WO2009081955A1 - 緑膿菌のiii型分泌装置構成タンパク質pa1698 - Google Patents
緑膿菌のiii型分泌装置構成タンパク質pa1698 Download PDFInfo
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- WO2009081955A1 WO2009081955A1 PCT/JP2008/073492 JP2008073492W WO2009081955A1 WO 2009081955 A1 WO2009081955 A1 WO 2009081955A1 JP 2008073492 W JP2008073492 W JP 2008073492W WO 2009081955 A1 WO2009081955 A1 WO 2009081955A1
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- pseudomonas aeruginosa
- antibody
- protein
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- amino acid
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
Definitions
- the present invention relates to an antibody against Pseudomonas aeruginosa type III secretion apparatus component protein PA1698, and a pharmaceutical composition comprising the antibody, a diagnostic agent for Pseudomonas aeruginosa infection, a therapeutic agent for Pseudomonas aeruginosa infection, and It relates to a detection kit.
- the present invention also relates to a vaccine composition comprising PA1698 protein or a peptide thereof as an antigen.
- Pseudomonas aeruginosa is a Gram-negative bacilli that is widely distributed in the natural environment, such as soil and water, but causes severe, lethal infections that are resistant to treatment. Its main target is an infectious patient with weakened defenses, commonly called a compromised host, including burns, organ transplants or cancer patients, and Pseudomonas aeruginosa It is the causative fungus. In addition, pulmonary infections caused by this bacterium are fatal in patients with cystic fibrosis.
- antibacterial agents having anti-Pseudomonas aeruginosa activity are mainly administered to these patients, there are many cases where a therapeutic effect is not sufficiently obtained due to drug resistance of Pseudomonas aeruginosa.
- vaccines and antibodies against Pseudomonas aeruginosa have been studied for a long time, but in the method using directly inactivated bacteria, various vaccines and antibodies must be prepared for each serotype of Pseudomonas aeruginosa. There were drawbacks.
- Pseudomonas aeruginosa infection by active or passive immunization using a protein derived from Pseudomonas aeruginosa having a common amino acid sequence among Pseudomonas aeruginosa is expected.
- proteins derived from Pseudomonas aeruginosa to vaccines include recombinant proteins fused with a part of outer membrane proteins OprF and OprI (JP-A-8-245699), type IV pilin protein (WO2004 / 099250) Etc. are known.
- anti-type IV pilin antibody (WO2004 / 099250 publication), anti-PA1706 (or PcrV) antibody (US6309651 publication, US6827935 publication), anti-PA5158 Antibodies (WO2007 / 049770), anti-PA0427 antibodies (WO2007 / 114340) and the like have been reported.
- proteins derived from bacterial cells commonly possessed by clinical isolates of Pseudomonas aeruginosa exhibiting various serotypes can be applied to the prevention, diagnosis or treatment of Pseudomonas aeruginosa infections as a “Pseudomonas aeruginosa common antigen”. There is always a need for it.
- PA1698 protein encoded by PA1698 (or PopN) gene is a protein constituting the type III secretion apparatus of Pseudomonas aeruginosa (Journal of Bacteriology, 2007, 189, 2599-2609) .
- a type III secretion device is a device used by pathogenic bacteria to transfer pathogenic factors directly into the host cytoplasm, and is composed of approximately 30 types of proteins.
- anti-PA1706 (or PcrV) antibody has been reported to have a protective effect on infection (US6309651 and US6827935).
- PA1698 protein is mutated between strains.
- this protein or partial peptide used as a vaccine component and antibodies produced from this protein or partial peptide are used to treat infection.
- drugs or diagnostics There are no examples of drugs or diagnostics.
- An object of the present invention is to provide an antibody and a vaccine composition that have the ability to substantially prevent or treat Pseudomonas aeruginosa infection and can cope with the diversity of clinical isolates derived from Pseudomonas aeruginosa infected patients. .
- Example 2 It was also found that there were only one or two amino acid mutations in the clinical isolates (Example 2). Furthermore, the antibody obtained by immunization with the PA1698 recombinant protein binds to the PA1698 protein (Examples 7 to 9), the antibody exhibits a cytotoxic inhibitory effect (Example 10), and the antibody It was confirmed (Examples 11 and 12) that a high infection protective effect was exhibited in a model mouse infected with Pseudomonas aeruginosa.
- PA1698 protein which is a type III secretion apparatus constituent protein of Pseudomonas aeruginosa is useful as a Pseudomonas aeruginosa common antigen, and an antibody against this antigen binds to Pseudomonas aeruginosa and has excellent protection against infection.
- the inventors have found that the effect can be exhibited, and have completed the present invention.
- the present invention relates to the following inventions.
- Antibody or functional fragment thereof that binds to PA1698 protein derived from Pseudomonas aeruginosa binds to PA1698 protein derived from Pseudomonas aeruginosa.
- ⁇ 2> The antibody or functional fragment thereof according to ⁇ 1>, wherein the P1698 protein derived from Pseudomonas aeruginosa is a protein described in (i) or (ii) below.
- a protein comprising the amino acid sequence represented by SEQ ID NO: 2.
- the amino acid sequence represented by SEQ ID NO: 2 comprising the amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2;
- ⁇ 3> The antibody or functional fragment thereof according to ⁇ 1>, which binds to the surface of Pseudomonas aeruginosa.
- ⁇ 4> The antibody or the functional fragment thereof according to ⁇ 1>, which has an activity of suppressing cytotoxic activity of Pseudomonas aeruginosa on human airway epithelial cells.
- ⁇ 5> The antibody or the functional fragment thereof according to ⁇ 1>, which has antibacterial activity in a patient infected with Pseudomonas aeruginosa.
- ⁇ 6> The antibody or the functional fragment thereof according to ⁇ 5>, which has antibacterial activity in infection with Pseudomonas aeruginosa in a patient in a state where neutrophils are decreased.
- ⁇ 7> The antibody or the functional fragment thereof according to ⁇ 1>, which binds to an epitope of an antibody produced by a hybridoma deposited under a deposit number of FERM BP-11055 or FERM BP-11056.
- an antigen composition comprising a protein antigen or peptide antigen capable of producing an antibody against PA1698 protein derived from Pseudomonas aeruginosa, and optionally one or more pharmaceutically acceptable carriers, diluents, And / or an adjuvant, which is used for the prevention or treatment of diseases associated with Pseudomonas aeruginosa.
- the amino acid sequence represented by SEQ ID NO: 2 comprising the amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2;
- ⁇ 12> comprising the antibody or functional fragment thereof according to any one of ⁇ 1> to ⁇ 7>, and optionally one or more pharmaceutically acceptable carriers and / or diluents.
- a diagnostic agent for Pseudomonas aeruginosa infection comprising the antibody or functional fragment thereof according to any one of ⁇ 1> to ⁇ 3>.
- a detection kit for Pseudomonas aeruginosa comprising the antibody according to any one of ⁇ 1> to ⁇ 3> or a functional fragment thereof.
- an antibody that recognizes PA1698 protein derived from Pseudomonas aeruginosa or a part thereof, or a functional fragment thereof is provided. It was found that the P. aeruginosa PA1698 protein to which the antibody according to the present invention binds has extremely high amino acid sequence conservation between strains regardless of serotype (Example 2). Accordingly, the P.
- aeruginosa PA1698 protein that is the target of the antibody according to the present invention is preferably a protein derived from the amino acid sequence PA01 strain shown in SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2 as 1 Alternatively, it is a protein having an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted or added, and having a function equivalent to that of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
- a substitution, deletion, insertion or addition of an amino acid sequence is generally 1 to 2 amino acids. Typically a substitution of 1-2 amino acids.
- the antibody according to the present invention is preferably obtained by administering (immunizing) an antigen composition comprising purified PA1698 protein as an antigen to an experimental animal in an amount that the antibody can induce. After purifying the antiserum obtained by collecting blood from a heart or an artery and separating it, it can be used as a pure antibody.
