• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    An improved herpes simplex virus subunit vaccine being effective against both of HSV type 1 and type 2 and having high safety and high effectiveness, which comprises as an active component a highly purified glycoprotein gB which is a component common to Herpes simplex virus type 1 and type 2, and a method for preparing the herpes simplex virus subunit vaccine and also a lyophilized preparation of the vaccine.
    公开号:SU1554755A3
    申请号:SU843786918
    申请日:1984-08-30
    公开日:1990-03-30
    发明作者:Кино Еитиро；Охтомо Нобуя
    申请人:Джуридикал Фаундейшн Дзе Кемо-Серо-Терапевтик Рисерч Институт (Фирма)；
    IPC主号:
    专利说明:

    one
    (21) 3786918 / 30-13
    (22) 08/30/84
    (31) 159641
    (32) 08/31/83
    (33) JP
    (46) 05.30.90. Kyul № 12
    (71) Juridicap Foundation Dze Kem-Sero-Therapist Research Institute (JP)
    (72) Eichiro Kino and Nobu Ohtomo (JP) (53) 615.372 (088.8)
    (56) The Human Heroes Viruses. Ed. by Nahraias et al. Elsevier. New York, 1981, p.p. 503-509,
    (54) METHOD FOR OBTAINING SIMPLY LONG VIRUS VIRUS OF THE VIRUS (57) The invention relates to virology and can be used in the manufacture of vaccines. The purpose of the invention is to increase the purity of the target product and the unification of the vaccine preparation. For the preparation of the vaccine, glycoprotein gB isolated from cells previously infected with simple lichen virus (VIL) is used: the glycoprotein is purified by affinity chromatography using monoclonal antibodies against p, B. The gel with adsorbed antibodies is equilibrated with phosphate buffer pH 7.2-7.4, containing 0.05% by volume of polyoxyethyleneoylphenyl ether. After adsorption, the gB gel is washed with the same buffer and the glycoprotein is eluted with a solution containing a ZM solution of potassium thiocyanate and 0.05 vol, 7, polyoxyethylene octylphenyl ether. The resulting purified gB preparation is dialyzed against phosphate buffer and adsorbed onto aluminum hydroxide. The vaccine can be lyophilized; it is effective against - IDP I and TI type.
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    This invention relates to virology and can be used in the manufacture of vaccines.
    The purpose of the invention is to increase the purity of the target product and to unify the vaccine preparation,
    Example 1. Preparation of monoclonal antibodies.
    Veto cells infected with KOS-gatamma of the common lichen virus (IDP) type I, 24 hours after infection, are dissolved in a phosphate buffer solution pH 7.2-7.4, containing 1 v / oG.% Polyoxyethylene octylphenyl ether (Triton X- 100), prg 4 ° (for 1 hour. The resulting solution is centrifuged for 1 hour at 100 thousand g and the supernatant is collected, which is a fraction of crude glycoprotein 0.1 ml of this fraction (protein content 100 mg / ml), was injected intradermally into the hind paw by 4-week-old female MYSHRY line BALB / C. Within a month after immunization, the animals extracted spleen removed, cut it into thin slices and suspended in Dulbecco MEM. The resulting suspension of cells in kontseptsl
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    radio cells of 10 cells / ml and separately prepared suspension of RE1 cells at a concentration of cells / ml is poured into a test tube and polyethylene glycol is added. 4000. The cell fusion reaction is carried out at 37 ° C for 5 minutes. The resulting cell hybrids are suspended in GAT medium, selecting a hybrid capable of producing antibodies to gB. The selected hybrid is administered intraperitoneally to 4-week-old female BALB / c mice treated with 2.6 -, - 10, 14-tetramethylpentadecane. 7–14 days after administration, ascites fluid is collected and a monoclonal antibody (monAT) specific for gB (IgG amount 10 mg / ml) is obtained.
    Example 2. Preparation of adsobent bound to monat,
    To the ascitic liquid containing monAT, prepared according to Example 1, a 50% ammonium sulfate solution is added at 4 ° to precipitate the IgG fraction. The immunoglobulin precipitate is dissolved in a buffer solution containing 0.05 M NaHCOs and 0.15 M NaCl pH 8.2.
     The resulting solution is dialyzed against the same buffer solution at 4 ° C for 24 hours, after which it is bound to activated CNBr Sepharose 4B at a concentration of 5 mg / ml. An adsorbent bound to MrnAT is obtained.
