WO2020203731A1 - 肺炎球菌表層タンパク質 - Google Patents
肺炎球菌表層タンパク質 Download PDFInfo
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- WO2020203731A1 WO2020203731A1 PCT/JP2020/013929 JP2020013929W WO2020203731A1 WO 2020203731 A1 WO2020203731 A1 WO 2020203731A1 JP 2020013929 W JP2020013929 W JP 2020013929W WO 2020203731 A1 WO2020203731 A1 WO 2020203731A1
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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
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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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- 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
- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
Definitions
- the present invention relates to a vaccine containing Streptococcus pneumoniae surface protein A (PspA) and PspA.
- PspA Streptococcus pneumoniae surface protein A
- PspA Streptococcus pneumoniae surface protein A
- Streptococcus pneumoniae is a clinically important upper respiratory tract infection pathogen along with influenza virus. It causes otitis media, pneumonia, bloodstream and meningitis, and causes serious diseases including death in children and adults. ..
- PCV pneumococcal conjugate vaccine
- PCV10 and PCV13 7-valent-, 10-valent-, and 13-valent-polysaccharide-conjugated pneumococcal vaccines
- this polysaccharide-based vaccine hardly induces an immune response to children due to the weak immunogenicity of T cell-independent polysaccharides, and has a protective effect against capsular serotype Streptococcus pneumoniae. Not shown. Furthermore, since intramuscular injection of vaccines mainly induces systemic anti-IgG antibody, these vaccines have problems such as being unable to induce a mucosal immune response against Streptococcus pneumoniae. There is.
- PspA Pneumococcal surface protein A
- Steptococcus pneumoniae Pneumococcal surface protein A
- Document 1 and Non-Patent Document 2 PspA is present on almost all types of Streptococcus pneumoniae, and PspA-based vaccines induce antibodies that induce cross-reactivity in mice and humans (Non-Patent Documents 3 to 5).
- PspA-specific mucosal and systemic antibodies are induced in pups (through the mother) and adult mice, and the induction of this antibody is mediated by Th1 and Th2 cytokine responses by CD4 + T cells.
- Th1 and Th2 cytokine responses CD4 + T cells.
- PspA is classified into three families (Families 1-3) and further into six subgroups called clade (clades 1-6). Among them, Streptococcus pneumoniae strains having PspA classified in Family 1 account for about 95% of the strains confirmed so far, and PspA classified in Family 1 is particularly important as a vaccine candidate. .. PspA derived from the D39 strain known as a universal antigen of Streptococcus pneumoniae is a typical vaccine antigen classified into Family 1 and Clade 2, and has high antigenicity and ability to induce a neutralizing antibody against Streptococcus pneumoniae. However, PspA derived from the D39 strain was easily deaminated at a pH near neutral, and had a problem in stability.
- an object of the present invention is to provide a D39-derived PspA mutant that does not undergo deamination even at a pH near neutral and retains its stability as a molecule.
- the present inventors analyzed the amino acid sequence of deaminated D39-derived PspA by the MS / MS method, and found that one amino acid of wild-type D39-derived PspA had a mutation. Regarding the position of this amino acid, it is clear that only asparagine (Asn) at position 254 is specifically changed to aspartic acid (Asp) in the entire amino acid sequence of wild-type D39-derived mature PspA represented by SEQ ID NO: 1. became. PspA (N254D) in which the 254 position of mature PspA derived from D39 was changed to aspartic acid is an extremely stable PspA, and there is no change in antigenicity and neutralizing antibody-inducing ability.
- the present invention is the following (1) to (5).
- the protein according to (1) above wherein a part of the amino acid sequence represented by SEQ ID NO: 2 is an ⁇ -helix region.
- the present invention provides a pneumococcal vaccine antigen that is stable as a molecule.
- the elution peak may be split into two, and the subsequent peak is a deamination compound. there were.
- the antigen provided by the present invention can be shown to be capable of preparing very stable formulations during the purification process and subsequent storage conditions.
- the present invention retains the same antigenicity and neutralizing antibody-inducing ability as the wild-type D39-derived PspA protein, and is more stable as a molecule (for example, less likely to cause amino acid mutation) than the wild-type D39-derived PspA.
