA is inserted.
- Insert two PspA PCR products from EF5668 (primers P3 and P4) or ATCC6303 (primers P5 and P6), respectively, to produce two expression vectors expressing D39 and EF5668-derived PspA fusion proteins PspA2 + 4, D39 and ATCC6303PspA fusion protein PspA2 + 5 did.
- a restriction enzyme EcoRI recognition sequence was added at the 5 ′ end side and a XhoI recognition sequence at the 3 ′ end side to the restriction enzyme EcoRI and XhoI recognition sites of the pET28a (+) vector into which the TIGR4-derived PspAspPCR product was inserted.
- a vector that expresses TIGR4 and WU2-derived PspA fusion protein PspA3 + 2 was prepared by inserting a PspA-PCR product derived from WU2 (primers P9 and P10).
- the base sequences of these three types of fusion genes (PspA2 + 4 gene, PspA2 + 5 gene and PspA3 + 2 gene) were confirmed by DNA sequencer, and the base sequences represented by SEQ ID NOs: 2, 4 and 5 were confirmed.
- Each of the produced fusion PspA protein expression vectors was transformed into E. coli BL21 (DE3), and this was subjected to shaking culture at 37 ° C. in an LB medium containing 30 ⁇ g / ml of kanamycin.
- IPTG final concentration 0.5 mM
- Fusion PspA protein was extracted from the collected cells and purified by Ni 2+ affinity chromatography and gel filtration using a polyhistidine tag present at the N-terminus. The purified fusion protein was confirmed to be the target fusion protein by SDS-PAGE (see FIG. 3A) and Western blotting using an anti-PspA antibody (see FIG. 3B).
- Example 2 Binding ability of IgG in fusion PspA protein immune serum to the surface of each PspA clade pneumococci]
- Immunization of mice with fusion PspA protein Fusion PspA protein (PspA2 + 4, PspA2 + 5 or PspA3 + 2) 0.1 ⁇ g and adjuvant (CpGK3 2.5 ⁇ g and Alum 5.0 ⁇ g) were prepared in LPS-free PBS, and 6 weeks old sputum C57 / BL6j mice were inoculated subcutaneously. Five mice were used per same immunization group. Inoculation was performed three times every other week. One week after the final immunization (third inoculation), blood was collected to obtain serum.
- Streptococcus pneumoniae was cultured in THY medium (Todd-Hewitt broth supplemented with 0.5% yeast extract), glycerol was added to the culture solution in the logarithmic growth phase to a final concentration of 25%, and this was stored frozen at ⁇ 80 ° C. I used something. After Streptococcus pneumoniae strain cultured overnight on blood agar medium for 4-5 hours on blood agar medium, Streptococcus pneumoniae was collected in PBS. About 10 7 CFU of pneumococcal solution 90 ⁇ l and 10 ⁇ l of immune serum (mixed serum of the same immune group) were reacted at 37 ° C. for 30 minutes. Further, after reacting with a FITC-labeled anti-mouse IgG goat antibody, washing and centrifugation were performed, and the fluorescence intensity bound to the cells was measured by a flow cytometry method.
- THY medium Todd-Hewitt broth supplemented with 0.5% yeast extract
- Example 3 Infectious protective effect in mouse lethal pneumonia model by fusion PspA protein immunization
- mice were immunized with the fusion PspA protein in the same manner as in Example 2. Mice treated with adjuvant alone served as negative controls. Two weeks after the final immunization (third inoculation), mice were inoculated nasally with the following pneumococcal strains expressing PspA clades 1-5 (see Table 3) to create a mouse lethal pneumonia model.
- BG9739 (clade 1) is 2 ⁇ 10 7 CFU
- WU2 (clade 2) is 2 ⁇ 10 7 CFU
- TIGR4 (clade 3) is 5 ⁇ 10 6 CFU
- KK1162 (clade 4) is 1.
- ⁇ 10 8 CFU, ATCC 6303 (clade 5), infected 5 ⁇ 10 5 CFU of a lethal bacterial mass.
- the number of animals in one group was 10 or 8 (PspA2 + 5 group of BG9739 infection group, negative control group of KK1162 infection group and PspA3 + 2 group, negative control group of ATCC 6303 infection group). Survival was observed over 2 weeks after pneumococcal infection of immunized mice. The difference in survival rate was analyzed using the Kaplan-Meier log-rank test. A significant difference was assumed when the P value was less than 0.05.
- mice immunized with PspA3 + 2 a significant improvement in survival rate was observed when any of the PspA clade 1-5 pneumococcal strains were infected.
- PspA2 + 4 and immunized mice with PspA2 + 5 significant improvement in survival was observed when PspA clade 2, 4 or 5 pneumococcal strains were infected.
- Example 4 Measurement of binding ability of IgG in immune serum to clinically isolated pneumococcal strain
- the identification method of the PspA family and clade of clinically isolated pneumococcal strains was performed as follows. That is, PCR was performed using the following primers LSM12 and SKH2 as templates for the genomic DNA of each clinically isolated pneumococcal strain, and the PCR products were sequenced.
- the base sequence of about 400 bp upstream of the proline-rich region was compared with the base sequence of PspA in which the family and clade were identified (reference: Pimenta FC, Ribeiro-Dias F, Brandileone MC et al. 2006). Genetic diversity of PspA types among nasopharyngeal isolates collected during an ongoing surveillance study of children in Brazil. J Clin Microbiol. 44: 2838-43.).