    Cleaning DV with Immune Verb Cells Infected with Type I IDPs (KO-gatamm), infected cells were harvested after 24 hours, dissolved in phosphate-buffered saline, pH 7.2-7.4, containing 0.05% v / v Triton X-100, and passed through a column filled with immunosorbent. The column is washed with the same buffer solution. The adsorbed gB was eluted with a ZM KSCN solution containing 0.05% v / v Triton X-100, dialyzed against the same buffer and concentrated by ultrafiltration. All operations are performed at
    4 ° C.
    Purified gB is subjected to electrophoretic analysis using an SDS-polyacrylamide gel. The main gB band is detected at about 90K.
    PRI me R 3. Getting the vaccine n purified gB,

    0
    five
    A solution of purified gB in a phosphate-buffered solution of pH 7.2-7.4 with 0.05% v / v Triton X-100, containing 30 μg / ml of protein, is mixed with an aqueous solution of aluminum chloride (in terms of aluminum hydroxide - in 8-fold i.- by weight in excess compared with the weight of protein gB). The pH of the mixture was adjusted with 1N NaOH to 6.7. An aluminum hydroxide gel is prepared with adsorbed gB protein. The resulting product was centrifuged, and the precipitate was dispersed in the initial buffer so that the concentration of gB was 30 µg / ml. Timersal is added to the preparation in an amount of 0.01% w / v. The mixture is poured with a volume of 2 ml, 1 ml each, and the ampoules are sealed. During the storage of the vaccine, a white mist precipitates out, with gentle shaking, a homogeneous white suspension is formed.
    Example 4. Preparation of a lyophilized vaccine.
    The aluminum gel precipitate with adsorbed gB obtained in Example 3 is dispersed in physiological saline (pH 7.2-7.4) containing 10% w / v lactose, 0.4% w / v L-glutaminate sodium , 0.4% w / v arginine, Ov8 w / v% gelatin and .0.05% w / v timmersal, protein concentration 30 µg / ml. The vaccine with a filler is poured into 1 ml in ampoules with a capacity of 2 ml and lyophilized according to the following scheme: pre-lyophilization at -50 ° C and atmospheric pressure for 6 hours, then first
    lyophilization at + 5 ° C under reduced pressure (0.04 Tbrr) for 15 minutes and second lyophilization at reduced pressure (0.05 Torr) for 8 hours. A lyophilized vaccine is obtained.
    权利要求:
    Claims (1)
    [1]
    When testing an adsorbed vaccine and an adsorbed - lyophilized vaccine in laboratory animals, it was established that the vaccines are reactive, non-toxic and immunogenic. The vaccine, regardless of its form, exhibits a high protective effect (100% survival rate of animals) in relation to both type I IDPs and type P. IDPs. Invention Formula
    The method of obtaining a subunit vaccine of lichen deprive, including
    cleaning a solution containing simple lichen glycoproteins using affinity chromatography followed by adsorption of the target product on aluminum gel, characterized in that, in order to increase the purity of the target product and standardize the vaccine preparation, affinity chromatography is carried out by passing the solution through a gel bound with a monoclonal antibody specific for glycoprotein gB and balanced by phosphate
    buffer-pH 7.2-7.4, containing 0.05% by volume of polyoxyethylene-octylphenyl ether, washing the gel with absorbate is carried out with the same buffer and elute the target product with an elution solution containing a ZM-solution of potassium thiocyanate and 0.05 %, polyoxyethylene octylphenyl ether, and, prior to adsorption on alumina, the target product is dialyzed against the buffer used for affinity chromatography.