- an excellent protein hereinafter referred to as “PspA protein of the present invention”. That is, the first embodiment of the present invention is the PspA protein of the present invention, which is the following protein (a) or (b).
- A A protein containing the amino acid sequence represented by SEQ ID NO: 2 and having pneumococcal vaccine antigenic activity, and a protein substantially the same as the protein.
- B A protein which is a part of the amino acid sequence represented by SEQ ID NO: 2 and contains aspartic acid at position 254 and has pneumococcal vaccine antigenic activity, and substantially the same as the protein.
- the PspA protein has a signal sequence (eg, GenBank accession no .: amino acid positions 1-31 of ABJ54172 (derived from D39 strain)) and an ⁇ -helix ( ⁇ -Helix) region (eg, ⁇ -Helix).
- Proline-rich region eg, GenBank accession no .: ABJ54172 amino acid positions 320-402
- choline binding region and C-terminal tail It consists of a (C-terminal tail) area (see Fig. 1).
- the signal sequence is cleaved to mature PspA (consisting of the ⁇ -helix region, proline-rich region, choline binding region and C-terminal tail region).
- the "pneumococcal vaccine antigenic activity” is an activity that induces immunity (humoral immunity and / or cell-mediated immunity) for protecting a living body from attack by pneumococcus. Whether or not a protein has “pneumococcal vaccine antigenic activity” can be easily confirmed by those skilled in the art (for example, by the method described in "4. Mouse nasal immunity” in Examples). it can).
- the "protein” in the above (a) is "a protein containing the amino acid sequence represented by SEQ ID NO: 2 and having pneumococcal vaccine antigen activity", and the “protein” in the above (b) is , "A part of the amino acid sequence represented by SEQ ID NO: 2, which contains aspartic acid at position 254 and has pneumococcal vaccine antigenic activity”.
- the "protein substantially identical to the protein” refers to one or several (preferably about 1 to 30, more preferably 1 to 1) in the amino acid sequence of the "protein". It consists of an amino acid sequence in which about 10 amino acids, more preferably 1 to 5) are deleted (excluding aspartic acid at position 254), substituted (excluding aspartic acid at position 254), inserted or added, and
- the protein is a protein having activity as a pneumoniae vaccine antigen.
- the "protein substantially identical to the protein” means that the amino acid at position 254 is aspartic acid, which is about 60% or more, preferably about 60% or more of the amino acid sequence of the "protein". About 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, most preferably about 99% amino acid sequences, and the protein is active as a pneumonia vaccine antigen. ..
- a part of the amino acid sequence represented by SEQ ID NO: 2 means, for example, a part or all of the ⁇ -helix region (however, including aspartic acid at position 254) and a proline-rich region.
- a region containing a part or all of the region is preferable, and examples thereof include a protein consisting of the amino acid represented by SEQ ID NO: 3.
- the second embodiment of the present invention is a nucleic acid (DNA or the like) encoding the "PspA protein of the present invention".
- the PspA protein of the present invention obtains a nucleic acid encoding this from a cDNA library or the like, integrates it into an appropriate expression vector, transforms or transfects an appropriate host cell with the expression vector, and uses this as an appropriate medium. It can be prepared by culturing in the medium, expressing the PspA protein of the present invention, and purifying it.
- Host cells for PspA protein expression include, for example, bacterial cells (eg, Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans , Pseudomonas putida, etc.), mold (eg, eg Penicillium camembertii , Acremonium chrysogenum, etc.), animal cells, plant cells, baculovirus / insect cells or yeast cells (eg, Saccharomyces cerevisiae and Pichia pastoris ) can be used and expressed in these cells.
- bacterial cells eg, Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans ,
- an expression vector for expressing the PspA protein a vector suitable for various host cells can be used.
- expression vectors include pBR322, pBR325, pUC118, pET (E. coli host), pEGF-C, pEGF-N (animal cell host), pVL1392, pVL1393 (insect cell host, baculovirus vector), pG. -1, Yep13 or pPICZ etc. (yeast cell host) can be used.