- LSM12 CCGGATCCAGCGTCGCTATCTTAGGGGCTGGTT (SEQ ID NO: 17)
- SKH2 CCACATACCGTTTTCTTGTTTCCAGCC (SEQ ID NO: 18)
- Example 5 Measurement of anti-PspA antibody titer in PspA protein immune serum
- a protein comprising the ⁇ -helix region and the proline-rich region of each PspA of clades 1 to 5 was prepared as a recombinant protein and used as an antigen protein.
- BG9739 was used as clade 1, D39 as clade 2, TIGR4 as clade 3, EF5668 as clade 4, and PspA derived from ATCC 6303 as clade 5.
- An expression vector for PspA antigen protein of each clade was prepared as follows.
- a DNA fragment encoding an ⁇ -helix and a proline-rich region in which a restriction enzyme NdeI recognition sequence was added to the 5 ′ end and an XhoI recognition sequence was added to the 3 ′ end was amplified by PCR.
- primers P11 and P12 were used for BG9739, primers P1 and P13 for D39, primers P7 and P14 for TIGR4, primers P15 and P4 for EF5668, and primers P7 and P6 for ATCC 6303.
- the obtained PCR product was inserted into the restriction enzyme cleavage sites NdeI and XhoI recognition sites of the pET28a (+) vector. These vectors were transformed into E.
- PspA antigen protein was extracted from the collected cells and purified by Ni 2+ affinity chromatography and gel filtration using a polyhistidine tag present at the N-terminus. The purified PspA antigen protein was confirmed to be the target protein by SDS-PAGE and Western blotting.
- mice Immunization of mice with antigen
- the PspA antigen protein (PspA2) of clade 2 obtained above, the PspA antigen protein (PspA3) of clade 3 and the fusion PspA protein (PspA3 + 2) prepared in Example 1 were used. used.
- Six-week-old spider C57 / BL6j mice were divided into the following 5 groups, 5 mice per group.
- PspA3 + PspA2 administration group (administered 0.05 ⁇ g of PspA3, 0.05 ⁇ g of PspA2 and adjuvant) PspA3 + 2 administration group (PspA3 + 2 0.1 ⁇ g and adjuvant administered) -PspA2 administration group (administered 0.1 ⁇ g of PspA2 and adjuvant) ⁇ PspA3 administration group (administered 0.1 ⁇ g of PspA3 and adjuvant) -PspA3 + 2 administration group (administer 4.0 ⁇ g of PspA3 + 2)
- As adjuvants 2.5 ⁇ g of CpGK3 and 5.0 ⁇ g of Alum were used.
- Antigen solution was prepared with LPS-free PBS and mice were inoculated subcutaneously. Inoculation was performed three times every other week. One week after the final immunization (third inoculation), blood was collected to obtain serum.
- each well was washed with PBST, alkaline phosphatase-labeled anti-mouse IgG goat antibody diluted 2000 times was added at 100 ⁇ l / well, and allowed to stand at room temperature for 45 minutes under light shielding. Thereafter, the absorbance at OD 405 nm was measured. The dilution factor at which the absorbance obtained by subtracting the absorbance of the negative control is 0.1 is indicated by Log 2 , and this was defined as the anti-PspA antibody titer contained in the serum.
- the fusion protein of PspA2 and PspA3 shows a high antibody titer against any clade when immunized at a high concentration without using an adjuvant. From this result, the fusion PspA protein has an equivalent or high antibody titer compared to administration as a separate protein, and even higher antibody titer can be obtained without using an adjuvant. It has been shown to be very useful.
- Example 6 Investigation of adjuvant (part 1)] As an adjuvant, the effect of combined use of CpG and Alum or single use of CpG was examined.
- Example 7 Investigation of adjuvant (part 2)] As an adjuvant, the effect of using Alum alone rather than the combined use of CpG and Alum was examined.
- the fusion PspA protein (PspA3 + 2) prepared in Example 1 was used as the antigen, and Alum was used as the adjuvant. The following three groups were provided. ⁇ PspA3 + 2 0.1 ⁇ g + Alum 5.0 ⁇ g ⁇ PspA3 + 2 0.1 ⁇ g + Alum 25.0 ⁇ g ⁇ PspA3 + 2 0.1 ⁇ g + Alum 50.0 ⁇ g
- Antigen solution was prepared with LPS-free PBS and mice were inoculated subcutaneously. Inoculation was performed three times every other week. One week after the final immunization (third inoculation), blood was collected to obtain serum.
