WO2009123119A1 - 合成ペプチドを含有する抗原薬物ビークルとこれを用いる粘膜ワクチン - Google Patents
合成ペプチドを含有する抗原薬物ビークルとこれを用いる粘膜ワクチン Download PDFInfo
- Publication number
- WO2009123119A1 WO2009123119A1 PCT/JP2009/056508 JP2009056508W WO2009123119A1 WO 2009123119 A1 WO2009123119 A1 WO 2009123119A1 JP 2009056508 W JP2009056508 W JP 2009056508W WO 2009123119 A1 WO2009123119 A1 WO 2009123119A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antigen
- vehicle
- drug
- vaccine
- acid
- Prior art date
Links
Classifications
-
- 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
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- 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
- A61K39/12—Viral antigens
-
- 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
- A61K39/385—Haptens or antigens, bound to carriers
-
- 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
-
- 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- 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
- A61P37/08—Antiallergic agents
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- A61K2039/541—Mucosal route
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6018—Lipids, e.g. in lipopeptides
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention relates to an antigen drug (AD) vehicle that enables nasal, transmucosal and transdermal administration, and a nasal / mucosal vaccine using the AD vehicle.
- AD antigen drug
- Patent Documents 1 and 2 describe in detail the drawbacks of conventional inactivated vaccines and toxoids, the current state of development of mucosal vaccines and immune adjuvants, and the like.
- the present inventors have addressed such a problem by using an antigen drug (AD) vehicle that is a complex of pulmonary surfactant protein B and / or pulmonary surfactant protein C and lipid, and a mucosa comprising the AD vehicle and an antigen.
- AD antigen drug
- Patent Document 1 A vaccine has been invented and a patent application has been filed (Patent Document 1). Furthermore, the inventors of the present application can convert between selective production of IgA antibody and production of both IgA and IgG antibodies by adjusting the weight ratio V / A of AD vehicle amount (V) and antigen amount (A). And a patent application has been filed for a mucosal vaccine having this mechanism of action (Patent Document 2). These Patent Documents 1 and 2 also disclose the effectiveness of fragments (peptides) of pulmonary surfactant proteins B and C.
- Patent Documents 3 to 8 are known as synthetic peptides related to pulmonary surfactant protein.
- International Publication WO 2005/097182 Publication International Publication WO 2007/018152 Japanese Patent Publication No. 3009690 JP 2004-305006
- a Special Table 2006-504635 International Publication WO 95/15980
- Special Table 2003-523348 International Publication WO 02/32451
- the present inventors have found that the antibody is a peptide having a size smaller than that of the partial peptides disclosed in Patent Documents 1 and 2.
- the present inventors have found a peptide having a strong induction or enhancement effect of production, in particular, the production of secretory IgA antibody alone, and the excellent and effective induction effect of both secretory IgA and blood IgG antibody production.
- the object of the present invention is to provide an antigen drug (AD) vehicle and a mucosal vaccine using such a novel synthetic peptide.
- AD antigen drug
- a first invention for solving the above-mentioned problems is a synthetic peptide comprising the following amino acid sequence: PVHLKRLm (where m is 11-15 or 16-20); or KnLm (where n is 4-8, m is 11-20) It is an antigen drug (AD) vehicle that is a complex of lipid and lipid.
- AD antigen drug
- the synthetic peptide is preferably a peptide consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 3.
- the lipid in this AD vehicle is preferably at least one of phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid More preferably, it is a mixture of three lipids of dipalmitoyl phosphatidylcholine, phosphatidylglycerol and palmitic acid.
- the second invention is a mucosal vaccine obtained by coexisting, contacting, capturing or adsorbing an antigen that induces mucosal immunity IgA in the antigen drug (AD) vehicle.
- the antigen in this mucosal vaccine is preferably an inactivated antigen or a detoxified toxin derived from an infectious disease pathogen.
- the third invention is an allergy preventive or therapeutic agent obtained by coexisting, contacting, capturing or adsorbing an allergen, an allergen epitope, or an allergen-derived antigen to the antigen drug (AD) vehicle.
- AD antigen drug
- the fourth invention is a method for preventing or treating infectious diseases, characterized in that the mucosal vaccine is administered at least twice.
- the fifth invention is a method for preventing or treating allergy, characterized in that the allergy preventing or treating agent is administered at least twice.
- the feature of the AD vehicle provided by the present invention lies in excellent and effective induction of secretory IgA antibody production alone and production of both secretory IgA and blood IgG antibodies.
- Application and general use of this AD vehicle will bring about the realization and widespread use of mucosal vaccines against various infectious diseases, allergy prevention and treatment agents, and transmucosal and transdermal administration of drugs.
- Nasal or mucosal vaccine is an immunization means that matches the actual condition of natural infection, and thus exhibits a significantly superior infection protection effect compared to conventional vaccines.
- nasal mucosal IgA and IgG induced by AD vehicles greatly improve medical care, health care and hygiene, and become a long-awaited gospel for workers in the medical care, health care, and hygiene fields of the world.
- biologics including conventional and future vaccines, toxoids, etc., and a wide variety of drugs, providing a means to add functions and performance that enable simple transmucosal administration and transdermal administration compared to injection. .
- the AD vehicle (Antigen and Drug Vehicle) of the present invention is a complex of a lipid and a synthetic peptide designed (designed) to enable transmucosal administration and transdermal administration of antigens and drugs.