- These expression vectors have replication origins, selectable markers and promoters suitable for each vector, and if necessary, enhancers, transcriptional concentrating sequences (terminators), ribosome binding sites, polyadenylation signals, etc. You may have.
- a base sequence for fusing and expressing a FLAG tag, His tag, HA tag, GST tag and the like may be inserted into the expression vector.
- the expression vector can be produced by a method known to those skilled in the art, and can also be appropriately produced by using a commercially available kit or the like.
- the cells or cultured cells are collected by a known method, suspended in an appropriate buffer, and ultrasonically, lysozyme and /.
- a soluble extract is obtained by centrifugation or filtration.
- cultured cells are used as a host, it is desirable to obtain the PspA protein expressed in the culture supernatant by collecting the supernatant. From the obtained extract or culture supernatant, the desired protein can be obtained by appropriately combining known separation / purification methods.
- Known separation and purification methods include methods that utilize solubility such as salting and solvent precipitation, methods that mainly utilize differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-PAGE, and ions.
- a method that utilizes a charge difference such as exchange chromatography, or a method that utilizes a specific affinity such as affinity chromatography (for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
- affinity chromatography for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
- Ni-NTA resin or Co-based resin when the polypeptide is expressed with the His tag
- anti-HA antibody resin when the polypeptide is expressed with the HA tag
- the polypeptide with the FLAG tag for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathi
- a third embodiment of the present invention is a vaccine containing the PspA protein of the present invention as an antigen for suppressing infection by Streptococcus pneumoniae (hereinafter, also referred to as "the pneumococcal vaccine of the present invention").
- the PspA protein of the present invention contained in the pneumococcal vaccine of the present invention may be contained alone, but is different from the PspA protein of the present invention (including PspA protein belonging to a different family and / or clade). It may be included in a form fused with all or part of (see, for example, WO2018102774).
- the pneumococcal vaccine of the present invention comprises one or more adjuvants, such as complete or incomplete Freund's adjuvant, cholera toxin, heat-resistant Escherichia coli toxin, aluminum hydroxide, potassium alum, saponin or derivatives thereof, muramyl dipeptide, minerals. It can include oil or vegetable oil, Novasome or nonionic block copolymer, DEAE dextran and the like. It may also contain a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier needs to be a compound that does not adversely affect the health of the vaccinated animal.
- a pharmaceutically acceptable carrier is, for example, sterile water or buffer.
- the pneumococcal vaccine of the present invention can be administered by a usual active immunization method, and may be administered by injection, orally, or by a transmucosal method such as nasal administration.
- the pneumococcal vaccine is an effective amount for the prevention or treatment of pneumococcal infection (amount sufficient to induce immunity in vivo against attack by pneumococcus), and is simply by a method suitable for the dosage form. It can be administered once or multiple times.
- the pneumococcal vaccine can be administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally, or mucosally (such as intranasally or sublingually).
- pneumococcal vaccine of the present invention can also be used in combination with other antigen components.
- the dose and frequency of administration of the pneumococcal vaccine may vary depending on the administration subject, but protective immunity can be induced by administering a vaccine containing several tens of ⁇ g of the antigen once to several times every few weeks.
- the pneumococcal vaccine of the present invention may contain nanogels so as to be suitable for nasal administration.
- the nanogel is a high molecular weight gel nanoparticle in which hydrophobic cholesterol is added as a side chain to a hydrophilic polysaccharide (for example, pullulan).
- the nanogel can be produced based on a known method, for example, the method described in WO2000012564. Specifically, first, a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms and OCN-R 1 NCO (in the formula, R 1 is a hydrocarbon group having 1 to 50 carbon atoms) are represented by di.
- the isocyanate compound is reacted to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon having 12 to 50 carbon atoms or sterol is reacted.
- the obtained isocyanato group-containing hydrophobic compound is reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide containing a hydrocarbon group or a steryl group having 12 to 50 carbon atoms.
- a highly pure hydrophobic group-containing polysaccharide can be produced.
- pullulan amylopectin, amylose, dextran, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, water-soluble cellulose and the like can be used, and pullulan is particularly preferable.