Abstract
Description
2000年に米国で小児用ワクチンとして無毒化ジフテリアトキシン(CRM197)をポリサッカライド抗原に結合させた7価肺炎球菌コンジュゲートワクチン(PCV7)が導入された。PCV7導入後にワクチン含有の7血清型の肺炎球菌による侵襲性感染症は明らかに減少したものの、小児および成人における非ワクチン含有血清型である血清型19AなどによるIPD症例の増加の問題が浮上してきた。このため、2010年にPCV7にさらに6血清型の莢膜多糖体を抗原として追加した13価肺炎球菌コンジュゲートワクチン(PCV13)が導入され、既に米国では小児、成人において承認されている。
非特許文献12では、ファミリー1クレード1のPspAとファミリー2クレード4のPspAとの融合タンパク質、およびファミリー1クレード1のPspAとファミリー2クレード3のPspAとの融合タンパク質のワクチン効果が検討されている。また、非特許文献13では、ファミリー1クレード2のPspAとファミリー2クレード4のPspAとの融合タンパク質のワクチン効果が検討されている。しかし、これら2つの文献に記載のPspA融合タンパク質については、クレード5および6のPspAを発現する肺炎球菌に対するワクチン効果や、広範囲の臨床分離肺炎球菌株に対するワクチン効果は評価されておらず、未だ実用化されたとの報告はない。
[1]少なくともファミリー1の肺炎球菌表面タンパク質A(ただし、肺炎球菌Rx1株由来の肺炎球菌表面タンパク質Aおよび肺炎球菌St435/96株由来の肺炎球菌表面タンパク質Aを除く)の全長またはそのフラグメントと、ファミリー2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントとを含む融合タンパク質を含有することを特徴とする肺炎球菌ワクチン。
[2]ファミリー1の肺炎球菌表面タンパク質Aがクレード2であることを特徴とする前記[1]に記載の肺炎球菌ワクチン。
[3]融合タンパク質が、以下の(1)~(3)のいずれかである前記[2]に記載の肺炎球菌ワクチン。
(1)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード3の肺炎球菌表面タンパク質Aとを含む融合タンパク質
(2)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード4の肺炎球菌表面タンパク質Aとを含む融合タンパク質
(3)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード5の肺炎球菌表面タンパク質Aとを含む融合タンパク質
[4]融合タンパク質が、以下の(4)~(6)のいずれかである前記[3]に記載の肺炎球菌ワクチン。
(4)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード3の肺炎球菌表面タンパク質Aからなる融合タンパク質
(5)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード4の肺炎球菌表面タンパク質Aからなる融合タンパク質
(6)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード5の肺炎球菌表面タンパク質Aからなる融合タンパク質
[5]肺炎球菌表面タンパク質Aのフラグメントが、少なくともプロリンリッチ領域の全部または一部を含むことを特徴とする前記[1]~[4]のいずれかに記載の肺炎球菌ワクチン。
[6]肺炎球菌表面タンパク質Aのフラグメントが、プロリンリッチ領域の全部または一部およびそれに隣接するα-へリックス領域の全部または一部からなることを特徴とする前記[5]に記載の肺炎球菌ワクチン。
[7]ファミリー1クレード2の肺炎球菌表面タンパク質Aが、D39、WU2、E134、EF10197、EF6796、BG9163およびDBL5からなる群より選択される肺炎球菌株由来であることを特徴とする前記[2]に記載の肺炎球菌ワクチン。
[8]ファミリー2クレード3の肺炎球菌表面タンパク質Aを発現する肺炎球菌が、TIGR4、BG8090またはAC122由来であり、ファミリー2クレード4の肺炎球菌表面タンパク質Aが、EF5668、BG7561、BG7817またはBG11703由来であり、ファミリー2クレード5の肺炎球菌表面タンパク質Aが、ATCC6303またはKK910由来であることを特徴とする前記[3]に記載の肺炎球菌ワクチン。
[9]融合タンパク質が、配列番号1、3もしくは5で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列からなることを特徴とする前記[1]に記載の肺炎球菌ワクチン。
[10]少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントと、クレード3、クレード4およびクレード5からなる群より選択されるファミリー2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントとを含有することを特徴とする肺炎球菌ワクチン。
[11]含有する肺炎球菌表面タンパク質Aが、以下の(i)~(iii)のいずれかである前記[10]に記載の肺炎球菌ワクチン。
(i)ファミリー1クレード2およびファミリー2クレード3のみ
(ii)ファミリー1クレード2およびファミリー2クレード4のみ
(iii)ファミリー1クレード2およびファミリー2クレード5のみ
[12]アジュバントを含むことを特徴とする前記[1]~[11]のいずれかに記載の肺炎球菌ワクチン。
[13]肺炎球菌以外の病原体に対するワクチン成分を含むことを特徴とする前記[1]~[12]のいずれかに記載の肺炎球菌ワクチン。