- Such a synthetic peptide is, for example, any of the following peptides.
- the parentheses are peptide abbreviations. Amino acid residues are indicated by single letter symbols. Sequence number 1 (SP-CL11): PVHLKRLLLLLLLLLLLLL Sequence number 2 (K6L16): KKKKKKLLLLLLLLLLLLLLLLLLLL Sequence number 3 (K6L11): KKKKKKLLLLLLLLLLLLL In SEQ ID NO: 1 (SP-CL11), 11 L (Leu) residues are added to amino acid sequence 7-12 in the amino acid sequence of lung surfactant protein C (SP-C) (PVHLKR). .
- SP-C lung surfactant protein C
- SEQ ID NO: 2 (K6L16) consists of 6 K (Lys) residues on the N-terminal side and 16 L residues on the C-terminal side.
- SEQ ID NO: 3 (K6L11) consists of 6 K (Lys) residues on the N-terminal side and 11 L residues on the C-terminal side.
- phospholipids contained in lung surfactant such as phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, phosphatidylglycerol, and the like.
- phosphatidylinositol phosphatidylethanolamine, phosphatidic acid, sphingomyelin and the like can be used.
- fatty acid lauric acid, myristic acid, palmitic acid, stearic acid, palmitooleic acid, oleic acid and the like can be used.
- lipids derived from aquatic animals such as whales and dolphins with active lung expansion can be used.
- Composition of AD vehicle The synthetic peptide is about 0.2 to about 12.0% by dry weight, and the lipid is about 88 to about 99.8% by dry weight.
- AD vehicle preparation example For example, 4 mg of synthetic peptide dissolved in methanol and 96 mg of lipid dissolved in chloroform-methanol mixture were mixed, dried under reduced pressure with an evaporator, suspended in 10% ethanol, Freeze-dry. This dried product is uniformly suspended in 5 mL of isotonic solution, for example, physiological saline or phosphate buffer (PBS). The obtained suspension is used as an AD vehicle (100 mg / 5 mL) solution. The vehicle is prepared each time it is used. In addition, an ultrasonic wave, a homogenizer, a mixer, a shaker, etc. can be used for suspension.
- isotonic solution for example, physiological saline or phosphate buffer (PBS).
- PBS physiological saline or phosphate buffer
- the obtained suspension is used as an AD vehicle (100 mg / 5 mL) solution.
- the vehicle is prepared each time it is used.
- lipid 96 mg for example, a mixture of phosphatidylcholine 64.5 mg, phosphatidylglycerol 22.7 mg, and palmitic acid 8.8 mg can be employed.
- Preparation of mucosal vaccine AD vaccine solution is added to the vaccine stock solution and mixed so that the dry weight ratio V / A of AD vehicle amount (V) to antigen amount (A) in the vaccine becomes a desired value.
- V / A 1
- the added amount of AD vehicle (50 mg / mL) solution prepared in (4) above is 50 ⁇ L.
- a homogenizer, a mixer, a shaker, a stirrer, etc. can be used.
- Antigens include bacteria-derived antigens, virus antigens, toxoids, etc. that have been highly purified for vaccines and that are more than about 90% pure, and allergens, proteins, sugars, etc. that are more than about 90% pure from cedar pollen, mites, etc. It means protein, high-molecular sugar, nucleic acid and the like.
- a value of the antigen mass a measured value can be used, or a calculated value from the purity, specific activity, molecular weight, antigen-antibody reaction, etc. of the antigen can be used.
- the dry weight (A) of the antigen in the mucosal vaccine of the present invention is about 0.1 to about 50 ⁇ g / kg, preferably about 0.3 to about 30 ⁇ g / kg.
- the V / A for preferentially and selectively inducing IgA antibody production is preferably about 0.1 to about 1.0.
- V / A for inducing production of both IgA and IgG antibodies can be in the range of about 1.0 to about 100, preferably about 5 to about 20.
- the resulting mucosal immune vaccine can efficiently induce IgA antibody production and / or IgG antibody production. it can.
- At least two vaccine administrations are performed. More preferably, three times of vaccine administration (primary immunization, secondary immunization, and tertiary immunization) are performed. Such multiple immunizations can significantly increase the antibody titers of IgA and IgG. Two or three vaccine administrations are performed at intervals of 1 to 3 weeks, preferably about 2 weeks.
- RK-SP-CL (SEQ ID NO: 5) has four more C-terminal L residues in SP-CL11 (SEQ ID NO: 1), and KR of the 5th-6th amino acid sequence of SP-CL11 is changed to RK It is reversed.
- AD vehicle Each of the synthetic peptides prepared in (1) above is added to three lipid mixtures (dipalmitoyl phosphatidylcholine: DPPC, phosphatidylglycerol: PG, palmitic acid: PA) to form a plate-like phospholipid membrane AD vehicle: SSF-2 (containing SP-C (1-35)), SSF-3 (containing SP-CL11), SSF-4 (containing K6L16), SSF-5 (containing RK-SP-CL) ) And SSF-6 (containing K6L11).
- the composition of the three lipid mixtures is PA, PG, PA (75:25:10, w / w / w). Each peptide was added in an amount corresponding to 0.6% mol of the lipid mixture.
- a mucosal vaccine consisting only of three lipid mixtures was prepared.