- Examples of the nanogel used in the third embodiment of the present invention include cationic cholesterol-substituted pullulan (referred to as cationic cholesterol-group-bearing pullulan: cCHP) and its derivatives.
- cCHP has a structure in which pullulan having a molecular weight of 30,000 to 200,000, for example, 100,000 molecular weight, is substituted with 1 to 10 cholesterols, preferably 1 to several cholesterols per 100 monosaccharides.
- the amount of cholesterol substituted in cCHP used in the present invention may be appropriately changed depending on the size of the antigen and the degree of hydrophobicity.
- an alkyl group (10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms) may be added.
- the nanogel used in the present invention has a particle size of 10 to 40 nm, preferably 20 to 30 nm. Nanogels are already widely available on the market and these commercially available products may be used.
- the nanogel used in the embodiments of the present invention is a nanogel in which a functional group having a positive charge, for example, an amino group has been introduced so that the vaccine can invade the surface of the negatively charged nasal mucosa.
- a method for introducing an amino group into a nanogel a method using cholesterol pullulan (CHPNH 2 ) having an amino group added can be mentioned. Specifically, CHP dried under reduced pressure is dissolved in dimethyl sulfoxide (DMSO), 1-1'carbonyldiimidazole is added thereto under a nitrogen stream, and the mixture is reacted at room temperature for several hours. Ethylenediamine is gradually added to the reaction solution, and the mixture is stirred for several hours to several tens of hours.
- DMSO dimethyl sulfoxide
- the resulting reaction solution is dialyzed against distilled water for several days.
- the reaction solution after dialysis is lyophilized to give a milky white solid.
- the degree of substitution of ethylenediamine can be evaluated by using elemental analysis, H-NMR, or the like.
- the pneumococcal vaccine of the present invention may contain a pharmaceutically acceptable known stabilizer, preservative, antioxidant and the like.
- the stabilizer include gelatin, dextran, sorbitol and the like.
- the preservative include thimerosal, ⁇ -propiolactone and the like.
- the antioxidant include ⁇ -tocopherol and the like.
- a fourth embodiment of the present invention is a method for preventing an infection caused by Streptococcus pneumoniae, which comprises administering the pneumococcal vaccine of the present invention to a patient.
- prevention means to prevent the infection in advance in a patient who may be infected with Streptococcus pneumoniae, and thereby, a treatment for the purpose of preventing the onset of Streptococcus pneumoniae infection in advance. That is.
- PspA 1-1 SEQ ID NO: 5
- PspA 1-2 SEQ ID NO: 6
- PspA 1-3 SEQ ID NO: 4
- PspA 1-3LB SEQ ID NO: 3
- the expressed PspA protein was extracted by a non-denaturing method according to a conventional method, and purified by LDS (Lithium Dodecyl Sulfate) -PAGE until it became uniform using ion exchange chromatography, butyl Sepharose, and the like.
- the PspA 1-3 molecule was suspended in 2 mg / ml PBS immediately after purification, and the pH was adjusted to a predetermined pH (pH 6.5, pH 7.0, pH 7.5, pH 6.5, pH 7.0, pH 7.5, 0.5M NaH 2 PO 4 or 0.5M Na 2 HP 4) .
- the pH was adjusted to 8.0 and pH 8.5), and the mixture was stored at 25 ° C. for a predetermined time (1 day, 2 days and 5 days).
- Each sample was electrophoresed at 200 V for 60 minutes on Native PAGE using 10 ⁇ l (1.0 ⁇ g / lane) and stained with Coomassie Blue (BioRad).
- the gel used was 7.5% Mini-Protein TGX Gel 12 well (BioRad), the treatment solution was Native Sample Buffer for Protein Gels (BIoRad), and the electrophoresis buffer was 10 x Premixed electrophoresis buffer (BioRad).
- Treatment under this condition showed a single band on all samples on LDS-PAGE and no change in apparent molecular weight (data not shown), but Native-PAGE showed no change in pH at 5 days of treatment. A band with high mobility was generated (arrow in FIG. 2). The protein corresponding to this band was PspA 1-3LB.