- the antibody according to the present invention includes a polyclonal antibody or a monoclonal antibody obtained by immunizing a mammal such as a mouse with the PA1698 protein as an antigen (including a monoclonal antibody produced by a hybridoma producing the monoclonal antibody of the present invention). And chimeric antibodies and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals and the like. When the antibody according to the present invention is administered as a medicine to humans, human antibodies are desirable from the viewpoint of reducing side effects.
- Human antibody refers to antibodies derived from humans in all regions. Human antibodies according to the present invention can be prepared using methods well known to those skilled in the art (for example, Intern. Rev. Immunol, 1995, 13, 65-93, J. Mol. Biol, 1991, 222, 581- 597, JP 10-146194, JP 10-155492, JP 2938569, JP 11-206387, JP 8-509612, JP 11-505107, etc. Can be referred).
- a “humanized antibody” is an antibody in which only the gene sequence of the antigen binding site (CDR; complementarity determining region) of a mouse antibody is transplanted (CDR grafting) into a human antibody gene.
- the humanized antibody according to the present invention can be prepared by a method well known to those skilled in the art (for example, refer to EP239400, WO90 / 07861, etc.).
- a “chimeric antibody” is an antibody in which a variable region of a certain antibody is linked to a constant region of a heterogeneous antibody. Specifically, an antibody is immunized with a mouse, and an antibody variable region (V region) that binds to the antigen is excised from the mouse monoclonal antibody gene and then combined with an antibody constant region (C region) gene derived from human bone marrow. can do.
- Chimeric antibodies according to the present invention can be prepared using methods well known to those skilled in the art (for example, JP-A-8-280387, U.S. Pat. No. 4816397, U.S. Pat. No. 4,816,567, U.S. Pat. No. 5,807,715). Issue gazette etc.).
- Monoclonal antibodies according to the present invention can be prepared using methods well known to those skilled in the art (eg, Antibodies A LABORATORY MANUAL Ed Harlow, David Lane Cold Spring Harbor Laboratory 1988, Monoclonal Antibody Experiment Manual (1987) Kodansha, Toyama). Edited by Shinji et al., Monoclonal Antibody Hybridoma and ELISA (1987) Kodansha, edited by Ikuzaki Ikuo et al. Polyclonal antibodies according to the present invention can also be generated using methods well known to those skilled in the art.
- the “functional fragment” according to the present invention means a part (partial fragment) of an antibody that specifically recognizes the protein according to the present invention.
- Specific examples include Fab, Fab ′, F (ab ′) 2 , variable region fragment (Fv), disulfide bond Fv, single chain antibody (scFv), and polymers thereof.
- the antibody of the present invention can be used for treatment or diagnosis of Pseudomonas aeruginosa infection or as a research reagent.
- Such antibodies include antibodies that recognize the PA1698 protein and bind to the surface of Pseudomonas aeruginosa.
- the absorbance of the whole cell ELISA test described in Example 8 is significantly higher than that of the control.
- the absorbance is preferably 0.3 or more, more preferably 0.5 or more, still more preferably 0.8 or more, and even more preferably 1.0 or more (for example, 1.2 or more, 1.4 or more).
- an antibody having an antibacterial activity or a functional fragment thereof is provided in a patient infected with Pseudomonas aeruginosa.
- a particularly preferred antibody is an antibody against a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or the protein comprising the amino acid sequence represented by SEQ ID NO: 2 having one or more conservative substitutions or the like A functional fragment.
- an antibody having antibacterial activity against Pseudomonas aeruginosa has been identified, those skilled in the art will identify a peptide region recognized by the antibody, bind to that region, and various antibodies exhibiting similar activity Can be created.
- a preferable example of such an antibody is an antibody that binds to an epitope of an antibody produced by a hybridoma commissioned under FERM BP-11055 or FERM BP-11056.
- the patient infected with Pseudomonas aeruginosa can be, for example, a patient whose neutrophils are in a reduced state due to administration of various drugs or radiation therapy.
- the antibody of the present invention is advantageous in that it can exert an effect on such a patient that easily develops to a serious infectious disease.
- the Pseudomonas aeruginosa infecting the patient can be multidrug resistant Pseudomonas aeruginosa.
- the antibodies of the present invention may also be effective against patients infected with multi-drug resistant Pseudomonas aeruginosa that cannot be treated with commonly used antibiotics.
- Pseudomonas aeruginosa is a major causative agent in chronic respiratory tract infections. According to the antibody of the present invention, since the cytotoxic activity of Pseudomonas aeruginosa on human airway epithelial cells can be suppressed, it can also be effective in the treatment of chronic respiratory tract infections.
- the inhibition rate of cell death of human airway epithelial cells is 10% or more, more preferably 15% or more, and further preferably 20% or more.
- examples of such antibodies include antibodies produced by hybridomas commissioned under FERM BP-11055 and FERM BP-11056.
- the present invention also provides a hybridoma that produces the antibody of the present invention.
- a hybridoma As a preferred hybridoma, the accession number FERM BP deposited on October 31, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture 305-8566) -11055 hybridoma (1698-1) and FERM BP-11056 hybridoma (1698-2).
- PA1698 protein derived from Pseudomonas aeruginosa can be used as a protein antigen, and an antigen composition containing such an antigen can be used as a vaccine for the prevention or treatment of diseases associated with Pseudomonas aeruginosa. Therefore, according to the present invention, there is provided a vaccine composition comprising an antigen composition capable of producing an antibody against P. aeruginosa-derived PA1698 protein.
- the “antigen composition” may be a composition containing a protein antigen as a sole component, or may be a composition comprising other components.
- prevention or treatment of a disease associated with Pseudomonas aeruginosa comprising the antigen composition and optionally one or more pharmaceutically acceptable carriers, diluents, and / or adjuvants.
- a disease associated with Pseudomonas aeruginosa comprising the antigen composition and optionally one or more pharmaceutically acceptable carriers, diluents, and / or adjuvants.
- Carriers used in vaccine compositions according to the present invention are selected based on the mode and route of administration, and standard pharmaceutical practice, and include carrier proteins such as bovine serum albumin (BSA), ovalbumin (OVA), Human serum albumin (HSA), hemocyanin (KLH: Keyhole limpet hemocyanin, etc.) derived from Lapas guy, solubilizer (eg, ethanol, polysorbate, Cremophor EL (registered trademark), etc.), isotonic agent, preservative, antioxidant Excipients (eg lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light anhydrous silicic acid, calcium carbonate, etc.), binders (eg starch, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy Methyl cellulose, gum arabic, etc.), lubricants (for example, , Magnesium stearate, talc
- glycerin, dimethylacetamide, 70% sodium lactate, surfactant, or basic substance eg sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, trisaminomethane, etc.
- basic substance eg sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, trisaminomethane, etc.
- a known KLH solution (125 mg / ml dissolved in 50% glycerol solution manufactured by Calbiotec) is coupled to the peptide according to the present invention ( coupling).
- the diluent used in the vaccine composition according to the present invention is selected based on the mode and route of administration and standard pharmaceutical practice, eg, water or saline, phosphate buffered saline, heavy weight. A carbonate solution etc. are mentioned.
- Adjuvants used in vaccine compositions according to the present invention are selected based on the mode and route of administration, and standard pharmaceutical practice, such as cholera toxin, E. coli heat labile enterotoxin (LT), liposomes, Or an immunostimulatory complex (ISCOM: immunostimulating complex) etc. are mentioned.
- Administration varies depending on the age, weight, sex, and general health status of the subject who may be infected by Pseudomonas aeruginosa, but either oral or parenteral (eg, intravenous, arterial, topical)
- the administration route can be any of these, but parenteral administration is preferred.
- Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art. By mixing the antigen composition according to the present invention with the above-mentioned pharmaceutically acceptable carrier, etc., conventional methods are obtained.
- Examples of the dosage form for oral administration include solid or liquid dosage forms, specifically, solvents, tablets, granules, powders, capsules and the like.
- dosage forms for parenteral administration include solvents, suspensions, ointments, creams, suppositories, ophthalmic solutions, nasal drops, ear drops and the like.
- flavoring agents and coloring agents can be added.