- SSF-1 a mucosal vaccine
- SSF-1 a mucosal vaccine
- a split-type influenza vaccine Suspension derived from embryonated eggs inoculated with influenza virus A Aichi / 68/2 / H3N2 (1 ⁇ 10 8 Plaque forming unit (PFU)) (Kawasaki Medical University, Department of Microbiology, Ouchi)
- a split-type influenza vaccine was created using the following procedure. ⁇ -propiolactone (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to a suspension of virus dialyzed overnight in 0.004M PBS (Takara Bio Inc., Japan, Tokyo, Shiga) to a final concentration of 8 nM.
- split influenza vaccine was added to 0.1 ⁇ g of the dry weight of the AD vehicle, mixed with a vortex mixer, and allowed to stand at room temperature for 1 hour for use.
- Animals 6-week-old female BALB / c mice were purchased from Nippon SLC Co., Ltd. (Shizuoka, Japan) and used. All animal experiments were performed at the Infectious Animal House (P2 level) at the Tokushima University School of Medicine Laboratory Animal Center, and were conducted according to the guidelines of the Animal Experiment Committee of the University of Tokushima School of Medicine.
- the mucosal vaccine of (4) above was diluted with PBS to give a 0.1 ⁇ g / 1 ⁇ L Phosphate buffered saline (PBS) solution corresponding to the dry weight.
- PBS Phosphate buffered saline
- One microliter per side was administered to both sides, and a total of 2 ⁇ L was administered nasally to the bilateral nasal cavity of mice anesthetized with ketalal (62.6 mg / Kg) and cerectal (12.4 mg / Kg).
- the control group received PBS in the same amount as the vaccine solution.
- Viral antigen HA alone was also administered.
- secondary immunization was performed in the same manner as the first immunization.
- HA alone and HA + SSF-4 the third immunization was performed two weeks after the second immunization.
- Preparation of mouse nasal cavity / alveolar lavage fluid and serum Nasal cavity / alveolar lavage fluid and serum were prepared two weeks after the second immunization, and virus-specific IgA and IgG were measured.
- IgA and IgG were measured in the same manner two weeks after the third immunization.
- mice were laparotomized under pentobarbital anesthesia, the trachea was opened, and 3 Fr (Atom Medical Co., Ltd., Tokyo, Japan) with Atom Vein Catheter was inserted into the lung, and 1 ml of physiological saline was injected. The liquid was collected. This was repeated 3 times, and a total of 3 mL was used as the alveolar lavage fluid.
- an atom vein catheter was inserted from the incised trachea toward the nasal cavity, 1 mL of physiological saline was injected, and the fluid that came out of the nose was collected. This solution was used as a nasal wash.
- ELISA assay was performed according to the method of Mouse ELISA quantitation kit of BETHYL LABORATORIES (Texas, USA). 96-well Nunc immunoplate (Nalgen Nunc International USA, New York) Each well was added 1 ⁇ g of vaccine and bovine serum albumin (BSA, SIGMA USA, MO) 1 ⁇ g / mL PBS solution 100 ⁇ L, followed by overnight solidification at 4 ° C It was.
- BSA bovine serum albumin
- the rinse vaccine solution was removed three times with a washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0).
- a washing solution 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0.
- Anti-influenza IgA and / or IgG production in the mucosal vaccine (HA + SSF-1 to HA + SSF-6) administration group in which SSF-1 to SSF-6 were mixed with the HA vaccine, respectively, compared to the HA vaccine alone administration group Was expensive.
- the production enhancement effect of IgG antibody was confirmed.
- SSF-1 and SSF-5 have almost the same antibody production enhancing action, RK-SP-CL with a partial amino acid sequence change of SSF-3 (SP-CL) is involved in the antibody production action. It was confirmed to be small.
- the treatment with the mucosal vaccine of the present invention is preferably at least twice, preferably three times.
- the antibody production enhancing effect of mucosal vaccine in a miniature pig model was tested.
- the mini pig was a 5-10 week old (3-7 kg) crown line (Japan Farm, Kagoshima).
- Two weeks before the first immunization nasal discharge samples were collected from minipigs by the following method, and confirmed to be anti-influenza antibody-negative individuals by ELISA, and used for inoculation tests.
- the antigen used was a formalin-inactivated ether split vaccine (HA in the table, referred to as vaccine in the text below: obtained from Osaka University Microbial Disease Research Society (Kagawa)), which was created using the influenza virus A New Caledonia strain (H1N1) as a raw material. .
- the amount of vaccination per mini pig was 24 ⁇ g in terms of hemagglutinin.
- AD vehicle (SP-C (1-35) -containing SSF-2, SP-CL11-containing SSF-3 and K6L16-containing SSF-4) was prepared in the same manner as in Example 1, and vaccine total protein weight 1 200 ⁇ l saline suspension mixed with AD vehicle 10 to the literature (Mizuno D, Ide-Kurihara M, Ichinomiya T, Kubo I, Kido H. in response to the influenza virus antigen. J Immunol. 2006; 176: 1122-30).
- This suspension was mucosal vaccine (HA + SSF-2, HA + SSF-3, HA + SSF-4, respectively) and sedated with a mixture of medetomidine (0.08 mg / Kg) and mitazolam (0.08 mg / Kg) Then, under anesthesia with ketalal (0.2 mg / Kg), 100 mg ⁇ L per nose was injected into both nasal cavities of the minipig using a rat oral sonde, which was used as a nasal vaccination.