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    同族专利:
    公开号 | 公开日
    JPS6051120A|1985-03-22|
    CA1244766A|1988-11-15|
    DE3485873D1|1992-09-24|
    KR850001700A|1985-04-01|
    EP0135841A3|1986-05-28|
    DE3485873T2|1993-03-25|
    AU571569B2|1988-04-21|
    EP0135841A2|1985-04-03|
    US4661349A|1987-04-28|
    EP0135841B1|1992-08-19|
    AU3254184A|1985-03-07|
    KR910005889B1|1991-08-06|
    AT79548T|1992-09-15|
    引用文献:
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    FI66020C|1977-09-19|1984-08-10|Merck & Co Inc|FRAMEWORK FOR ANTIGENIC IMMUNOGENIC SUBMITS OF HSV-1 AND HSV-2|
    US4452734A|1980-02-11|1984-06-05|Merck & Co., Inc.|Herpes subunit vaccine|
    US4374127A|1977-09-19|1983-02-15|Merck & Co., Inc.|Herpes sub unit vaccine|
    IT1117218B|1979-05-18|1986-02-17|Depa Sas|PROCEDURE FOR THE PRODUCTION OF A PRODUCT FOR THE DIAGNOSIS OF TUMORS OF THE SOFT PARTS OR CARCINOMES AND PRODUCT OBTAINED|
    US4341754A|1980-01-24|1982-07-27|Vanderbilt University|Diagnostic reagent for herpes simplex virus encephalitis|
    US4337242A|1980-02-05|1982-06-29|Merck & Co., Inc.|Vaccine stabilizer containing L-glutamic acid and L-arginine|
    US4430437A|1980-08-27|1984-02-07|The United States Of America As Represented By The Department Of Health And Human Services|Test methods employing monoclonal antibodies against Herpes simplex virus types 1 and 2 nucleocapsids proteins|
    US4540669A|1980-09-11|1985-09-10|Merck & Co., Inc.|Herpes simplex type I subunit vaccine|
    US4317811A|1980-09-11|1982-03-02|Merck & Co., Inc.|Herpes simplex type 1 subunit vaccine|
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    US4762708A|1982-02-18|1988-08-09|University Patents, Inc.|Materials and methods for herpes simplex virus vaccination|
    US4535057A|1982-07-26|1985-08-13|Amf Incorporated|Immunoassay employing monoclonal herpes simplex antibody and biotin-avidin detection system|
    DE3228501A1|1982-07-30|1984-02-02|Behringwerke Ag, 3550 Marburg|METHOD FOR PRODUCING A HERPES ANTIGENT, MEANS THAT ARE SUITABLE FOR ITS PRODUCTION, AND METHOD FOR THE PRODUCTION THEREOF AND THE USE OF THIS ANTIQUE|
    DE3461928D1|1983-06-23|1987-02-12|Person Stanley|Immunologically reactive non-glycosylated amino acid chains of glycoprotein b of herpes virus types 1 and 2|
    US4533496A|1984-05-08|1985-08-06|Monsanto Company|Method of isolating monoclonal antibodies from hybridoma cultures|US5244792A|1984-04-06|1993-09-14|Chiron Corporation|Expression of recombinant glyoprotein B from herpes simplex virus|
    US5171568A|1984-04-06|1992-12-15|Chiron Corporation|Recombinant herpes simplex gb-gd vaccine|
    US5612041A|1984-07-17|1997-03-18|Chiron Corporation|Recombinant herpes simplex gD vaccine|
    US4689225A|1984-11-02|1987-08-25|Institut Merieux|Vaccine for cytomegalovirus|
    US4790987A|1985-11-15|1988-12-13|Research Corporation|Viral glycoprotein subunit vaccine|
    EP0289550B1|1986-10-20|1996-04-10|Chiron Corporation|Vaccine for use in the therapeutic treatment of hsv|
    US5149529A|1988-04-08|1992-09-22|Board Of Trustees Of Leland Chiron Corporation|Compositions and treatment for herpes simplex|
    US5151267A|1988-07-15|1992-09-29|University Of Saskatchewan|Bovine herpesvirus type 1 polypeptides and vaccines|
    US5585264A|1988-07-15|1996-12-17|University Of Saskatchewan|Nucleotide sequences encoding recombinant bovine herpesvirus type-1 GI, GIII and GIV polypeptides|
    US5081010A|1989-02-09|1992-01-14|Eastman Kodak Company|Extraction composition, test kit and their use to extract or determine herpes simplex viral antigen|
    WO1990013652A1|1989-05-12|1990-11-15|Triton Diagnostics, Inc.|Herpes simplex virus type 2-glycoprotein g proteins and polypeptides|
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    CA2088600C|1990-08-02|1999-11-16|Rae L. Burke|Herpes simplex virus vp16 vaccines|
    US5334503A|1991-10-08|1994-08-02|Eastman Kodak Company|Test kit and method for the detection of microorganisms associated with periodontal diseases using surfactant mixture as extraction composition|
    US6156319A|1994-07-25|2000-12-05|The Trustees Of The University Of Pennsylvania|Soluble herpesvirus glycoprotein complex vaccine|
    US5807557A|1994-07-25|1998-09-15|The Trustees Of The University Of Pennsylvania|Soluble herpesvirus glycoprotein complex|
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    US20040265800A1|2003-06-30|2004-12-30|Sysmex Corporation|Sample pretreatment solution for immunological test and method for using the same|
    WO2020113197A1|2018-11-30|2020-06-04|Vaccine Stabilization Institute|Viral formulations containing amino acids|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP58159641A|JPS6051120A|1983-08-31|1983-08-31|Herpes simplex subunit vaccine|
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