本発明は、少なくともファミリー1のPspAの全長またはそのフラグメントと、ファミリー2のPspAの全長またはそのフラグメントとを含む融合タンパク質を含有する肺炎球菌ワクチンを提供する。ただし、前記非特許文献13に記載のファミリー1クレード2のPspAとファミリー2クレード4のPspAとの融合タンパク質に用いられた肺炎球菌Rx1株由来のPspA(ファミリー1クレード2)を含む融合タンパク質は本発明から除かれる。また、前記非特許文献12に記載の融合タンパク質において、ファミリー1クレード1のPspAとして用いられた肺炎球菌St435/96株(非特許文献12およびMiyaji EN et al., Infect Immun. 70: 5086-5090, 2002.のTABLE 1参照)由来のPspA(ファミリー1クレード1)を含む融合タンパク質も本発明から除かれる。肺炎球菌St435/96株のPspAをコードする遺伝子の一部およびそのアミノ酸配列は、アクセッション番号AY082387でGenBank等のデータベースに登録されている。本明細書の以下の記載において、「ファミリー1のPspA」にはRx1株およびSt435/96株由来のPspAは含まれない。
(1)少なくともファミリー1クレード2のPspAとファミリー2クレード3のPspAとを含む融合タンパク質
(2)少なくともファミリー1クレード2のPspAとファミリー2クレード4のPspAとを含む融合タンパク質
(3)少なくともファミリー1クレード2のPspAとファミリー2クレード5のPspAとを含む融合タンパク質
さらに好ましくは以下の(4)~(6)の融合タンパク質である。
(4)ファミリー1クレード2のPspAとファミリー2クレード3のPspAからなる融合タンパク質
(5)ファミリー1クレード2のPspAとファミリー2クレード4のPspAからなる融合タンパク質
(6)ファミリー1クレード2のPspAとファミリー2クレード5のPspAからなる融合タンパク質
また、ファミリー2のPspAはクレード3であることが好ましい。したがって、本発明のワクチンとしては、上記(1)または(4)の融合タンパク質を含有することが好ましい。
さらに、本発明の融合タンパク質は、配列番号1、3もしくは5で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列からなることが好ましい。なかでも配列番号5で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列からなる融合タンパク質がより好ましい。
配列番号3で表されるアミノ酸配列は、D39由来PspAとATCC6303由来PspAとの融合タンパク質であり、N末端側からポリヒスチジンタグを含むベクター由来配列、D39のPspAのアミノ酸配列(Accession No. ABJ54172, 619aa)の第32位~第401位、EcoRI認識塩基配列由来配列、ATCC6303のPspAの部分アミノ酸配列(Accession No. AF071820, 461aa)の第32位~第461位が順に連結したアミノ酸配列である。
配列番号5で表されるアミノ酸配列は、TIGR4由来PspAとWU2由来PspAとの融合タンパク質であり、N末端側からポリヒスチジンタグを含むベクター由来配列、TIGR4のPspAのアミノ酸配列(Accession No. AAK74303, 744aa)の第32位~第524位、EcoRI認識塩基配列由来配列、WU2のPspAの部分アミノ酸配列(Accession No. AAF27710, 415aa)の第32位~第409位が順に連結したアミノ酸配列である。
本発明のワクチンがアジュバントを含む場合、アジュバントの配合量は、本発明の融合タンパク質に対する免疫応答を非特異的に高める量であれば特に限定されず、アジュバントの種類等により適宜選択すればよい。例えば、アルミニウムアジュバント(水酸化アルミニウム)およびCpGを併用する場合、本発明の融合タンパク質に対して質量比でアルミニウムアジュバントが約1~100倍量、CpGが約1~50倍量を配合することが好ましい。
経口投与用の液体製剤は、一般的に用いられる希釈剤に溶解、懸濁又は乳化されて製剤化される。希釈剤としては、例えば、精製水、エタノール、それらの混液等が挙げられる。さらにこの液剤は、湿潤剤、懸濁化剤、乳化剤、甘味剤、風味剤、芳香剤、保存剤、緩衝剤等を含有していてもよい。
本発明のワクチンの投与回数および投与間隔は特に限定されない。例えば、単回投与でもよく、約2日~約8週間の間隔で複数回投与してもよい。
ワクチン投与量は、投与対象、投与方法などにより異なるが、1回投与量を約0.01μg~約10mgとすることが好ましく、約0.1μg~約1mgとすることがより好ましく、約1μg~約0.1mgとすることがさらに好ましい。
本発明には、本発明のワクチンの有効量を動物に投与することを含む、肺炎球菌感染症の予防または治療方法が含まれる。
1)単一の融合タンパク質抗原のみで広範なタイプの肺炎球菌株に対して効果を示す。
2)タンパク質抗原であることから、キャリアータンパク質との融合ステップが不要であり生産コストが安価である。
3)タンパク質抗原であることから、キャリアータンパク質非存在状態で、小児および成人のどちらに対しても感染防御免疫を誘導できる。
4)単一の融合タンパク質を用いており、複数の抗原を混合する必要性がない。
5)融合タンパク質(ワクチン抗原)1種の精製を行うだけでよく、製造プロセスが容易であり、製造コストが抑制できる。
(i)ファミリー1クレード2およびファミリー2クレード3のみ
(ii)ファミリー1クレード2およびファミリー2クレード4のみ
(iii)ファミリー1クレード2およびファミリー2クレード5のみ
配列番号26で表されるアミノ酸配列は、WU2由来PspA(部分アミノ酸配列を配列番号32に、コードする遺伝子の塩基配列を配列番号33にそれぞれ示す)のα-へリックス領域の全長とプロリンリッチ領域の全長からなるタンパク質であり、WU2のPspAの部分アミノ酸配列(配列番号32)の第32位~第409位に該当する。
配列番号27で表されるアミノ酸配列は、TIGR4由来PspA(アミノ酸配列を配列番号34に、コードする遺伝子の塩基配列を配列番号35にそれぞれ示す)のα-へリックス領域の全長とプロリンリッチ領域の全長からなるタンパク質であり、TIGR4のPspAのアミノ酸配列(配列番号34)の第32位~第524位に該当する。