- ketalal 0.2 mg / Kg
- Booster inoculation (secondary immunization) was performed 3 weeks after the first vaccination, and nasal juice samples and serum samples from the jugular vein were sampled every week from the first administration to the 5th week. Sampling during the vaccination week (0, 3 weeks) was performed 2 days before vaccination.
- the nasal discharge sample was collected by wiping the nasal cavity of both minipigs with a cotton swab and washing the swab in 2 mL of raw food to collect the nasal discharge. Each sample was stored at ⁇ 80 ° C. until used for testing.
- the third immunization is performed from the second immunization to the second week, and then the second immunization to the second week (first immunization to the seventh week). Went.
- the anti-influenza IgA and IgG antibody titers contained in each sample were measured by partially modifying the ELISA of Example 1.
- the detection of anti-influenza-specific antibody is performed by immobilizing the vaccine antigen used for nasal inoculation on a plate, using a serial dilution of minipig nasal juice sample 4 times and serum sample 10 times to 2 times. Measurements were performed. Absorbance exceeding the standard value of [average value of 450 nm absorbance + 2 x standard deviation] of the 4-fold diluted nasal juice sample of the saline administration group as the control group and the 10-fold diluted serum sample is shown. The maximum dilution ratio was taken as the IgA or IgG antibody titer contained in each sample.
- the antibody titer after the third immunization (7 weeks after the first immunization) for HA + SSF-2 and HA + SSF-4 further increased the nasal IgA antibody titer, and in the case of HA + SSF-4 serum IgG antibody titer also increased greatly.
- This result strongly shows the effectiveness of the third immunization because the antibody titers of nasal IgA and serum IgG are greatly decreased when measuring up to the seventh week only with the second immunization (week 7 *). Is.
- This result also confirmed that it is preferable to administer the vaccine at least twice, preferably 3 times in the treatment using the mucosal vaccine of the present invention.
Abstract
Description
PVHLKRLm(ただしmは11-15または16-20);または
KnLm(ただしnは4-8、mは11-20)
と脂質との複合体である抗原薬物(AD)ビークルである。
(1)合成ペプチド
PVHLKRLm(ただしmは11-15または16-20)またはKnLm(ただしnは4-8、mは11-20)のアミノ酸配列からなるペプチドである。すなわち、PVHLKRLmは、PVHLKRLのC末側にm個のL(Leu)残基が連続しており、KnLmはN末側のn個のK(Lys)残基とC末側のm個のL残基が連続している。なお、PVHLKRLmにおけるm=16は特許文献2の配列番号27よして公知のペプチドである、本願発明からは除外される。
配列番号1(SP-CL11):PVHLKRLLLLLLLLLLL
配列番号2(K6L16):KKKKKKLLLLLLLLLLLLLLLL
配列番号3(K6L11):KKKKKKLLLLLLLLLLL
なお、配列番号1(SP-CL11)は、肺サーファクタントプロテインC(SP-C)のアミノ酸配列における第7-12番アミノ酸配列(PVHLKR)に11個のL(Leu)残基が付加されている。配列番号2(K6L16)は、N末側の6個のK(Lys)残基とC末側の16個のL残基とからなる。配列番号3(K6L11)は、N末側の6個のK(Lys)残基とC末側の11個のL残基とからなる。
(2)脂質
リン脂質としては、肺サーファクタントが含有するリン脂質、例えばホスファチジルコリン、ジパルミトイルホスファチジルコリン、ホスファチジルセリン、ホスファチジルグリセロール等の使用が望ましい。その他、ホスファチジルイノシトール、ホスファチジルエタノールアミン、ホスファチジン酸、スフィンゴミエリン等を用いることができる。また、脂肪酸としては、ラウリル酸、ミリスチン酸、パルミチン酸、ステアリン酸、パルミトオレイン酸、オレイン酸等を用いることができる。更に、肺の膨張が活発なクジラ、イルカ等の水棲動物に由来の脂質を用いることができる。
(3)ADビークルの組成
合成ペプチドは、約0.2~約12.0乾燥重量%、脂質は約88~約99.8乾燥重量%である。
(4)ADビークルの調製例