- Peptide Map To identify molecular changes from PspA 1-3 to PspA 1-3LB, 10 ⁇ g trypsin (sigma, sigma,) for 0.5 mg / ml of PspA 1-3 and PspA 1-3LB immediately after purification, respectively. Sequence grade) was added and digested at 37 ° C. for 24 hours at pH 8.5. 50 ⁇ l of the treated sample was introduced into reverse phase HPLC (Waters C18 1.7 ⁇ m 2.1 x 100 mm), and the separation temperature was 50 ° C., 0.1%.
- the pneumococcal surface protein of the present invention and a part thereof can be used as an antigen for a pneumococcal vaccine. Therefore, the present invention is expected to be utilized in the field of preventive medicine for infectious diseases.
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Abstract
Description
このような肺炎球菌による感染を防御する手段として、近年、成人向けの7価-、10価-、および13価-多糖結合型肺炎球菌ワクチン(PCV(pneumococcal conjugate vaccine)7、PCV10およびPCV13)が開発され、筋肉内注射によって投与されている。しかし、この多糖体ベースのワクチンは、T細胞非依存的な多糖の免疫原性が弱いため、小児に対する免疫応答をほとんど誘導せず、また、莢膜血清型の肺炎球菌にしか感染防御効果を示さない。さらに、ワクチンの筋肉内注射は、主として、全身性の抗IgG抗体を誘導するため、これらのワクチンは肺炎球菌に対して、粘膜免疫の応答を誘導することができないなどの問題点を有している。
肺炎球菌のユニバーサル抗原として知られているD39株由来のPspAは、ファミリー1、クレード2に分類される代表的なワクチン抗原であり、高い抗原性と肺炎球菌中和抗体誘導能を持つ。しかし、D39株由来のPspAは中性付近のpHで脱アミノ化されやすく、安定性に問題があった。
このアミノ酸の位置は、配列番号1で表される野生型D39由来成熟PspAの全アミノ酸配列中、254位のアスパラギン(Asn)のみが特異的にアスパラギン酸(Asp)に変わっていることが明らかとなった。D39由来成熟PspAの254位がアスパラギン酸に変化したPspA(N254D)は、極めて安定なPspAであって、抗原性、中和抗体誘導能にも変化がない。そして、このようなアミノ酸の変化は、例えば、ファミリー2、クレード3のEF3296肺炎球菌株のPspAや、ファミリー2、クレード4のEF5668肺炎球菌株のPspAには見られなかった。
以上の知見に基づいて本発明は完成された。
(1)以下の(a)または(b)のタンパク質。
(a)配列番号2で表されるアミノ酸配列を含み、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質、
(b)配列番号2で表されるアミノ酸配列の一部であって、当該一部には254位のアスパラギン酸が含まれており、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質
(2)前記配列番号2で表されるアミノ酸配列の一部がα-ヘリックス領域であることを特徴とする上記(1)に記載のタンパク質。
(3)配列番号3で表されるアミノ酸配列の全部または一部であることを特徴とする上記(2)に記載のタンパク質。
(4)上記(1)ないし(3)のいずれかに記載のタンパク質をコードするDNA。
(5)上記(1)ないし(3)のいずれかに記載のタンパク質を抗原または抗原の一部として含む肺炎球菌ワクチン。
すなわち、本発明の第1の実施形態は、本発明のPspAタンパク質であって、以下の(a)または(b)のタンパク質である。
(a)配列番号2で表されるアミノ酸配列を含み、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質、