配列番号28で表されるアミノ酸配列は、EF5668由来PspA(アミノ酸配列を配列番号36に、コードする遺伝子の塩基配列を配列番号37にそれぞれ示す)のα-へリックス領域の全長とプロリンリッチ領域の全長からなるタンパク質であり、EF5668のPspAのアミノ酸配列(配列番号36)の第32位~第454位に該当する。
配列番号29で表されるアミノ酸配列は、ATCC6303由来PspA(部分アミノ酸配列を配列番号38に、コードする遺伝子の塩基配列を配列番号39にそれぞれ示す)のα-へリックス領域の全長とプロリンリッチ領域の全長からなるタンパク質であり、ATCC6303のPspAのアミノ酸配列(配列番号38)の第32位~第461位に該当する。
本発明は、本発明の融合タンパク質をコードするポリヌクレオチドを提供する。ポリヌクレオチドは、RNA(例えば、mRNA)の形態、またはDNAの形態(例えば、cDNAまたはゲノムDNA)で存在することができる。ポリヌクレオチドは、二本鎖でもよく一本鎖でもよい。二本鎖の場合は、二本鎖DNA、二本鎖RNAまたはDNAと RNAとのハイブリッドのいずれであってもよい。一本鎖の場合は、コード鎖(センス鎖)または非コード鎖(アンチセンス鎖)のいずれであってもよい。また、本発明のポリヌクレオチドは、その5’側または3’側でタグ標識(タグ配列またはマーカー配列)をコードするポリヌクレオチドに融合されていてもよい。さらに、非翻訳領域(UTR)の配列やベクター配列(発現ベクター配列を含む)などの配列を含むものであってもよい。
本発明は、上記本発明の融合タンパク質を製造するために使用される発現ベクターを提供する。本発明の発現ベクターは、本発明の融合タンパク質をコードするポリヌクレオチドを含むものであれば特に限定されないが、RNAポリメラーゼの認識配列を有するプラスミドベクター(pSP64、pBluescriptなど)が好ましい。発現ベクターの作製方法としては、プラスミド、ファージ、またはコスミドなどを用いる方法が挙げられるが特に限定されない。ベクターの具体的な種類は限定されず、宿主細胞中で発現可能なベクターを適宜選択することができる。すなわち、宿主細胞の種類に応じて、確実に本発明のポリヌクレオチドを発現させるために適宜プロモーター配列を選択し、これと本発明のポリヌクレオチドを各種プラスミド等に組み込んだベクターを発現ベクターとして用いればよい。本発明の発現ベクターを用いて形質転換された宿主を、培養、栽培または飼育した後、培養物などから慣用的な手法(例えば、濾過、遠心分離、細胞の破砕、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーなど)に従って、本発明の融合タンパク質を回収、精製することができる。
本発明は、上記本発明の発現ベクターが導入された形質転換体を提供する。本発明の形質転換体は、細胞、組織または器官だけでなく、生物個体をも含む。また、形質転換の対象となる生物も特に限定されるものではなく、上記宿主細胞として例示した各種微生物、植物または動物が挙げられる。本発明の形質転換体は、本発明の融合タンパク質の製造に好適に使用することができる。これらの形質転換体は、本発明の融合タンパク質を安定的に発現するものであることが好ましいが、一過性に発現するものでもよい。
[1]少なくともファミリー1の肺炎球菌表面タンパク質Aの全長またはそのフラグメントと、ファミリー2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントとを含むことを特徴とする融合タンパク質。
[2]以下の(1)~(3)のいずれかである前記[1]に記載の融合タンパク質。
(1)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード3の肺炎球菌表面タンパク質Aからなる融合タンパク質
(2)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード4の肺炎球菌表面タンパク質Aからなる融合タンパク質
(3)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード5の肺炎球菌表面タンパク質Aからなる融合タンパク質
[3]ファミリー2の肺炎球菌表面タンパク質Aがクレード3であることを特徴とする前記[1]または[2]に記載の融合タンパク質。
[4]肺炎球菌表面タンパク質Aのフラグメントが、少なくともプロリンリッチ領域の全部または一部を含むことを特徴とする前記[1]に記載の融合タンパク質。
[5]肺炎球菌表面タンパク質Aのフラグメントが、プロリンリッチ領域の全部または一部およびそれに隣接するα-へリックス領域の全部または一部からなることを特徴とする前記[4]に記載の融合タンパク質。
[6]配列番号1、3もしくは5で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列からなることを特徴とする前記[1]に記載の融合タンパク質。
[7]前記[1]~[6]のいずれかに記載の融合タンパク質をコードするポリヌクレオチド。
[8]前記[7]に記載のポリヌクレオチドを含む発現ベクター。
[9]前記[8]に記載の発現ベクターが導入された形質転換体。
[10]肺炎球菌ワクチンを製造するための前記[1]~[6]のいずれかに記載の融合タンパク質の使用。
[11]前記[1]~[6]のいずれかに記載の融合タンパク質を含むことを特徴とする肺炎球菌ワクチン。
[12]さらにアジュバントを含むことを特徴とする前記[11]に記載の肺炎球菌ワクチン。
[13]前記[1]~[6]のいずれかに記載の融合タンパク質の有効量を動物に投与することを含む、肺炎球菌感染症の予防または治療方法。
[14]肺炎球菌感染症の予防または治療に使用するための前記[1]~[6]のいずれかに記載の融合タンパク質。
表3に示した肺炎球菌のうち、D39、TIGR4、EF5668、ATCC6303およびWU2由来のPspAを用いて、図2(A)、(B)および(C)に示した3種類の融合PspAタンパク質を作製した。全ての遺伝子クローニング操作はE.coli DH5αを用いて行った。E.coli DH5αはLB培地(1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl)で培養を行い、必要時には、終濃度30μg/mlとなるようカナマイシンをLB培地に添加した。