例えば、メタノールに溶解した合成ペプチドを4 mg、クロロホルム-メタノール混液に溶解した脂質を96 mgを混合し、エバポレーターで減圧乾固した後、10%エタノールで懸濁し、凍結乾燥する。この乾燥物を5 mLの等張液、例えば生理的食塩水やリン酸緩衝液(PBS)中に均一に懸濁させる。得られた懸濁液は、ADビークル(100 mg/5 mL)液として使用に供する。該ビークルは、使用時にその都度、調製する。尚、懸濁には、超音波、ホモジナイザー、ミキサー、振盪器等を用いることができる。
(5)粘膜ワクチンの調製
ワクチン中の抗原量(A)に対するADビークル量(V)の乾燥重量比V/Aが所望の値になるよう、ワクチン原液にADビークル液を添加混合し調製する。例えば、抗原含有量が50 mg/mLのワクチン原液50 μLに対し、重量比V/A=1を採用すると、上記(4)で調製したADビークル(50 mg/mL)液の添加混合量は50 μLである。尚、均一に混合するには、ホモジナイザー、ミキサー、振盪器、攪拌器等を用いることができる。
(1)合成ペプチドの調製
以下のペプチドを公知の方法により化学合成した。
SP-CL11:PVHLKRLLLLLLLLLLL(配列番号1)
K6L16:KKKKKKLLLLLLLLLLLLLLLL(配列番号2)
K6L11:KKKKKKLLLLLLLLLLL(配列番号3)
SP-C(1-35):FGIPCCPVHLKRLLIVVVVVVLIVVVIVGALLMGL(配列番号4)
RK-SP-CL:PVHLRKLLLLLLLLLLLLLLLL(配列番号5)
SP-C(1-35)(配列番号4)はヒト肺サーファクタントプロテインCの第1-35番アミノ酸配列に相当し、特許文献1の配列番号21と同一である。RK-SP-CL(配列番号5)はSP-CL11(配列番号1)のC末側L残基数がさらに4個多く、またSP-CL11の第5-6番アミノ酸配列のKRがRKに逆転している。
(2)ADビークルの調製
前記(1)で調製した合成ペプチドのそれぞれを、3種脂質混合物(ジパルミトイルホスファチジルコリン:DPPC、ホスファチジルグリセロール:PG、パルミチン酸:PA)に加えて板状のリン脂質膜を作成し、ADビークル:SSF-2(SP-C(1-35)含有)、SSF-3(SP-CL11含有)、SSF-4(K6L16含有)、SSF-5(RK-SP-CL含有)およびSSF-6(K6L11含有)を調製した。3種脂質混合物の組成は、PA、PG、PA(75:25:10、w/w/w)である。また、各ペプチドは脂質混合物の0.6%モルに相当する量を加えた。
(3) スプリット型インフルエンザワクチンの作製
インフルエンザウイルスA Aichi/68/2/H3N2株を接種した発育鶏卵由来浮遊液(1×108 Plaque forming unit (PFU))(川崎医科大学・微生物学教室 大内正信教授より供与された)を用いて以下の操作でスプリット型インフルエンザワクチンの作成を行った。0.004M PBS(タカラバイオ株式会社 日本・東京・滋賀)で一晩透析されたウイルス浮遊液にβプロピオラクトン(和光純薬株式会社 日本・大阪)を液量の0.05%、最終濃度8 nMになるように添加し、氷浴中で18時間インキュベートした。その後、37℃で1.5時間インキュベートすることで、βプロピオラクトンの加水分解を行った。その後、終濃度0.1%となるようにTween20(和光純薬株式会社)を加え、さらにTweenと等量のジエチルエーテル(和光純薬株式会社)を加え、4℃で2時間転倒混和した。この液を2000 rpm、5分間遠心分離することにより水層を回収した。さらにAutomatic Environmental SpeedVac System(SAVANT INSTRUMENTS, INC. アメリカ・ニューヨーク)を用いて水層よりジエチルエーテルの除去を行った。これをMillex 0.45 μmフィルター(MILLIPORE アメリカ・マサチューセッツ)で濾過し、不活化スプリット型インフルエンザワクチン(HA)として用いた。なおβプロピオラクトンの変わりに、ホルマリンによる不活化スプリット型インフルエンザワクチンも同様に使用できる。
(4)粘膜ワクチンの調製
上記(3)で作製したスプリット型インフルエンザワクチン(HA)に、前記(2)で調製したADヴィークル(SSF-1~SSF-6)を混合し、粘膜ワクチン(HA+SSF-1~HA+SSF-6)を調製した。すなわち、各ADビークルをワクチン投与に必要な濃度で用時PBSに懸濁し、室温、5分間の超音波処理により均一な懸濁液とした。これにADビークルの乾燥重量で0.1μgに対してスプリット型インフルエンザワクチンを0.1μg加え、ボルテックスミキサーで混合したのち、室温で1時間静置して使用した。
(5)動物
6週齢、メスBALB/cマウスを日本エスエルシー株式会社(日本・静岡)から購入して用いた。全ての動物実験は徳島大学医学部実験動物センターの感染動物舎(P2レベル)で行われ、徳島大学医学部動物実験委員会のガイドラインに従って行われた。
(6)免疫法
ワクチンの経鼻投与においては、上記(4)の粘膜ワクチンを乾燥重量相当量として0.1 μg/1μL Phosphate buffered saline(PBS)溶液になるようにPBSで希釈し、これを1匹当たり片側1μLづつを両側に投与して、合計2μLをケタラール(62.6 mg/Kg)及びセラクタール(12.4 mg/Kg)で麻酔したマウスの両側鼻腔に点鼻投与した。対照群にはワクチン液と同量のPBSを投与した。また、ウイルス抗原のHA単独投与も行った。4週間後に初回免疫と同様の方法によって2次免疫を行った。さらに、HA単独とHA+SSF-4については2次免疫後2週間後に3次免疫を行った。
(7)マウス鼻腔・肺胞洗浄液および血清の調製
2次免疫後2週間目のマウスの、鼻腔・肺胞洗浄液および血清を調製して、ウイルス特異的なIgA、IgGの測定を行った。また、HA単独とHA+SSF-4については3次免疫から2週間後に同様にしてIgA、IgGの測定を行った。
(8)抗インフルエンザ抗体の定量
鼻腔、肺胞洗浄液および血清中の抗インフルエンザIgA、IgG含有量を、ELISA assayにより定量した。ELISA assayはBETHYL LABORATORIES社(アメリカ・テキサス)のMouse ELISA quantitation kitの方法に従って行った。96ウェルNuncイムノプレート(Nalgen Nunc International アメリカ・ニューヨーク)各ウェルにワクチン1μg、ウシ血清アルブミン(BSA, SIGMA アメリカ・ミズーリ)1μg/mL PBS溶液100μLを加え、4℃で一晩固層化反応を行った。その後洗浄液(50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0)で3回すすぎワクチン液を除去した。各ウェルに0.15 M NaCl、1% BSAを含む50 mM Tris-HCl緩衝液(pH 8.0)200μLを加え、室温で1時間ブロッキング反応を行った。各ウェルを洗浄液で3回すすいだのち、サンプル結合緩衝液(50 mM Tris, 0.15 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0)にて適量に希釈した鼻洗浄液・肺洗浄液あるいは血清を100μL加え、室温で2時間反応させた。Goat anti-mouse IgAまたはIgG-horse rADish peroxidase(HRP)(BETHYL LABORATORIES INC.)を二次抗体として用い、TMB Microwell Peroxidase Substrate System(Kirkegaard & Perry Laboratories, Inc. アメリカ・メリーランド)を用いて発色反応を行った。各ウェルに100μL、2 M H2SO4(和光純薬株式会社)を添加することによって反応を停止し、450 nmの吸光度をSPECTRAmax PLUS 384で測定した。定量のためのスタンダードとして、上記肺洗浄液から精製した抗インフルエンザIgAおよびIgG 10 ngについて同様にして得られた吸光度を用いた。