(b)配列番号2で表されるアミノ酸配列の一部であって、当該一部には254位のアスパラギン酸が含まれており、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質
上記(a)における「当該タンパク質」とは、「配列番号2で表されるアミノ酸配列を含み、肺炎球菌ワクチン抗原活性を有するタンパク質」のことであり、上記(b)における「当該タンパク質」とは、「配列番号2で表されるアミノ酸配列の一部であって、当該一部には254位のアスパラギン酸が含まれており、肺炎球菌ワクチン抗原活性を有するタンパク質」のことである。
発現用ベクターの作製は、当業者に公知の手法により実施することができ、適宜、市販のキットなどを使用して行うこともできる。
本発明の肺炎球菌ワクチンに含まれる本発明のPspAタンパク質は、単独で含まれていてもよいが、本発明のPspAタンパク質とは異なるPspAタンパク質(異なるファミリーおよび/またはクレードに属するPspAタンパク質を含む)の全部または一部と融合された形態で含まれていてもよい(例えば、WO2018102774などを参照のこと)。
本発明の肺炎球菌ワクチンは、通常の能動免疫法で投与することができ、注射により投与しても、経口、または経鼻などの経粘膜方法で投与してもよい。また、肺炎球菌ワクチンは、肺炎球菌感染の予防または治療に対して、有効な量(肺炎球菌による攻撃に対し、生体内において免疫を誘導するに足りる量)で、剤形に適合した方法による単回または複数回投与することができる。肺炎球菌ワクチンは、皮内、皮下、筋肉内、腹腔内、静脈内、経口的に、または、粘膜(鼻腔内または舌下など)に投与することができる。また、本発明の肺炎球菌ワクチンは、他の抗原成分と混合して用いることもできる。
肺炎球菌ワクチンの投与量、投与回数は投与対象により変わり得るが、抗原を数10μg 含むワクチンを1週間から数週間に一度の頻度で、数回投与することによりに防御免疫を誘導し得る。
具体的には、まず、炭素数12~50の水酸基含有炭化水素またはステロールと、OCN-R1 NCO(式中、R1は炭素数1~50の炭化水素基である)で表されるジイソシアナート化合物を反応させて、炭素数12~50の水酸基含有炭化水素またはステロールが1分子反応したイソシアナート基含有疎水性化合物を製造する。得られたイソシアナート基含有疎水性化合物と多糖類とを反応させ、炭素数12~50の炭化水素基またはステリル基を含有する疎水性基含有多糖類を製造する。次に、得られた生成物をケトン系の溶媒で精製することにより、純度の高い疎水性基含有多糖類を製造することができる。
ここで、多糖類としては、プルラン、アミロペクチン、アミロース、デキストラン、ヒドロキシエチルデキストラン、マンナン、レバン、イヌリン、キチン、キトサン、キシログルカンまたは水溶性セルロース等が利用可能であり、特に、プルランが好ましい。
ここで、「予防」とは、肺炎球菌に感染するおそれがある患者において、その感染を予め阻止することを意味し、これによって肺炎球菌感染症の発症を予め阻止することを目的とする処置のことである。
以下に実施例を示してさらに本発明の説明を行うが、実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。
各種PspAタンパク質、PspA 1-1(配列番号5)、PspA 1-2(配列番号6)、PspA 1-3(配列番号4)およびPspA 1-3LB(配列番号3)を大腸菌発現系等発現させた(詳細は、WO2018102774を参照のこと)。発現したPspAタンパク質を定法により非変性法で抽出しイオン交換クロマト、ブチルセファロース等を使用し、LDS(Lithium Dodecyl Sulfate)-PAGEで均一になるまで精製した。
この条件の処理で、LDS-PAGEではすべての検体で単一バンドを示し見かけの分子量には変化がなかったが(データは示さない)、Native-PAGEでは、処理5日でpHによらず、移動度が大きいバンドが生じた(図2の矢印)。このバンドに対応するタンパク質をPspA 1-3LBとした。データは示さないが、この現象は精製直後のPspA 1-1およびPspA 1-2においても同様に観察されたが、PspA 2(EF3296株由来)およびPspA 3(EF5668株由来)では観察されず、肺炎球菌D39株由来のPspAに特異的に起こる現象であると考えられた。