(1)融合PspAタンパク質によるマウスの免疫
融合PspAタンパク質(PspA2+4、PspA2+5またはPspA3+2)0.1μgとアジュバント(CpGK3 2.5μgおよびAlum 5.0μg)をLPSフリーのPBSで調製し、6週齢の♀のC57/BL6jマウスに皮下接種した。同じ免疫グループあたり5匹のマウスを用いた。接種は、1週間おきに合計3回行った。最終免疫(3回目の接種)から1週間後に採血し、血清を得た。
肺炎球菌は、PspAクレード1~6の6種類を用いた。具体的には、クレード1の菌株としてBG9739、クレード2の菌株としてWU2、クレード3の菌株としてTIGR4、クレード4の菌株としてKK1162、クレード5の菌株としてATCC6303、クレード6の菌株としてBG6380を用いた(表3参照)。肺炎球菌は、THY培地(Todd-Hewitt broth supplemented with 0.5% yeast extract)で培養し、対数増殖期の培養液にグリセロールが終濃度25%になるよう添加し、これを-80℃で冷凍保存したものを使用した。
血液寒天培地で一晩培養した肺炎球菌株を、血液寒天培地で4~5時間継代培養した後、肺炎球菌をPBSにて収集した。約107CFUの肺炎球菌溶液90μlと10μlの免疫血清(同じ免疫グループの混合血清)を37℃で30分間反応させた。さらにFITC標識抗マウスIgGヤギ抗体と反応させた後、洗浄遠心し、菌体に結合する蛍光強度をフローサイトメトリー法で測定した。
結果を図4に示した。結合率は、菌数10000個当たりの免疫血清中IgG結合菌数を%で表した。PspA3+2の免疫血清は、クレード1~5のいずれの肺炎球菌株に対しても高い結合性を示した。PspA2+4およびPspA2+5の免疫血清は、クレード3のTIGR4に対しては若干低い結合性を示したが、これ以外のクレードの肺炎球菌株に対しては、高い結合性を示した。クレード6のBG6380に対しては、PspA2+5が高い結合性を示した。
(1)実験方法
実施例2と同様の方法でマウスに融合PspAタンパク質の免疫を行った。アジュバント単独投与マウスを陰性コントロールとした。最終免疫(3回目の接種)の2週間後にマウスにPspAのクレード1~5を発現する以下の肺炎球菌株(表3参照)を経鼻接種し、マウス致死的肺炎モデルを作製した。マウス一匹に対して、BG9739(クレード1)は2×107CFU、WU2(クレード2)は2×107CFU、TIGR4(クレード3)は5×106CFU、KK1162(クレード4)は1×108CFU、ATCC6303(クレード5)は5×105CFUの致死的な菌量を感染させた。1群の匹数は10匹または8匹(BG9739感染グループのPspA2+5群、KK1162感染グループの陰性コントロール群およびPspA3+2群、ATCC6303感染グループの陰性コントロール群)とした。
免疫マウスへの肺炎球菌感染後、2週間にわたってその生存率を観察した。生存率の違いについてはKaplan-Meier log-rank testを用いて解析を行った。P値が0.05より小さい場合に有意差があるとした。
結果を図5に示した。図5中の*はワクチン投与群におけるマウスの生存率をアジュバント投与群と比較し、有意差が示され、P値が0.05より小さい場合を表し、**は同様の比較で有意差が示され、P地が0.01より小さい場合を表す。(A)はBG9739(クレード1)、(B)はWU2(クレード2)、(C)はTIGR4(クレード3)、(D)はKK1162(クレード4)、(E)はATCC6303(クレード5)の結果である。PspA3+2による免疫マウスでは、PspAクレード1~5の肺炎球菌株のいずれを感染させた場合でも有意な生存率の改善が認められた。PspA2+4による免疫マウスおよびPspA2+5による免疫マウスでは、PspAクレード2、4または5の肺炎球菌株を感染させた場合に有意な生存率の改善が認められた。
(1)実験方法
実施例2で得られた各融合PspAタンパク質の免疫血清を用いて、免疫血清中IgGの臨床分離肺炎球菌株への結合能を測定した。対象菌株を臨床分離肺炎球菌株に変更した以外、実施例2と同様に行った。
なお、臨床分離肺炎球菌株のPspAファミリーおよびクレードの同定方法は、以下のように行った。すなわち、下記のプライマーLSM12およびSKH2を用いて各臨床分離肺炎球菌株のゲノムDNAをテンプレートとしてPCRを行い、PCR産物のシークエンス解析を行った。プロリンリッチ領域の上流約400bpの塩基配列を、ファミリーおよびクレードが同定されているPspAの塩基配列と比較し、同定を行った(参考文献:Pimenta FC, Ribeiro-Dias F, Brandileone MC et al. 2006. Genetic diversity of PspA types among nasopharyngeal isolates collected during an ongoing surveillance study of children in Brazil. J Clin Microbiol. 44:2838-43.)。
LSM12:CCGGATCCAGCGTCGCTATCTTAGGGGCTGGTT(配列番号17)
SKH2:CCACATACCGTTTTCTTGTTTCCAGCC(配列番号18)
前述したように、肺炎球菌菌体表層に存在するPspAタンパク質に対する免疫血清中IgGの結合が、感染防御効果に必須であることから、肺炎球菌へのIgG結合能を基準に様々な臨床分離肺炎球菌株に対する各融合PspAタンパク質のカバー率を評価した。
結果を図6に示した。図6中、括弧内は使用した臨床分離肺炎球菌株の血清型およびPspAクレードを示す。(A)のPspA2+4による免疫血清、(B)のPspA2+5による免疫血清、(C)のPspA3+2による免疫血清のいずれにおいても、ほぼ全ての臨床分離肺炎球菌株(97.0%)に対するIgG結合能(結合率10%以上)を示した。