(9)結果
抗インフルエンザ抗体の定量結果を表1に示す。HAワクチン単独投与群に比べ、SSF-1からSSF-6をそれぞれHAワクチンと混合した粘膜ワクチン(HA+SSF-1~HA+SSF-6)投与群で抗インフルエンザIgAおよび/またはIgGの産生量が高かった。特に「SP-CL11を含有するSSF-3」、「K6L16を含有するSSF-4」及び「K6L11を含有するSSF-6」で、天然のSP-C(1-35)に匹敵する強いIgAおよびIgG抗体の産生増強作用が確認された。また、SSF-1とSSF-5の抗体産生増強作用がほぼ同等なことから、SSF-3(SP-CL)の一部アミノ酸配列を変えたRK-SP-CLの抗体産生作用への関与は小さいことが確認された。
(1)方法の概略
ミニブタは5-10週齢(3-7 kg)のクラウン系統(ジャパンファーム・鹿児島)を用いた。ミニブタは初回免疫2週間前に下記の方法で鼻汁サンプルを採取し、ELISA法にて抗インフルエンザ抗体陰性の個体であることを確かめて接種試験に用いた。抗原はインフルエンザウイルスAニューカレドニア株(H1N1)を原料として作成されたホルマリン不活化エーテルスプリットワクチン (表中HA、以下文中ではワクチンと称す:阪大微生物病研究会(香川)より入手)を使用した。ミニブタ1頭あたりのワクチン接種量はヘマグルチニン量に換算して24μgとした。
(2)結果
結果は表2に示したとおりである。血中、鼻汁の抗体価は、共に2次免疫を行った後、1、2週にかけて著しく増加した。5週目の最終抗体価で比較すると、ワクチン抗原のみの経鼻接種によって誘導された抗インフルエンザ抗体価は、鼻汁IgA、血清IgGでそれぞれ28、66であったのに対し、各ADビークル混合粘膜ワクチン接種群においては、IgAで448~784抗体価に、IgGで832~1280抗体価にまで誘導された。また、検定した3種のADビークルSP-C(1-35)、K6L16、およびSP-CL11の効果はいずれも有効で、ADビークル間に統計学的有意差は認められなかった。
Claims (9)
- 以下のアミノ酸配列からなる合成ペプチド:
PVHLKRLm(ただしmは11-15または16-20);または
KnLm(ただしnは4-8、mは11-20)
と脂質との複合体である抗原薬物(AD)ビークル。 - 合成ペプチドが、配列番号1から3のいずれかのアミノ酸配列からなる請求項1の抗原薬物(AD)ビークル。
- 脂質が、ホスファチジルコリン、ジパルミトイルホスファチジルコリン、ホスファチジルセリン、ホスファチジルグリセロール、ホスファチジルイノシトール、ホスファチジルエタノールアミン、ホスファチジン酸、ラウリル酸、ミリスチン酸、パルミチン酸、ステアリン酸およびオレイン酸の少なくとも1種である請求項1の抗原薬物(AD)ビークル。
- 脂質が、ジパルミトイルホスファチジルコリン、ホスファチジルグリセロールおよびパルミチン酸の3種脂質混合物である請求項3の抗原薬物(AD)ビークル。
- 請求項1から4のいずれかに記載の抗原薬物(AD)ビークルに、粘膜免疫IgAを誘導する量の抗原を共存、接触、捕捉又は吸着させることにより得られる粘膜ワクチン。
- 抗原が伝染病病原体に由来の不活化抗原又は無毒化毒素である請求項5の粘膜ワクチン。
- 請求項1から4のいずれかに記載の抗原薬物(AD)ビークルに、アレルゲン、アレルゲンエピトープ、又はアレルゲン由来抗原を共存、接触、捕捉又は吸着させることにより得られるアレルギー予防剤又は治療剤。
- 請求項5に記載の粘膜ワクチンを、少なくとも2回投与することを特徴とする感染症の予防又は治療方法。
- 請求項7に記載のアレルギー予防又は治療剤を、少なくとも2回投与することを特徴とするアレルギーの予防又は治療方法。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2720277A CA2720277C (en) | 2008-04-02 | 2009-03-30 | Antigen-and-drug vehicle comprising synthetic peptide, and mucosal vaccine using the same |
US12/936,075 US8287887B2 (en) | 2008-04-02 | 2009-03-30 | Antigen-and-drug vehicle comprising synthetic peptide, and mucosal vaccine using the same |
JP2010505901A JP5473899B2 (ja) | 2008-04-02 | 2009-03-30 | 合成ペプチドを含有する抗原薬物ビークルとこれを用いる粘膜ワクチン |
CN2009801115156A CN101980721B (zh) | 2008-04-02 | 2009-03-30 | 含有合成肽的抗原药物赋形剂和使用其的粘膜疫苗 |
EP09728744.5A EP2281574B1 (en) | 2008-04-02 | 2009-03-30 | Antigen-and-drug vehicle comprising synthetic peptide, and mucosal vaccine using the same |
HK11105932.7A HK1151733A1 (en) | 2008-04-02 | 2011-06-13 | Antigen-and-drug vehicle comprising synthetic peptide, and mucosal vaccine using the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008-096244 | 2008-04-02 | ||
JP2008096244 | 2008-04-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009123119A1 true WO2009123119A1 (ja) | 2009-10-08 |
Family
ID=41135493