PspA 1-3からPspA 1-3LBへの分子変化を特定するために、精製直後のPspA 1-3およびPspA 1-3LB、各々、0.5 mg/mlに対し、10 μgのトリプシン(sigma、Sequence grade)を加え、37℃、24時間、pH 8.5で消化処理を行い、処理したサンプルの50 μlを逆相HPLC(Waters C18 1.7 μm 2.1 x 100mm)に導入し、分離温度50℃、0.1% TFA (100%)、0.1%TFA-40% acetonitrile(55%)の110 min Linear-Gradient溶出、0.2ml/min、220nm(Detection)で分析した。
その結果、溶出時間が異なるピークが検出された(図3)。そこで、本ピークを分取しPPSQ-21A Protein sequencer(Shimadzu)によりN-末分析を行ったところ、AAEENDNVE(配列番号7)という配列がPspA 1-3LBに観測された。この結果から、PspA 1-3LBアミノ酸配列において、特異的な脱アミノ化が起きている可能性が示唆された。
次に、PspA 1-3LBの全アミノ酸配列分析を行うため、上記2.と同様にtrypsin消化を行い、LC-MS/MS(HPLC acquity UPLC (Waters:C4-1.7 μm、2.1 x 100mm)-MS: Orbitrap Fusion Tribrid(Thermo)MS1:Orbitrap, MS2: Ion Trapを用いて配列分析を実施した。
その結果、302アミノ酸残基中、208残基(69%)のアミノ酸の配列を同定し、さらにwhole-MS分析も実施し(Intact体33569.03および1脱アミノ体33569.93)、各理論値と一致したのでN254D以外に脱アミノ化が起きている可能性は極めて低いと結論した。
4-1.方法
4-1-1.抗原のナノゲル化(ワクチンの準備)
cCHPナノゲルと各PspAタンパク質(PspA 1-1、PspA 1-2、PspA 1-3および PspA 1-3LB)を分子比1:1で混合し、40℃のヒートブロックで1時間インキュベーションした。
4-1-2.マウスへの経鼻免疫
各cCHP-PspA溶液を、Balb/cマウスの7週齢メスにそれぞれ経鼻投与した。投与抗原量は、1匹、1回あたり各PspAタンパク量として10 μgを投与した。経鼻免疫は1週間隔で計3回実施した。
4-1-3.免疫マウスからの血清サンプル調製
最終投与から1週間で上昇してくる血清中のPspA特異的IgGを測定するために、免疫マウスの眼窩静脈から採血を行った。採取した血液サンプルを4℃、7,000rpmで遠心後、上清を回収し血清サンプルとした。
4-1-4.抗原特異的IgGの測定
ELISAプレートに各抗原を終濃度1 μg/mlでコーティングし、一晩反応後、ウェルを洗浄した。非特異的反応を避けるため、1% BSA含有PBSで25℃、1時間ウェルをブロッキングし、さらに洗浄後、28から段階希釈した血清サンプルを作製し、それぞれウェルに添加し、25℃で2時間反応させた。ウェルを洗浄後、HRP標識の抗マウスIgGを加えて25℃で1時間半反応させた。さらに洗浄後、HRPの基質であるTMB(3, 3', 5, 5'-tetramethylbenzidine)溶液を添加して発色させた。2N 硫酸で反応停止後、直ちにマイクロプレートリーダーで吸光度(450nm)を測定し、エンドポイント法で抗体価を算出した。
cCHP-PspA 1-1、cCHP-PspA 1-2、cCHP-PspA 1-3またはcCHP-PspA 1-3LBの経鼻投与により、マウス血清中において、それぞれの抗原に特異的なIgGが誘導されてくることが分かった。また、各PspA抗原間での抗体価に大きな差異はなかった(図4)。
Claims (5)
- 以下の(a)または(b)のタンパク質。
(a)配列番号2で表されるアミノ酸配列を含み、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質、
(b)配列番号2で表されるアミノ酸配列の一部であって、当該一部には254位のアスパラギン酸が含まれており、肺炎球菌ワクチン抗原活性を有するタンパク質、および当該タンパク質と実質的に同一のタンパク質 - 前記配列番号2で表されるアミノ酸配列の一部がα-ヘリックス領域であることを特徴とする請求項1に記載のタンパク質。
- 配列番号3で表されるアミノ酸配列の全部または一部であることを特徴とする請求項2に記載のタンパク質。
- 請求項1ないし3のいずれかに記載のタンパク質をコードするDNA。
- 請求項1ないし3のいずれかに記載のタンパク質を抗原または抗原の一部として含む肺炎球菌ワクチン。
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