- a biodegradable polymer eg, poly-D, L-lactide-co-glycolide, polyglycolide, etc.
- a bulking matrix eg, US Pat. No. 5,417,986. No. 4,675,381 and U.S. Pat. No. 4,450,150).
- the dosage of the vaccine composition according to the present invention may be determined by, for example, the type of vaccine antigen, whether or not an adjuvant is administered together with the antigen, the type of adjuvant to be co-administered, the mode and frequency of administration, and the desired effect (for example, Generally, the vaccine composition of the present invention is administered in an amount of 1 ⁇ g to 100 mg per adult dose, depending on whether it is prophylactic or therapeutic. When an adjuvant is administered with this vaccine, it is generally administered in an amount of 1 ng to 1 mg per administration per adult. The administration is repeated if necessary according to the determination of the inventor. For example, administration for initialization can be followed by administration for 3 enhancements every week. Alternatively, boosting injections can be made with the same formulation 8-12 weeks after the first immunization and a second boosting 16-20 weeks.
- Pseudomonas aeruginosa is a pathogen of opportunistic infections that can be fatal as the host's resistance declines, and because it is resistant to antibiotics, it is also a major cause of nosocomial infections is there.
- the antibody of the present invention actually has an infection-protecting effect (Example 11).
- the antibody according to the present invention In a mouse Pseudomonas aeruginosa susceptibility model in which neutrophils were reduced by administration of cyclophosphamide monohydrate, it was confirmed that the antibody according to the present invention actually has an infection-protecting effect (Example 12). Further, one of the reasons why the antibody according to the present invention has an infection-protecting effect is to suppress cytotoxicity caused by Pseudomonas aeruginosa (Example 10). Therefore, the antibody according to the present invention is useful for the prevention or treatment of diseases associated with Pseudomonas aeruginosa.
- diseases associated with Pseudomonas aeruginosa include systemic infection diseases caused by Pseudomonas aeruginosa infection including multidrug resistant Pseudomonas aeruginosa, such as sepsis, meningitis, endocarditis and the like.
- otitis media In the otolaryngology area, otitis media, sinusitis, in the respiratory area, pneumonia, chronic respiratory tract infection, catheter infection, in the surgical area, postoperative peritonitis, postoperative peritonitis such as postoperative biliary tract, in the ophthalmic area , Eyelid abscess, lacrimal abscess, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, in urology, urinary tract infections including complicated urinary tract infections, catheter infections, perianal abscesses, etc. Can be mentioned. In addition to these, there are severe burns, burns including airway burns, pressure ulcer infections, cystic fibrosis, and the like.
- a method for preventing or treating a disease associated with Pseudomonas aeruginosa which comprises the step of administering a prophylactically or therapeutically effective amount of the antibody according to the present invention to mammals including humans.
- Diagnostic agent for Pseudomonas aeruginosa infection As shown in Examples below, it was confirmed that the antibody according to the present invention binds to Pseudomonas aeruginosa (Example 8). From these results, it was suggested that the antibody according to the present invention can detect the presence of Pseudomonas aeruginosa. Therefore, the antibody according to the present invention can be used as a diagnostic agent for Pseudomonas aeruginosa infection.
- a method for diagnosing Pseudomonas aeruginosa infection using the antibody according to the present invention is provided.
- the diagnostic method according to the present invention collects biological samples such as sputum, lung lavage fluid, pus, tears, blood, urine, etc. from mammals including humans that may be infected with Pseudomonas aeruginosa, and then collects the collected samples and the present invention. It can be carried out by contacting an antibody and determining whether an antigen-antibody reaction has occurred.
- kits for detecting the presence of Pseudomonas aeruginosa at least comprising at kit antibody according to the invention is provided.
- the antibody contained in the detection kit according to the present invention may be labeled.
- This detection kit can detect the presence of Pseudomonas aeruginosa using an antigen-antibody reaction.
- the detection kit according to the present invention may further contain various reagents for carrying out the antigen-antibody reaction, for example, secondary antibodies used in the ELISA method, coloring reagents, buffers, instructions, and / or instruments, if desired. Can do.
- various reagents for carrying out the antigen-antibody reaction for example, secondary antibodies used in the ELISA method, coloring reagents, buffers, instructions, and / or instruments, if desired. Can do.
- compositions or agent according to the present invention uses the antibody according to the present invention as an active ingredient, preferably a purified antibody composition and optional ingredients such as physiological saline, sucrose aqueous solution or phosphate buffer. You may use with the form of the composition containing this.
- the pharmaceutical composition according to the present invention may be formed into a liquid or lyophilized form as required, and optionally a pharmaceutically acceptable carrier such as a stabilizer, preservative, isotonic agent (isotonic). agent) or the like.
- a pharmaceutically acceptable carrier such as a stabilizer, preservative, isotonic agent (isotonic). agent) or the like.
- the pharmaceutically acceptable carrier examples include mannitol, lactose, saccharose, human albumin and the like in the case of a lyophilized preparation.
- physiological saline, water for injection, phosphate Buffers, aluminum hydroxide, etc. can be mentioned as examples. However, it is not limited to these.
- Administration varies depending on the age, weight, sex, and general health condition of the subject, but may be administered by any route of oral administration or parenteral administration (eg, intravenous administration, arterial administration, topical administration). However, parenteral administration is preferred.
- the dosage of the pharmaceutical composition varies depending on the age, weight, sex, general health condition, degree of Pseudomonas aeruginosa infection, and components of the antibody composition to be administered.
- the antibody composition according to the present invention is generally administered to an adult at 0.1 to 1000 mg, preferably 1 to 100 mg per kg body weight per day.
- the pharmaceutical composition according to the present invention is preferably administered in advance to a patient who may be infected by Pseudomonas aeruginosa.
- the antibody titer of ascites a culture solution containing the target antibody, or purified antibody is measured and diluted appropriately with PBS (phosphate buffer containing physiological saline), etc., and 0.1% sodium azide or the like as a preservative Add.
- PBS phosphate buffer containing physiological saline
- an antibody obtained by adsorbing the antibody of the present invention on latex or the like is used after obtaining an antibody titer and appropriately diluting it, and adding a preservative.
- the antibody of the present invention bound to latex particles is one of the preferable dosage forms as a diagnostic agent.
- a suitable resin material for example, latex such as polystyrene, polyvinyltoluene, polybutadiene or the like is suitable.
- Example 1 GeneChip R analysis> A GeneChip R expression analysis system (Affymetrix GeneChip R P. aeruginosa genome array) was used as a method for searching for genes expressed in a human serum-added medium. Using Pseudomonas aeruginosa PAO1 strain at 37 ° C in two culture conditions, namely, Luria-Bertani (LB) medium (manufactured by Nacalai Tesque) supplemented with 0% and 50% human serum (the final LB medium composition is uniform) Until the absorbance at 595 nm reaches 1.0, extract total RNA using the RNeasy Protect Bacteria Mini kit (QIAGEN GmbH) according to the method of the package insert, and use the 2100 Bioanalyzer (Agilent Technologies) Quantification was performed.
- LB Luria-Bertani
- QIAGEN GmbH extract total RNA using the RNeasy Protect Bacteria Mini kit (QIAGEN GmbH) according to the method of the package insert
- PA4761 (DnaK or HSP70), which is a housekeeping protein, was determined to be “Present” indicating that the transcript was detected regardless of the presence or absence of serum in any culture condition, and the gene was expressed. It was shown that.
- PA2018 (MexY) (J. Bacteriology, 2005, which is induced by ribosome inhibitors such as tetracycline and aminoglycoside antibiotics in the transmembrane protein that forms a drug efflux pump in association with PA5158 (OpmG) and PA2019 (MexX). 187, 5341-5346) were determined as “Absent” under the present conditions in which these drugs were not present, indicating that the gene was not expressed.
- the PA1698 gene was determined to be “Present” regardless of the presence or absence of serum under any condition.