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2009/056508 WO2009123119A1 (ja) | 2008-04-02 | 2009-03-30 | 合成ペプチドを含有する抗原薬物ビークルとこれを用いる粘膜ワクチン |
Country Status (8)
Country | Link |
---|---|
US (1) | US8287887B2 (ja) |
EP (1) | EP2281574B1 (ja) |
JP (1) | JP5473899B2 (ja) |
KR (1) | KR101614344B1 (ja) |
CN (1) | CN101980721B (ja) |
CA (1) | CA2720277C (ja) |
HK (1) | HK1151733A1 (ja) |
WO (1) | WO2009123119A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011108521A1 (ja) * | 2010-03-02 | 2011-09-09 | 国立大学法人徳島大学 | 粘膜ワクチン |
WO2013031827A1 (ja) | 2011-08-29 | 2013-03-07 | 国立大学法人徳島大学 | Rsv粘膜ワクチン |
WO2020045155A1 (ja) | 2018-08-30 | 2020-03-05 | 国立大学法人徳島大学 | 免疫寛容誘導剤及びアレルギー性疾患の治療又は予防剤 |
WO2022168889A1 (ja) * | 2021-02-03 | 2022-08-11 | 応用酵素医学研究所株式会社 | 微粒子粉末剤型粘膜ワクチン |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH039690B2 (ja) | 1985-08-22 | 1991-02-12 | Furukawa Electric Co Ltd | |
WO1995015980A1 (fr) | 1993-12-08 | 1995-06-15 | Tokyo Tanabe Company Limited | Nouveau peptide synthetique, tensioactif pulmonaire contenant ledit peptide et remede contre le syndrome de souffrance respiratoire |
WO2002032451A1 (en) | 2000-10-18 | 2002-04-25 | Intercell Biomedizinische Forschungs- Und Entwicklungs Ag | Vaccine composition comprising an antigen and a peptide having adjuvant properties |
JP2003523348A (ja) | 2000-02-16 | 2003-08-05 | ノースウェスタン ユニバーシティ | ポリペプトイド肺サーファクタント |
JP2004305006A (ja) | 2003-04-01 | 2004-11-04 | Japan Science & Technology Agency | 人工調製肺サーファクタント |
WO2005097182A1 (ja) | 2004-04-05 | 2005-10-20 | The University Of Tokushima | 経粘膜及び経皮投与を可能にする抗原薬物ヴィークル、これを用いる粘膜免疫の誘導方法、粘膜ワクチン及びdds |
JP2006504635A (ja) | 2002-05-17 | 2006-02-09 | キエシ・フアルマチエウテイチ・ソチエタ・ペル・アチオニ | 再構成サーファクタントの調製用の改善された合成脂質混合物 |
WO2007018152A1 (ja) | 2005-08-05 | 2007-02-15 | The University Of Tokushima | IgA抗体の選択的産生からIgA及びIgG両抗体産生への切換えを可能にする抗原薬物ビークルとこれを用いる経鼻・粘膜ワクチン |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7611863B2 (en) * | 2004-01-08 | 2009-11-03 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Selective staining of biomembranes using voltage-sensitive dyes |
-
2009
- 2009-03-30 WO PCT/JP2009/056508 patent/WO2009123119A1/ja active Application Filing
- 2009-03-30 JP JP2010505901A patent/JP5473899B2/ja active Active
- 2009-03-30 CN CN2009801115156A patent/CN101980721B/zh active Active
- 2009-03-30 EP EP09728744.5A patent/EP2281574B1/en active Active
- 2009-03-30 CA CA2720277A patent/CA2720277C/en active Active
- 2009-03-30 US US12/936,075 patent/US8287887B2/en active Active
- 2009-03-30 KR KR1020107021908A patent/KR101614344B1/ko active IP Right Grant
-
2011
- 2011-06-13 HK HK11105932.7A patent/HK1151733A1/xx unknown
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH039690B2 (ja) | 1985-08-22 | 1991-02-12 | Furukawa Electric Co Ltd | |
WO1995015980A1 (fr) | 1993-12-08 | 1995-06-15 | Tokyo Tanabe Company Limited | Nouveau peptide synthetique, tensioactif pulmonaire contenant ledit peptide et remede contre le syndrome de souffrance respiratoire |
JP2003523348A (ja) | 2000-02-16 | 2003-08-05 | ノースウェスタン ユニバーシティ | ポリペプトイド肺サーファクタント |
WO2002032451A1 (en) | 2000-10-18 | 2002-04-25 | Intercell Biomedizinische Forschungs- Und Entwicklungs Ag | Vaccine composition comprising an antigen and a peptide having adjuvant properties |