(1)各クレードのPspA抗原タンパク質の作製
クレード1~5の各PspAのα-ヘリックス領域およびプロリンリッチ領域からなるタンパク質を組み換えタンパク質として作製し、抗原タンパク質とした。クレード1としてBG9739、クレード2としてD39、クレード3としてTIGR4、クレード4としてEF5668、クレード5としてATCC6303由来のPspAを使用した。各クレードのPspA抗原タンパク質の発現ベクターを、以下のように作製した。
抗原には、上記により得られたクレード2のPspA抗原タンパク質(PspA2)、クレード3のPspA抗原タンパク質(PspA3)および実施例1で作製した融合PspAタンパク質(PspA3+2)を使用した。6週齢の♀のC57/BL6jマウスを1群あたり5匹ずつ以下の5群に分けた。
・PspA3+PspA2投与群(PspA3 0.05μg、PspA2 0.05μgおよびアジュバントを投与)
・PspA3+2投与群(PspA3+2 0.1μgおよびアジュバントを投与)
・PspA2投与群(PspA2 0.1μgおよびアジュバントを投与)
・PspA3投与群(PspA3 0.1μgおよびアジュバントを投与)
・PspA3+2投与群(PspA3+2 4.0μgを投与)
アジュバントにはCpGK3 2.5μgおよびAlum 5.0μgを用いた。LPSフリーのPBSで抗原溶液を調製し、マウスに皮下接種した。接種は、1週間おきに合計3回行った。最終免疫(3回目の接種)から1週間後に採血し、血清を得た。
各クレードの精製PspA抗原タンパク質を5μg/mlに調製し、96ウェルプレートに100μl/ウェルずつ添加した。これを一晩4℃に静置し、抗原をプレートにコーティングした。プレートをPBST(0.05% Tween20含有PBS)で洗浄し、段階希釈した免疫血清サンプルを50μl/ウェルずつ添加し、37℃で30分間静置した。続いて、各ウェルをPBSTで洗浄し、2000倍に希釈したアルカリホスファターゼ標識抗マウスIgGヤギ抗体を100μl/ウェルずつ添加し、室温、遮光下で45分間静置した。その後、OD405nmの吸光度を測定した。陰性コントロールの吸光度を引いた吸光度が0.1になる希釈倍率をLog2で示し、これを血清中に含まれる抗PspA抗体価とした。
結果を図7に示した。PspA2単独ではクレード3に対する抗体価が低く、PspA3単独ではクレード1、2、4および5に対する抗体価が低くかった。PspA2とPspA3を併用すると、クレード1~5のいずれに対しても高い抗体価を示した。PspA2とPspA3の融合タンパク質(PspA3+2)は、PspA2とPspA3の併用よりいずれのクレードに対しても同等もしくは高い抗体価を示した。さらにPspA2とPspA3の融合タンパク質(PspA3+2)は、アジュバントを併用しなくても高濃度で免疫することにより、いずれのクレードに対しても高い抗体価を示すことが明らかになった。この結果から、融合PspAタンパク質は、別々のタンパク質としての投与と比較して同等あるいは高い抗体価が得られ、さらにアジュバントを併用しなくても高い抗体価が得られるので、肺炎球菌ワクチンの抗原として非常に有用であることが示された。
アジュバントとして、CpGおよびAlumの併用またはCpGの単独使用における効果について検討した。
(1)マウスの免疫
抗原には実施例1で作製した3種類の融合PspAタンパク質(PspA2+4、PspA2+5、PspA3+2)を使用し、アジュバントにはCpGおよびAlumの併用またはCpGの単独を使用した。以下の6群を設けた。
・PspA2+4 0.1μg+CpGK3 2.5μg
・PspA2+4 0.1μg+CpGK3 2.5μg+Alum 5.0μg
・PspA2+5 0.1μg+CpGK3 2.5μg
・PspA2+5 0.1μg+CpGK3 2.5μg+Alum 5.0μg
・PspA3+2 0.1μg+CpGK3 2.5μg
・PspA3+2 0.1μg+CpGK3 2.5μg+Alum 5.0μg
LPSフリーのPBSで抗原溶液を調製し、マウスに皮下接種した。接種は、1週間おきに合計3回行った。最終免疫(3回目の接種)から1週間後に採血し、血清を得た。
実施例5と同様の方法でELISAを行い、血清中に含まれる抗PspA抗体価を求めた。
(3)結果
結果を図8に示した。図8中の*は、同じクレードのPspA抗原に対するAlum含有群の抗体価がAlum非含有群に比べ有意に高く、P値が0.05より小さい場合を表し、**は同様の比較で有意に高く、P値が0.01より小さい場合を表す。PspA2+4では、Alum非含有条件でクレード2,4,5に対する抗体価が高く、クレード1,3に対する抗体価が低かったが、Alumの添加によってクレード1,3に対する抗体価の上昇が認められた。PspA2+5ではAlum非含有条件で特にクレード1,3に対する抗体価が低く、Alumの添加によってもクレード3に対する抗体価は低値のままであった。PspA3+2ではAlum非含有条件でクレード1に対する抗体価が低値であったが、Alumの添加によって全てのクレードに対する高い抗体価が認められた。この結果から、本条件下では、Alumの添加によってPspAに対する特異抗体価の上昇が確認され、また、融合型PspAが、全てのクレードのPspAに対する高い特異抗体価を誘導することが明らかとなった。
アジュバントとして、CpGとAlumの併用ではなく、Alumを単独で用いた場合の効果について検討した。
(1)マウスの免疫
抗原には実施例1で作製した融合PspAタンパク質(PspA3+2)を使用し、アジュバントにはAlumを使用した。以下の3群を設けた。
・PspA3+2 0.1μg+Alum 5.0μg
・PspA3+2 0.1μg+Alum 25.0μg
・PspA3+2 0.1μg+Alum 50.0μg
LPSフリーのPBSで抗原溶液を調製し、マウスに皮下接種した。接種は、1週間おきに合計3回行った。最終免疫(3回目の接種)から1週間後に採血し、血清を得た。
実施例5と同様の方法でELISAを行い、血清中に含まれる抗PspA抗体価を求めた。
(3)結果