- Example 2 Analysis of PA1698 gene in clinical isolates>
- the strain used was 93 strains of Pseudomonas aeruginosa isolated from various clinical materials at clinical facilities nationwide (stored in Yokohama Research Institute, Meiji Seika Co., Ltd.). These strains are derived from blood, urine, sputum, pus, pharyngeal mucus, etc., and the serotype is based on the serological classification determined by the Type Review Committee sponsored by the Pseudomonas aeruginosa Research Association (1975). Groups, B groups, E groups, G groups, I groups, M groups, etc. are included.
- Genomic DNA was prepared from the obtained cells using DNeasy Tissue kit (QIAGEN GmbH) according to the method of the package insert.
- the PCR product was purified by a MultiScreen PCR plate (manufactured by Millipore Corporation) and then subjected to a sequencing reaction. Based on the genome sequence of the PAO1 strain (NC_002516), a primer (SEQ ID NO: 5 to SEQ ID NO: 7) that can sequence each PCR product was designed, and BigDye Terminator v1.1 Cycle Sequencing kit (Applied) was used for the sequencing reaction. BioSystems) was used. The sequencing reaction was performed with GeneAmp PCR System 9700 (Applied BioSystems) according to the attached instructions.
- sequence reaction product was purified on a MultiScreen-HV plate (Millipore Corporation) filled with Sephadex G-50 Fine DNA Grade (Amersham Biosciences AB) previously swollen with water, and then Applied Biosystems 3730 DNA Analyzer (Applied BioSystems Was used to analyze the polynucleotide sequence.
- Example 3 Cloning of PA1698 gene DNA fragment> A DNA fragment containing the full length of 867 bases of the amino acid coding region of the Pseudomonas aeruginosa PA1698 gene (SEQ ID NO: 1) was cloned using the vector pIVEX2.4d (Roche Diagnostics) by the following method, and the expression vector pET15b (Novagen Incorporated).
- the DNA fragment to be cloned was amplified from the genomic DNA of Pseudomonas aeruginosa PAO1 strain by PCR (DNA Thermal Cycler 480; manufactured by Perkin-Elmer). Pyrobest (manufactured by Takara Shuzo Co., Ltd.) is used as the DNA polymerase, 5% dimethyl sulfoxide is added to the reaction solution, and the PCR primer is a primer containing a base for adding restriction enzyme sites NotI (GCGGCCGC) and BamHI (GGATCC) (sequence) No. 8 and SEQ ID No. 9) were used.
- PCR temperature conditions were 94 ° C for 2 minutes, 25 cycles of 94 ° C for 30 seconds, 60 ° C for 1 minute and 72 ° C for 2 minutes.
- the PCR product was purified using MinElute PCR Purification Kit (Qiagen) and cleaved with restriction enzymes NotI (New England Biolabs) and BamHI (Toyobo). PIVEX2.4d was cut with NotI and BamHI.
- the DNA fragments generated by the cleavage were separated by agarose gel electrophoresis and extracted and purified using QIAquick Gel Extraction Kit (manufactured by Qiagen).
- the PCR product cleaved with NotI-BamHI and pIVEX2.4d were ligated with T4 DNA ligase (Ligation High, manufactured by Toyobo Co., Ltd.) to transform Escherichia coli DH5 ⁇ strain (Competent High DH5 ⁇ , manufactured by Toyobo Co., Ltd.).
- the pIVEX2.4d plasmid (pIVEX-PA1698-1) containing the PA1698 gene fragment was purified using QIAprep Spin Miniprep Kit (Qiagen), and BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) was used. A cycle sequence reaction was performed, and the nucleotide sequence of the inserted portion was confirmed (3730 DNA Analyzer, Applied Biosystems / HITACHI).
- a PCR primer (SEQ ID NO: 10) containing a base for adding a restriction enzyme site NdeI (CATATG) to the start codon (ATG) part and a position downstream of the insertion part Amplify the fragment containing the insert using a PCR primer (SEQ ID NO: 11) that matches the T7 terminator to be cleaved, and cleave with restriction enzyme NdeI (New England Biolabs) and BamHI with a restriction enzyme site immediately after the stop codon did.
- PET15b was cleaved with NdeI and BamHI.
- Example 4 Expression and purification of PA1698 recombinant protein>
- E. coli BL21 (DE3) strain incorporating the T7 RNA polymerase gene and a pET vector expression system having a T7 promoter (Novagen) were used.
- E. coli expression vector pET-PA1698-1 is a plasmid encoding PA1698 protein in which a His-tag (six consecutive histidines) is fused downstream of the T7 promoter (see Example 3).
- BL21 (DE3) strain was treated with calcium chloride (see Molecular Cloning 2nd edition, Sambrook et al. (1989)) and transformed with pET-PA1698-1.
- the transformant was cultured overnight in LB medium containing 50 ⁇ g / ml ampicillin, suspended in fresh medium at a dilution of 200-fold, cultured at 37 ° C. for 4 hours, and IPTG was added at a final concentration of 0.5 mM to induce expression. The culture was further continued for 3 hours. Cells were collected by centrifugation and frozen at -20 ° C.
- Cells are lysed with B-PER Bacterial Protein Extraction Reagent (manufactured by Pierce), the insoluble fraction containing the expressed protein is recovered, and the final concentration is 100 ⁇ g / ml lysozyme (egg white lysozyme, Seikagaku Corporation) After the treatment, it was washed with Dulbecco's phosphate buffered saline (PBS) supplemented with 1% Triton X-100.
- PBS Dulbecco's phosphate buffered saline
- Ni chelate chromatography using His-tag was used for protein purification.
- the insoluble protein expressed and prepared was solubilized with a lysis buffer (PBS supplemented with 8M urea, 5 mM imidazole, 200 mM NaCl and 0.05% NP-40).
- the dissolved protein was bound to Ni-NTA Agarose (Qiagen) and washed with 40 volumes of lysis buffer.
- His-tagged protein was eluted and collected with elution buffer (PBS with 8M urea, 300 mM imidazole, 200 mM NaCl). .
- 2.3 mg of protein was finally recovered from 230 ml of E. coli.
- Example 5 Immunization of antigen and preparation of antiserum>
- strain PA103 ATCC29260
- Several colonies are suspended in LB medium and then cultured overnight at 37 ° C with shaking and washed with PBS. After resuspension, formalin was added to 1%, and inactivated bacteria that had been inactivated for 24 hours or more were used.
- male BN rats purchased from Japan Charles River
- Formalin-inactivated bacteria and PA1698 recombinant protein were each immunized at 20 ⁇ g / animal.
- whole blood was collected from the abdominal aorta or carotid artery, allowed to stand at room temperature for 1 hour, centrifuged (1500 G, 20 minutes), and about 5 ml of the supernatant was collected per rat as antiserum.
- Example 6 Purification of IgG fraction from antiserum and ascites> Purification of the IgG fraction from rat antiserum and ascites used the method of Harlow & Lane, 288-318, Chapter 8, Antibodies, A Laboratory Manual, Cold Spring Harbor (1988). Rat antiserum or rat-mouse hybridoma was propagated in the peritoneal cavity of the mouse, and the obtained ascites was centrifuged at 10,000 xg for 20 minutes, and 2 volumes of 60 mM sodium acetate (pH 4.0) was added to the supernatant after removing insolubles. The pH was adjusted to 4.8 with 1N hydrochloric acid.
- caprylic acid 0.06 volume was gradually added to the antiserum or ascites sample at room temperature and stirred for 30 minutes to produce insoluble matter. After removing the precipitate by centrifugation at 13,000 ⁇ g for 10 minutes, it was passed through a 0.45 ⁇ m filter. The obtained sample was concentrated using Amicon Ultra-15 (Millipore), and finally replaced with a PBS ( ⁇ ) solution to obtain a final sample.
- rat antiserum 40 ml
- mouse ascites fluid 75 mL, 35 mL
- rat MAbs produced from hybridomas with the accession numbers of the National Institute of Advanced Industrial Science and Technology (AIST), FERM BP-11055 and FERM BP-11056 Purification was performed, and 102 mg, 14 mg, and 3 mg of protein were recovered as IgG fractions. Protein quantification was evaluated by DC protein assay (Bio-Rad) based on Lowry method, and the purity of IgG was evaluated by SDS-PAGE.