JP2006504635A (ja) | 2002-05-17 | 2006-02-09 | キエシ・フアルマチエウテイチ・ソチエタ・ペル・アチオニ | 再構成サーファクタントの調製用の改善された合成脂質混合物 |
JP2004305006A (ja) | 2003-04-01 | 2004-11-04 | Japan Science & Technology Agency | 人工調製肺サーファクタント |
WO2005097182A1 (ja) | 2004-04-05 | 2005-10-20 | The University Of Tokushima | 経粘膜及び経皮投与を可能にする抗原薬物ヴィークル、これを用いる粘膜免疫の誘導方法、粘膜ワクチン及びdds |
WO2007018152A1 (ja) | 2005-08-05 | 2007-02-15 | The University Of Tokushima | IgA抗体の選択的産生からIgA及びIgG両抗体産生への切換えを可能にする抗原薬物ビークルとこれを用いる経鼻・粘膜ワクチン |
Non-Patent Citations (6)
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011108521A1 (ja) * | 2010-03-02 | 2011-09-09 | 国立大学法人徳島大学 | 粘膜ワクチン |
CN103002908A (zh) * | 2010-03-02 | 2013-03-27 | 国立大学法人德岛大学 | 粘膜疫苗 |
JP5804278B2 (ja) * | 2010-03-02 | 2015-11-04 | 国立大学法人徳島大学 | 粘膜ワクチン |
WO2013031827A1 (ja) | 2011-08-29 | 2013-03-07 | 国立大学法人徳島大学 | Rsv粘膜ワクチン |
JPWO2013031827A1 (ja) * | 2011-08-29 | 2015-03-23 | 国立大学法人徳島大学 | Rsv粘膜ワクチン |
US9463236B2 (en) | 2011-08-29 | 2016-10-11 | Tokushima University | RSV mucosal vaccine |
WO2020045155A1 (ja) | 2018-08-30 | 2020-03-05 | 国立大学法人徳島大学 | 免疫寛容誘導剤及びアレルギー性疾患の治療又は予防剤 |
WO2022168889A1 (ja) * | 2021-02-03 | 2022-08-11 | 応用酵素医学研究所株式会社 | 微粒子粉末剤型粘膜ワクチン |
Also Published As
Publication number | Publication date |
---|---|
CA2720277A1 (en) | 2009-10-08 |
HK1151733A1 (en) | 2012-02-10 |
JP5473899B2 (ja) | 2014-04-16 |
JPWO2009123119A1 (ja) | 2011-07-28 |
KR20110002841A (ko) | 2011-01-10 |
EP2281574A4 (en) | 2011-03-23 |
US8287887B2 (en) | 2012-10-16 |
EP2281574B1 (en) | 2014-07-09 |
CN101980721A (zh) | 2011-02-23 |
EP2281574A1 (en) | 2011-02-09 |
CN101980721B (zh) | 2013-03-13 |
US20110110971A1 (en) | 2011-05-12 |
CA2720277C (en) | 2017-01-17 |
KR101614344B1 (ko) | 2016-04-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5028627B2 (ja) | IgA抗体の選択的産生からIgA及びIgG両抗体産生への切換えを可能にする抗原薬物ビークルとこれを用いる経鼻・粘膜ワクチン | |
JP4137640B2 (ja) | 新規プロテオソーム−リポサッカリドワクチンアジュバント | |
JP4843792B2 (ja) | 経粘膜及び経皮投与を可能にする抗原薬物ヴィークル、これを用いる粘膜免疫の誘導方法、粘膜ワクチン及びdds | |
EP0791064B1 (fr) | Procede pour ameliorer l'immunogenicite d'un compose immunogene ou d'un haptene et application a la preparation de vaccins | |
JP2003522802A (ja) | プロテオソーム・インフルエンザ・ワクチン | |
US9540420B2 (en) | Mucosal vaccines | |
JP5473899B2 (ja) | 合成ペプチドを含有する抗原薬物ビークルとこれを用いる粘膜ワクチン | |
TW201427687A (zh) | 黏膜疫苗用佐劑 | |
KR20090016659A (ko) | 비로좀을 기제로 한 비강내 인플루엔자 백신 | |
JP6085886B2 (ja) | Rsv粘膜ワクチン | |
WO2021206103A1 (ja) | 皮下投与型ワクチン | |
WO2019045090A1 (ja) | インフルエンザhaスプリットワクチンの製造方法 | |
WO2006019183A1 (ja) | 粘膜免疫、その感作剤を用いる粘膜ワクチン及び予防接種システム |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200980111515.6 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09728744 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2010505901 Country of ref document: JP |
|
ENP | Entry into the national phase |
Ref document number: 20107021908 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 6935/DELNP/2010 Country of ref document: IN Ref document number: 2720277 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2009728744 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12936075 Country of ref document: US |