結果を図9に示した。Alum単独の使用でも、クレード1~5のいずれに対しても高い抗体価を示した。また、投与量を併用の場合と同じ5.0μgにしても、併用の場合とほぼ同等の抗体価を示した。
日本で分離された成人侵襲性肺炎球菌感染症由来の肺炎球菌73株におけるPspAファミリーおよびクレードの分布を調べた。PspAファミリーおよびクレードの同定は、実施例4に記載の方法と同様に、プロリンリッチ領域の上流約400bpの塩基配列を、ファミリーおよびクレードが同定されているPspAの塩基配列と比較することにより行った。
Claims (13)
- 少なくともファミリー1の肺炎球菌表面タンパク質A(ただし、肺炎球菌Rx1株由来の肺炎球菌表面タンパク質Aおよび肺炎球菌St435/96株由来の肺炎球菌表面タンパク質Aを除く)の全長またはそのフラグメントと、ファミリー2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントとを含む融合タンパク質を含有することを特徴とする肺炎球菌ワクチン。
- ファミリー1の肺炎球菌表面タンパク質Aがクレード2であることを特徴とする請求項1に記載の肺炎球菌ワクチン。
- 融合タンパク質が、以下の(1)~(3)のいずれかである請求項2に記載の肺炎球菌ワクチン。
(1)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード3の肺炎球菌表面タンパク質Aとを含む融合タンパク質
(2)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード4の肺炎球菌表面タンパク質Aとを含む融合タンパク質
(3)少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード5の肺炎球菌表面タンパク質Aとを含む融合タンパク質 - 融合タンパク質が、以下の(4)~(6)のいずれかである請求項3に記載の肺炎球菌ワクチン。
(4)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード3の肺炎球菌表面タンパク質Aからなる融合タンパク質
(5)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード4の肺炎球菌表面タンパク質Aからなる融合タンパク質
(6)ファミリー1クレード2の肺炎球菌表面タンパク質Aとファミリー2クレード5の肺炎球菌表面タンパク質Aからなる融合タンパク質 - 肺炎球菌表面タンパク質Aのフラグメントが、少なくともプロリンリッチ領域の全部または一部を含むことを特徴とする請求項1~4のいずれかに記載の肺炎球菌ワクチン。
- 肺炎球菌表面タンパク質Aのフラグメントが、プロリンリッチ領域の全部または一部およびそれに隣接するα-へリックス領域の全部または一部からなることを特徴とする請求項5に記載の肺炎球菌ワクチン。
- ファミリー1クレード2の肺炎球菌表面タンパク質Aが、D39、WU2、E134、EF10197、EF6796、BG9163およびDBL5からなる群より選択される肺炎球菌株由来であることを特徴とする請求項2に記載の肺炎球菌ワクチン。
- ファミリー2クレード3の肺炎球菌表面タンパク質Aを発現する肺炎球菌が、TIGR4、BG8090またはAC122由来であり、ファミリー2クレード4の肺炎球菌表面タンパク質Aが、EF5668、BG7561、BG7817またはBG11703由来であり、ファミリー2クレード5の肺炎球菌表面タンパク質Aが、ATCC6303またはKK910由来であることを特徴とする請求項3に記載の肺炎球菌ワクチン。
- 融合タンパク質が、配列番号1、3もしくは5で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列からなることを特徴とする請求項1に記載の肺炎球菌ワクチン。
- 少なくともファミリー1クレード2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントと、クレード3、クレード4およびクレード5からなる群より選択されるファミリー2の肺炎球菌表面タンパク質Aの全長またはそのフラグメントとを含有することを特徴とする肺炎球菌ワクチン。
- 含有する肺炎球菌表面タンパク質Aが、以下の(i)~(iii)のいずれかである請求項10に記載の肺炎球菌ワクチン。
(i)ファミリー1クレード2およびファミリー2クレード3のみ
(ii)ファミリー1クレード2およびファミリー2クレード4のみ
(iii)ファミリー1クレード2およびファミリー2クレード5のみ - アジュバントを含むことを特徴とする請求項1~11のいずれかに記載の肺炎球菌ワクチン。
- 肺炎球菌以外の病原体に対するワクチン成分を含むことを特徴とする請求項1~12のいずれかに記載の肺炎球菌ワクチン。
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2013
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US9938326B2 (en) | 2018-04-10 |
JP6072210B2 (ja) | 2017-02-01 |
EP2910251A4 (en) | 2016-05-25 |
JP2016047842A (ja) | 2016-04-07 |
JP5854537B2 (ja) | 2016-02-09 |
EP2910251A1 (en) | 2015-08-26 |
HK1211839A1 (en) | 2016-06-03 |
CN104812405A (zh) | 2015-07-29 |
JPWO2014045621A1 (ja) | 2016-08-18 |
EP2910251B1 (en) | 2019-11-20 |
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US20150320851A1 (en) | 2015-11-12 |
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