- Example 7 ELISA test>
- the PA1698 recombinant protein was dissolved in PBS supplemented with 8M urea, and a 96-well nickel plate (HIS-Select High Sensitivity (HS) Nickel Coated Plates, Sigma 0.5 ⁇ g of protein per well was allowed to bind to the plate at room temperature.
- a washing buffer PBS supplemented with 0.05% Tween 20, 5 mM imidazole and 500 mM NaCl
- a blocking buffer washing buffer supplemented with 0.5% gelatin
- Samples were placed in wells and allowed to react at room temperature. After washing with a washing buffer, a secondary antibody (peroxidase-labeled goat anti-rat IgG antibody, diluted 10,000 times, manufactured by Sigma) was added and washed after the reaction. After reaction by adding a chromogenic substrate (TMB Microwell Peroxidase Substrate System, manufactured by KPL), the enzyme reaction was stopped with 1M phosphoric acid, and the absorbance at 450 nm was measured.
- a secondary antibody peroxidase-labeled goat anti-rat IgG antibody, diluted 10,000 times, manufactured by Sigma
- the absorbance was 0.490 when PA1698 recombinant protein-immunized rat antiserum (diluted 10,000 times) was used as a sample, whereas the pre-immune serum (10,000-fold diluted) as a negative control had an absorbance of 0.061. It was. This indicates that the PA1698 recombinant protein-immunized rat antiserum contains an antibody that binds to the PA1698 recombinant protein as an immunogen.
- Example 8 Whole cell ELISA test> Whole cell ELISA was prepared by dispensing 100 ⁇ L of the PA103 strain cultured overnight in LB medium to a 96-well ELISA plate (MaxiSorp Type, NUNC), immobilizing at 4 ° C for 1 hour, and washing buffer. After washing with (0.05% Tween20-containing TBS), blocking with a blocking buffer (2% bovine serum albumin-containing TBS), the PA1698 recombinant protein immunity obtained in Examples 5 and 6 was diluted with PBS as the primary antibody sample. Rat antiserum or purified IgG fraction was added and reacted at 37 ° C. for 1 hour.
- a secondary antibody peroxidase-labeled goat anti-rat IgG antibody, diluted 5000 times, manufactured by Sigma
- a chromogenic substrate TMB Microwell Peroxidase substrate System, manufactured by KPL
- TMB Microwell Peroxidase substrate System manufactured by KPL
- the absorbance of the antiserum of PA1698 recombinant protein-immunized rat was 1.058, whereas the absorbance of the pre-immune serum that was a negative control was 0.729.
- This result indicates that the antibody (IgG) recognizing the PA1698 protein present on the cell surface or culture supernatant of Pseudomonas aeruginosa is contained in the PA1698 recombinant protein-immunized rat antiserum.
- anti-PA1698 IgG which is a purified IgG fraction obtained from PA1698 recombinant protein-immunized rat antiserum
- an IgG fraction 50 ⁇ g / g purified from control rat serum obtained by administering only an adjuvant as a negative control.
- the absorbance of 0.1 well was 0.147, whereas that of anti-PA1698 IgG (50 ⁇ g / well) was 0.460. This indicates that the IgG fraction contains an antibody (IgG) that recognizes Pseudomonas aeruginosa.
- Example 9 Production of monoclonal antibody (MAb)>
- MAb monoclonal antibody
- myeloma cells (P3X63Ag8U1 cells) in the logarithmic growth phase pre-cultured in RPMI-1640 medium containing 10% FCS (fetal bovine serum) at 5% CO 2 , relative humidity 100% and 37 ° C are in RPMI-1640 medium And the mixture was mixed so that the ratio of spleen cells to myeloma cells was 4: 1 as described above. The mixed cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to sufficiently loosen the cells.
- FCS fetal bovine serum
- the absorbance in the ELISA that detects binding to the wells adsorbed with the PA1698 recombinant protein was 0.056 in the negative control 10% FCS-RPMI medium, whereas the National Institute of Advanced Industrial Science and Technology (AIST)
- the culture supernatant of the hybridoma whose accession number of the Patent Organism Depositary is FERM BP-11055 has an absorbance of 0.828
- the culture supernatant of the hybridoma whose accession number is FERM BP-11056 has an absorbance of 0.811. From the above results, it was revealed that MAb binding to the PA1698 recombinant protein was produced.
- the rat MAb IgG subclass was determined by heavy antibody and light chain using a monoclonal antibody isotyping kit (RMT1, Dainippon Pharmaceutical Co., Ltd.). As a result, the heavy chain was IgG2a and IgG2a, respectively, and the light chain was all ⁇ . It was judged.
- Anti-1698-1 IgG (MAb) result The absorbance of anti-1698-1 IgG (MAb), which is an IgG fraction obtained by purifying from the ascites of mice administered with a hybridoma whose accession number is FERM BP-11055, is 0.871. On the other hand, the absorbance of the IgG fraction (50 ⁇ g / well) purified from the serum of the control rat that was able to administer only the negative control adjuvant was 0.084. From this result, it was revealed that the IgG fraction contained an antibody (IgG) that recognizes Pseudomonas aeruginosa.
- anti-1698-2 IgG Absorbance of anti-1698-2 IgG (MAb), which is an IgG fraction obtained by purifying from the ascites of mice administered with a hybridoma whose accession number is FERM BP-11056, is 1.415
- the absorbance of the IgG fraction (50 ⁇ g / well) purified from the serum of the control rat that was able to administer only the negative control adjuvant was 0.084. From this result, it was revealed that the IgG fraction contained an antibody (IgG) that recognizes Pseudomonas aeruginosa.
- Example 10 Protective effect of PA1698 antibody against cytotoxic activity of Pseudomonas aeruginosa PA103 strain on human airway epithelial cells (Beas2B cells)> Beas2B cells were dispensed into a 96-well culture plate at a cell number of 5 ⁇ 10 4 / well and cultured in a 5% CO 2 incubator at 37 ° C. for 2 days. After culturing, the plate was washed twice with PBS ( ⁇ ), and 50 ⁇ L of 16% BSA-added (dissolved and neutralized with KOH) DMEM / F12 medium was added.
- LDH lactate dehydrogenase
- anti-PA1698 IgG 2.5 mg / mL
- anti-1698-1 IgG MAb
- anti-1698-2 IgG (MAb) (0.625 mg / mL), an IgG fraction obtained by purifying from mouse ascites to which a hybridoma with the accession number FERM BP-11056 was administered, suppressed 22.5% cell death. .
- Example 11 Protective ability of PA1698 recombinant protein immunized rat antiserum against systemic infection of PA103 strain in normal mice> Evaluation in a normal mouse systemic infection model was carried out as follows: 1.7 ⁇ 10 5 cfu / mouse of PA103 strain suspended in 500 ⁇ l of 5% mucin-containing physiological saline in 4-week-old CD-1 male mice (purchased from Charles River Japan) 34LD 50 ) was inoculated intraperitoneally, and immediately after that an antiserum sample diluted 2.5-fold with physiological saline was administered from the tail vein at 10 ml / kg, and the protective activity against the infection was determined by viability after 7 days.
- Example 12 Protective ability of PA1698 recombinant protein immunized rat antiserum against systemic infection of PA103 strain in neutropenic mice> Evaluation in the neutropenic mouse systemic infection model was prepared by preparing 12.5 mg / mL (physiological saline) of cyclophosphamide (hereinafter referred to as CY; manufactured by Sigma-Aldrich). Was administered intraperitoneally a total of 3 times on day-5, -2, and 0 at a dose of 125 mg / kg to reduce neutrophils in peripheral blood.
- CY cyclophosphamide
- the PA1698 protein which is the target of the antibody according to the present invention, was found to have extremely high storage stability between strains regardless of serotype or the like. For this reason, the antibody according to the present invention can react with various clinical isolates, and can exhibit excellent effects as a pharmaceutical or diagnostic agent for the prevention or treatment of Pseudomonas aeruginosa infection. In addition, since the antibody according to the present invention can also have an effect of suppressing damage to human airway epithelial cells caused by Pseudomonas aeruginosa, chronic antibodies such as diffuse panbronchiolitis (DPB) and cystic fibrosis (CF) are used. Expected to have efficacy against respiratory tract infections.
- DPB diffuse panbronchiolitis
- CF cystic fibrosis
- the vaccine composition of the present invention can exhibit excellent preventive and therapeutic effects against Pseudomonas aeruginosa infection by inducing the antibody of the present invention in vivo.
- the antibody and vaccine composition according to the present invention can greatly contribute to prevention, treatment or diagnosis against Pseudomonas aeruginosa infection.
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Abstract
Description
(i)配列番号:2で表されるアミノ酸配列を含んでなるタンパク質。
(ii)配列番号:2で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含んでなり、かつ配列番号:2で表されるアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。
(i)配列番号:2で表されるアミノ酸配列を含んでなるタンパク質。
(ii)配列番号:2で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含んでなり、かつ配列番号:2で表されるアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。
本発明によれば、緑膿菌由来のPA1698タンパク質またはその一部を認識する抗体またはその機能的断片が提供される。本発明による抗体が結合する緑膿菌のPA1698タンパク質は、血清型等によらず株間でのアミノ酸配列の保存性が極めて高いことが見出された(実施例2)。従って、本発明による抗体の標的となる緑膿菌のPA1698タンパク質は、好ましくは、配列番号:2に記載のアミノ酸配列PA01株由来のタンパク質、あるいは配列番号:2で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含んでなり、かつ配列番号:2で表されるアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質である。アミノ酸配列の置換、欠失、挿入もしくは付加は、一般的には、1~2アミノ酸である。典型的には、1~2アミノ酸の置換である。
緑膿菌由来のPA1698タンパク質は、タンパク質抗原として用いることができ、このような抗原を含む抗原組成物は、緑膿菌に関連する疾患の予防または治療のためのワクチンとして用いることができる。従って、本発明によれば、緑膿菌由来のPA1698タンパク質に対する抗体を産生することができる抗原組成物を含んでなるワクチン組成物が提供される。ここで「抗原組成物」は、タンパク質抗原を唯一の構成要素とする組成物でもよく、また他の成分を含んでなる組成物であってもよい。
緑膿菌に関連する疾患
緑膿菌は宿主の抵抗力の低下につれて致命的な結果となる日和見感染の病原菌であり、また、抗生物質に抵抗性であるため、院内感染の主要な原因菌でもある。後記実施例により示されるように、ムチン投与によりマクロファージの機能を低下させたマウス緑膿菌易感染性モデルにおいて、本発明の抗体が、実際に感染防御効果を有すること(実施例11)、また、Cyclophosphamide monohydrate投与により好中球を減少させたマウス緑膿菌易感染性モデルにおいて、本発明による抗体が実際に感染防御効果を有することが確認された(実施例12)。また、本発明による抗体が感染防御効果を有する理由の一つとして、緑膿菌による細胞障害性を抑制することが挙げられる(実施例10)。従って、本発明による抗体は、緑膿菌に関連する疾患の予防または治療に有用である。
後記の実施例において示されるように、本発明による抗体は緑膿菌と結合することが確認された(実施例8)。これらの結果から、本発明による抗体は、緑膿菌の存在を検出することができることが示唆された。したがって、本発明による抗体は、緑膿菌感染症診断剤として用いることができる。
本発明によれば、緑膿菌の存在を検出するためのキットであって、本発明による抗体を少なくとも含んでなるキットが提供される。本発明による検出キットに含まれる抗体は、標識したものであってもよい。この検出キットは、抗原抗体反応を利用して、緑膿菌の存在を検出することができる。
本発明による医薬組成物または用剤は、本発明による抗体を有効成分として用い、好ましくは、精製した抗体組成物と任意の成分、例えば生理食塩水、葡萄糖水溶液又は燐酸塩緩衝液などを含有する組成物の形態で使用しても良い。
ヒト血清添加培地で発現している遺伝子を探索する手法として、GeneChipR発現解析システム(Affymetrix社製GeneChipRP. aeruginosaゲノムアレイ)を用いた。緑膿菌PAO1菌株を用いて、2通りの培養条件、すなわち0%、50%ヒト血清添加Luria-Bertani(LB)培地(ナカライテスク社製)(最終のLB培地組成は均一)で37℃にて595nmの吸光度が1.0になるまで振とう培養し、RNeasy Protect Bacteria Miniキット(QIAGEN GmbH社製)を用い、添付文書の方法に従って、全RNAを抽出し、2100バイオアナライザー(Agilent Technologies社製)により定量を行った。その後、GeneChipRの添付文書の方法に従って実験を行った。遺伝子発現データの解析はMicroarray Suite 5.0(Affymetrix社製)により行い、シグナルならびにディテクションを計算した。このとき、全プローブセットのシグナルの平均値が1000となるように補正を行った。実験は独立に2回実施した。
使用菌株は、全国の臨床施設において各種臨床材料より分離された緑膿菌93株(明治製菓株式会社 横浜研究所に保管)を試験に供した。これらの株は血液、尿、喀痰、膿、咽頭粘液などに由来しており、血清型は緑膿菌研究会主催の型別検討委員会の決定(1975年)による血清学的分類に基づくA群、B群、E群、G群、I群、M群などが含まれている。
臨床分離の緑膿菌93株について、ミューラーヒントン培地(ベクトン・ディッキンソン社製)で37℃にて一晩培養し、低速遠心によって集菌した。得られた菌体からDNeasy Tissueキット(QIAGEN GmbH社製)を用い、添付文書の方法に従って、ゲノムDNAを調製した。
調製したゲノムDNAを鋳型に、PA1698遺伝子を含む領域をPCRにより増幅した。具体的には、緑膿菌PAO1菌株のゲノム配列(NCBIのデータベースにおけるアクセッション番号:NC_002516)をもとに、PA1698遺伝子を特異的に増幅するプライマーセット(配列番号:3、配列番号:4)を設計し、Takara ExTaq(タカラバイオ株式会社製)で添付の説明書に従い、GeneAmp PCR System 9700(Applied BioSystems社製)を用いてPCRを実施した。PCRにて増幅されたDNA断片は、アガロースゲル電気泳動により、目的のサイズ(1128塩基対)であることを確認した。
PCR産物は、MultiScreen PCRプレート(Millipore Corporation社製)にて精製後、シーケンス反応に供した。PAO1株のゲノム配列(NC_002516)をもとに、各PCR産物をシーケンシングできるプライマー(配列番号:5~配列番号:7)を設計し、シーケンス反応にはBigDye Terminator v1.1 Cycle Sequencingキット(Applied BioSystems社製)を用いた。シーケンス反応は添付の説明書に従い、GeneAmp PCR System 9700(Applied BioSystems社製)で実施した。シーケンス反応産物は、あらかじめ水で膨張させたSephadex G-50 Fine DNA Grade(Amersham Biosciences AB社製)をつめたMultiScreen-HVプレート(Millipore Corporation社製)で精製後、Applied Biosystems 3730 DNA Analyzer(Applied BioSystems社製)を用いてポリヌクレオチド配列の解析を行った。
緑膿菌PA1698遺伝子(配列番号:1)のアミノ酸コード領域867塩基の全長を含むDNA断片を、以下の方法により、ベクターpIVEX2.4d(Roche Diagnostics社)を用いてクローニングし、発現ベクターpET15b(Novagen社)に組み込んだ。
組換えタンパク質の発現には、T7 RNAポリメラーゼ遺伝子が組み込まれた大腸菌BL21(DE3)菌株とT7プロモーターを有するpETベクターの発現系(Novagen社製)を使用した。大腸菌発現ベクターpET-PA1698-1は、T7プロモーターの下流にHis-タグ(6個の連続したヒスチジン)が融合したPA1698タンパク質をコードするプラスミドである(実施例3参照)。BL21(DE3)菌株を塩化カルシウムで処理(Molecular Cloning第2版、Sambrook他(1989)参照)し、pET-PA1698-1により形質転換した。形質転換体を50μg/mlアンピシリンを含むLB培地で一晩培養し、新しい培地に200倍希釈で懸濁して37℃で4時間培養後、IPTGを最終濃度0.5mMで添加して発現誘導し、さらに3時間培養した。細胞を遠心で回収し、-20℃で凍結した。細胞をタンパク質抽出試薬B-PER Bacterial Protein Extraction Reagent(Pierce社製)で溶解し、発現タンパク質を含む不溶性画分を回収し、最終濃度100μg/mlのリゾチーム(卵白リゾチーム、生化学工業社製)で処理後、1%Triton X-100を添加したダルベッコリン酸緩衝塩類溶液(PBS)で洗浄した。
緑膿菌はPA103菌株(ATCC29260)をミューラーヒントン寒天培地上にて一晩37℃で培養し、数個のコロニーをLB培地に懸濁後、一晩37℃で振とう培養し、PBSで洗浄、再懸濁した後、1%となるようにホルマリンを加え、24時間以上不活化処理した不活化菌を使用した。
ラット抗血清および腹水からのIgG画分の精製は、Ha rlow & Lane, 288-318, Chapter 8, Antibodies, A Laboratory Manual, Cold Spring Harbor (1988)らの方法を使用した。ラット抗血清あるいはラット-マウスハイブリドーマをマウス腹腔で増殖させ、得た腹水を10,000xgで20分間遠心し不溶物を除いた上清に、2容量の60mM酢酸ナトリウム(pH4.0)を添加した後、1N塩酸によりpHを4.8に調製した。抗血清あるいは腹水試料に対して0.06容量のカプリル酸を室温にて除々に添加し30分間攪拌し不溶物を生成させた。13,000xgで10分間遠心し沈殿を除いた後、0.45μmのフィルターを通過させた。得られた試料は、Amicon Ultra-15 (Millipore)を用い濃縮後、最終的にPBS(-)溶液に交換し最終標品とした。本方法によりラット抗血清40mlあるいは独立行政法人産業技術総合研究所特許生物寄託センターの受託番号がFERM BP-11055、FERM BP-11056であるハイブリドーマから産生されたラットMAbを含むマウス腹水75mL、35mLより精製を行い、IgG画分として102mg、14mg、3mgのタンパク質を回収した。タンパク定量はLowry法に基づくDC Protein Assay(Bio-Rad社製)、IgGの純度はSDS-PAGEにより評価した。
PA1698組換えタンパク質に結合する抗体をELISA法で検出するため、PA1698組換えタンパク質を、8M尿素を添加したPBSに溶解し、96穴ニッケルプレート(HIS-Select High Sensitivity (HS) Nickel Coated Plates、Sigma社製)にウェルあたり0.5μgのタンパク質を入れ、室温でプレートに結合させた。洗浄バッファー(0.05%Tween 20、5mMイミダゾールおよび500mM NaClを添加したPBS)で洗浄し、ブロッキングバッファー(0.5%ゼラチンを添加した洗浄バッファー)でブロッキング後、実施例5あるいは6で得られた抗体を含む試料をウェルに入れ、室温で反応させた。洗浄バッファーで洗浄後、2次抗体(ペルオキシダーゼ標識ヤギ抗ラットIgG抗体、10000倍希釈、Sigma社製)を入れ、反応後に洗浄した。発色基質(TMB Microwell Peroxidase Substrate System、KPL社製)を添加して反応後、1Mリン酸で酵素反応を停止し、450nmの吸光度を測定した。
Whole cell ELISAはLB培地にて終夜培養したPA103菌株の菌液を96ウェルELISAプレート(MaxiSorp Type、NUNC社製)にウェルあたり100μL分注し、4℃、1時間で固相化後、洗浄バッファー(0.05%Tween20含有TBS)で洗浄し、ブロッキングバッファー(2%ウシ血清アルブミン含有TBS)でブロッキング後、1次抗体サンプルとして、PBSで希釈した実施例5および6で得られたPA1698組換えタンパク質免疫ラット抗血清または精製IgG画分を加え、37℃で1時間反応させた。洗浄後、2次抗体(ペルオキシダーゼ標識ヤギ抗ラットIgG抗体、5000倍希釈、Sigma社製)を添加し、室温で1時間反応後に洗浄した。発色基質(TMB Microwell Peroxidase substrate System、KPL社製)を添加して暗所で反応後、1Mリン酸溶液で酵素反応を停止し、450nmの吸光度を測定した。
PA1698組換えタンパク質の実施例5における最終免疫の1週間後、ラットから麻酔下後、無菌的に脾臓を摘出した。得られた脾臓をRPMI-1640培地(Gibco社製)で洗浄した後、スライドグラスに脾臓を挟み、すり潰し微小片として供試脾細胞を得た。得られた脾細胞はRPMI-1640培地で1000rpm、5分間遠心し洗浄した。一方、10%FCS(ウシ胎児血清)を含むRPMI-1640培地で5%CO2、相対湿度100%、37℃で予め培養して対数増殖期にあるミエローマ細胞(P3X63Ag8U1細胞)をRPMI-1640培地で遠心洗浄し、先に述べた脾細胞とミエローマ細胞の比が4:1になるように混合した。混合した細胞を1000rpmで5分間遠心し、上清を捨て細胞を充分にほぐした。この細胞を含む遠心管にポリエチレングリコール(M.W. 1000 和光純薬社製)2g、RPMI-1640培地2mLおよびDMSO(ナカライテスク社製)0.2mLから成る溶液1mLを静かに加え、遠心管をゆっくり回転させ細胞を混合させた。1分後、遠心管をゆっくり回転させながらRPMI-1640培地15mLを3分かけ加えた。1000rpmで5分間遠心後、上清を捨て充分に細胞をほぐした後、脾細胞として1.6×106/mLとなるようHAT培地(Gibco社製)にて調整し、96穴マイクロプレート(住友ベークライト社製)に0.2mLずつ分注した。5%CO2、相対湿度100%、37℃で約1~2週間培養後、ウェル中に生育しているハイブリドーマが顕微鏡下で観察された。
PA1698組換えタンパク質に結合する抗体を実施例7に記載したELISA法で検出した。また、緑膿菌に結合する抗体を実施例8に記載したwhole cell ELISA法で検出した。
スクリーニングの結果、目的抗体を産生していると判定されたハイブリドーマを5個/0.2mLあるいは20個/0.2mLとなるようBM-Condimed H1 Hybridoma Cloning Supplement(Roche Diagnostics社製)を5%含む10%FCS/HT(Gibco社製)培地にて調整し各ウェルに0.2mLずつ分注した。1~2週間後にクローンの生育が顕微鏡下で観察できた。スクリーニングの項で述べた方法で分析し、目的抗体を産生しているクローンを選択した。再度上記の方法でハイブリドーマを1個/0.2mLあるいは2個/0.2mLとなるようBM-Condimed H1 Hybridoma Cloning Supplementを5%含む10%FCS/HT(Gibco社製)培地にて調整し、96穴マイクロプレートの各ウェルに0.2mLずつ分注した。1~2週間後にスクリーニングの項で述べた方法で分析し目的の抗体を産生する単クローンを選択し、独立行政法人産業技術総合研究所特許生物寄託センターの受託番号がFERM BP-11055、FERM BP-11056であるハイブリドーマを得た。
96穴マイクロプレートで充分増殖させた目的クローンは48穴プレート、12穴プレート、50mL、250mLフラスコに徐々にスケールアップして10%FCS-RPMI培地で培養した。このようにして得られた細胞の培養上清に産生されているMAbを実施例7に記載したELISA法で検出した。
BALB/c-nu/nuマウス(日本チャールスリバー社より購入)に独立行政法人産業技術総合研究所特許生物寄託センターの受託番号がFERM BP-11055、FERM BP-11056であるハイブリドーマを1×107/mouseとなるように腹腔内投与し、1~2週間後に腹水を採取した。腹水中に含まれるMAbは実施例6に記載した方法で精製し、得られた精製IgG画分をそれぞれ、抗1698-1 IgG(MAb)、抗1698-2 IgG(MAb)と命名した。このラットMAbのIgGのサブクラスは、モノクローナル抗体アイソタイピングキット(RMT1、大日本製薬社製)によって重鎖および軽鎖を判定した結果、重鎖はそれぞれIgG2a、IgG2aであり、軽鎖は全てκと判定された。
Beas2B細胞を96穴培養用プレートに5×104/wellの細胞数になるように分注し、2日間37℃の5%CO2インキュベーター内で培養した。培養後、PBS(-)で2度洗浄し、16%BSA添加(溶解後KOHで中和した)DMEM/F12培地を50μL加えた。そこへPBSで希釈したPA1698組換えタンパク質免疫ラット抗血清から得られた精製IgG画分である抗PA1698 IgGあるいは抗1698-1 IgG(MAb)、抗1698-2 IgG(MAb)と、緑膿菌PA103菌株5×108cfu/mLを25μLずつ加え37℃、5%CO2インキュベーター内で4時間培養した。4時間後、培養上清中のLDH(乳酸脱水素酵素)量を細胞死の指標として測定した。0.2%TritonX100で細胞を可溶化したときのLDH量を100%細胞死量とし、緑膿菌PA103菌株を添加しないときのLDH量を0%細胞死量として計算した。
正常マウス全身感染モデルでの評価は、4週齡のCD-1雄性マウス(日本チャールズリバー社より購入)に5%ムチン含有生理食塩水500μlに懸濁したPA103菌株の1.7x105cfu/マウス(34LD50)を腹腔内に接種し、直後に生理食塩水で2.5倍希釈した抗血清サンプルを10ml/kgで尾静脈より投与し、7日後の生死で感染防御活性を判定した。
好中球減少マウス全身感染モデルでの評価は、cyclophosphamide(以下、CYとする。Sigma-Aldrich社製)の12.5mg/mL(生理食塩水)を調製し、4週齡のCD-1雄性マウスに125mg/kgの投与量でday-5、-2、0に合計3回腹腔内投与して末梢血中の好中球を減少させた。その後、生理食塩水250μlに懸濁したPA103菌株の9.5x104cfu/マウス(70LD50)を腹腔内に接種し、直後にサンプルを10mL/kgで尾静脈より投与し、7日後の生死で感染防御活性を判定した。
Claims (15)
- 緑膿菌由来のPA1698タンパク質に結合する抗体またはその機能的断片。
- 緑膿菌由来のPA1698タンパク質が、以下の(i)または(ii)に記載のタンパク質である、請求項1に記載の抗体またはその機能的断片。
(i)配列番号:2で表されるアミノ酸配列を含んでなるタンパク質。
(ii)配列番号:2で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含んでなり、かつ配列番号:2で表されるアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。 - 緑膿菌表面に結合する、請求項1に記載の抗体またはその機能的断片。
- 緑膿菌がヒト気道上皮細胞に及ぼす細胞障害活性を抑制する活性を有する、請求項1に記載の抗体またはその機能的断片。
- 緑膿菌が感染している患者において抗菌活性を有する、請求項1に記載の抗体またはその機能的断片。
- 好中球が減少している状態における患者の緑膿菌の感染において抗菌活性を有する、請求項5に記載の抗体またはその機能的断片。
- FERM BP-11055またはFERM BP-11056の受託番号のもと寄託されたハイブリドーマにより産生される抗体のエピトープに結合する、請求項1に記載の抗体またはその機能的断片。
- FERM BP-11055またはFERM BP-11056の受託番号のもと寄託されたハイブリドーマ。
- 緑膿菌由来のPA1698タンパク質に対する抗体を産生することができるタンパク質抗原またはペプチド抗原を含んでなる抗原組成物と、場合によっては1種以上の薬学的に許容される担体、希釈剤、および/またはアジュバントとを含んでなる、緑膿菌に関連する疾患の予防または治療に用いられるワクチン組成物。
- 緑膿菌由来のPA1698タンパク質が、以下の(i)または(ii)に記載のタンパク質である、請求項9に記載のワクチン組成物。
(i)配列番号:2で表されるアミノ酸配列を含んでなるタンパク質。
(ii)配列番号:2で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含んでなり、かつ配列番号:2で表されるアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。 - 緑膿菌に関連する疾患が、緑膿菌感染に起因する全身感染疾患である、請求項9に記載のワクチン組成物。
- 請求項1~7のいずれか一項に記載の抗体またはその機能的断片と、場合によっては1種以上の薬学的に許容される担体、および/または希釈剤とを含んでなる、緑膿菌に関連する疾患の予防または治療に用いられる医薬組成物。
- 緑膿菌に関連する疾患が、緑膿菌感染に起因する全身感染疾患である、請求項12に記載の医薬組成物。
- 請求項1~3のいずれか一項に記載の抗体またはその機能的断片を含んでなる、緑膿菌感染症診断剤。
- 請求項1~3のいずれか一項に記載の抗体またはその機能的断片を含んでなる、緑膿菌の検出キット。
Priority Applications (6)
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CN2008801228677A CN101970468A (zh) | 2007-12-25 | 2008-12-24 | 绿脓杆菌的ⅲ型分泌系统构成蛋白质pa1698 |
CA2709500A CA2709500A1 (en) | 2007-12-25 | 2008-12-24 | Type iii secretion system component protein pa1698 of pseudomonas aeruginosa |
AU2008342152A AU2008342152B2 (en) | 2007-12-25 | 2008-12-24 | Component protein PA1698 for type-III secretion system of Pseudomonas aeruginosa |
EP08864708A EP2236515A4 (en) | 2007-12-25 | 2008-12-24 | COMPONENT PROTEIN PA1698 FOR PSEUDOMONAS AERUGINOSA TYPE-III SECRETION SYSTEM |
JP2009547118A JPWO2009081955A1 (ja) | 2007-12-25 | 2008-12-24 | 緑膿菌のiii型分泌装置構成タンパク質pa1698 |
US12/810,459 US20100291070A1 (en) | 2007-12-25 | 2008-12-24 | Type iii secretion system component protein pa1698 of pseudomonas aeruginosa |
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JP2007-331653 | 2007-12-25 | ||
JP2007331653 | 2007-12-25 |
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PCT/JP2008/073492 WO2009081955A1 (ja) | 2007-12-25 | 2008-12-24 | 緑膿菌のiii型分泌装置構成タンパク質pa1698 |
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US (1) | US20100291070A1 (ja) |
EP (1) | EP2236515A4 (ja) |
JP (1) | JPWO2009081955A1 (ja) |
KR (1) | KR20100100941A (ja) |
CN (1) | CN101970468A (ja) |
AU (1) | AU2008342152B2 (ja) |
CA (1) | CA2709500A1 (ja) |
SG (1) | SG186682A1 (ja) |
WO (1) | WO2009081955A1 (ja) |
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EP1946768A4 (en) * | 2005-10-28 | 2009-11-11 | Meiji Seika Kaisha | OUTER COAT PROTEIN PA5158 OF PSEUDOMONAS AERUGINOSA |
CN105424929B (zh) * | 2015-11-24 | 2017-08-08 | 四川夹金山逢春养殖科技有限公司 | 一种铜绿假单胞菌抗体检测试剂盒及检测方法 |
CN113122507B (zh) * | 2021-04-27 | 2023-03-24 | 江南大学 | 一种快速检测铜绿假单胞菌的双抗体夹心elisa方法 |
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Also Published As
Publication number | Publication date |
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EP2236515A1 (en) | 2010-10-06 |
AU2008342152A1 (en) | 2009-07-02 |
US20100291070A1 (en) | 2010-11-18 |
SG186682A1 (en) | 2013-01-30 |
AU2008342152B2 (en) | 2013-06-27 |
CN101970468A (zh) | 2011-02-09 |
JPWO2009081955A1 (ja) | 2011-05-06 |
KR20100100941A (ko) | 2010-09-15 |
EP2236515A4 (en) | 2012-10-31 |
CA2709500A1 (en) | 2009-07-02 |
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