CS233279B1 - Method of subunit vaccine preparation against tick encephalitis - Google Patents
Method of subunit vaccine preparation against tick encephalitis Download PDFInfo
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- CS233279B1 CS233279B1 CS836759A CS675983A CS233279B1 CS 233279 B1 CS233279 B1 CS 233279B1 CS 836759 A CS836759 A CS 836759A CS 675983 A CS675983 A CS 675983A CS 233279 B1 CS233279 B1 CS 233279B1
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- tick
- subunit vaccine
- encephalitis
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- 238000000034 method Methods 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 229940031626 subunit vaccine Drugs 0.000 title abstract description 5
- 206010014599 encephalitis Diseases 0.000 title abstract description 4
- 229960005486 vaccine Drugs 0.000 claims abstract description 15
- 241000700605 Viruses Species 0.000 claims abstract description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001085 differential centrifugation Methods 0.000 claims abstract description 4
- 210000002308 embryonic cell Anatomy 0.000 claims abstract description 4
- 208000004006 Tick-borne encephalitis Diseases 0.000 claims description 6
- 241000710771 Tick-borne encephalitis virus Species 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 230000000644 propagated effect Effects 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 abstract description 2
- 239000013049 sediment Substances 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101100338513 Mus musculus Hdac9 gene Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 101100338514 Xenopus laevis hdac9 gene Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 229960003239 encephalitis vaccine Drugs 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Vynález sa týká sposobu pripravy subjednotkovéj vakcíny ku klieštovej encefalitidě. Podstatou vynálezu je, že· termosenzitívny kmen virusu klieštovej encefalitidy Skalica Cl. gl. 53 sa pomnožuje na tkanivových kulturách kuřácích embryonálnych buniek, virus sa semipurifikuje diferenciálnou centrifugáciou, sediment se resuspenduje do fyziologického roztoku,, štiepi sa tri krát éterom, ktorý sa odstráni, vakcína sa lyofilizuje. Vynález ma použitie v humánně j a veterinárnej praxi.The invention relates to a method of preparation a subunit vaccine to a tongs encephalitis. It is an object of the invention that: a thermosensitive strain of the tick virus encephalitis Skalica Cl. gl. 53 is growing on tissue cultures of smokers embryonic cells, the virus does semipurifies by differential centrifugation the sediment is resuspended in physiological saline solution, cleaves three times the ether to be removed, the vaccine lyophilized. The invention has utility in humans and veterinary practice.
Description
Vynález sa týká sposobu*přípravy subjednotkovej vakcíny proti kliešťovej encefalitidě.The invention relates to a method for preparing a subunit vaccine against tick-borne encephalitis.
Ε. N. Levkovič a tiež Ch. Kunz připravili inaktivovaně vakcíny proti kliešťovej encefalitidě na kuřácích embryonálnych buňkách s následnou inaktiváciou formaldehydom> /5. N. levkovič: Biology of the tick-borne encephalitis complex. Czechoslovak Acad. Sci. 317 (1962); Ch. Kunz: Arzneim. Porsch. Drug. Res. 28, 10, 1806, (1978)J· Nevýhodou inaktivováných vakcín je ich příprava s plné virulentným kmeňom virusu kliešťovej encefalitidy. Mayer a Mitrová připravili živú atenuovanú vakcínu s kmeňom Langat /Mayer a Mitrová: Biological works. Slovák Aoad. Sci. Bratislava, (1977)J· Nevýhodou atenuovanej vakcíny je, že kmen Langat revertoval a nemóže sa použiť k príprave žívej očkovacej látky.Ε. N. Levkovic and also Ch. Kunz prepared inactivated tick-borne encephalitis vaccines on smoking embryonic cells followed by formaldehyde inactivation> / 5. N. Levkovič: Biology of the tick-borne encephalitis complex. Czechoslovak Acad. Sci. 317 (1962); Ch. Kunz: Arzneim. Porsch. Drug. Res. 28, 10, 1806, (1978). A disadvantage of inactivated vaccines is their preparation with a full virulent strain of tick-borne encephalitis virus. Mayer and Mitr prepared a live attenuated vaccine with the Langat / Mayer and Mitr strain: Biological works. Slovak Aoad. Sci. Bratislava, (1977) J A disadvantage of an attenuated vaccine is that the Langat strain has been reversed and cannot be used to prepare a live vaccine.
Uvedené nevýhody v podstatnej miere odstraňuje sposob přípravy subjednotkovej vakcíny proti kliešťovej encefalitidě, ktorého podstata spočívá v tom, že termosenzitívny kmeň virusu kliešťovej encefalitidy -Cl. gl. 53? sa pomnožuje na tkanivových kultúrach kuřácích embryonálnych buniek; virus sa semipurifikuje diferenciálnou centrifugáciou, resuspenduje do fyziologického roztoku,, štiepi sa 3 krát éterom, ktorý sa odstráni, vakcína sa vycíri a lyofilizuje.These disadvantages are substantially eliminated by the method of preparing a subunit vaccine against tick-borne encephalitis, which is based on the fact that the thermosensitive strain of tick-borne encephalitis -Cl virus. gl. 53? is propagated on tissue cultures of smoking embryonic cells; the virus is semi-purified by differential centrifugation, resuspended in saline, digested 3 times with ether which is removed, the vaccine is clarified and lyophilized.
Výhoda navrhovaného sposobu přípravy subjednotkovej vakcí- 2 233 279 ny je, žs sa pracuje s termosenzitívnym kmeňom virusu kliešťovej encefalitidy, ktorý je nepatogenný po podkožnom podaní bielym dospělým myšiam, čo je významné z hiadiska bezpečnosti pri práci.An advantage of the proposed method of preparing the subunit vaccine 2 233 279 is that the thermosensitive strain of tick-borne encephalitis virus is non-pathogenic after subcutaneous administration to white adult mice, which is important for safety at work.
II
Vakcína obsahuje subjednotky virusu, ktoré indukujú tvorbu protilátok a je zbavená balastných látok. Tento sposob přípravy je ekonomicky nenáročný. Vakcínu možno skladovat po lyofilizácii.The vaccine contains virus subunits that induce antibody production and are free of ballast substances. This method of preparation is economically undemanding. The vaccine may be stored after lyophilization.
Příklad 1Example 1
Pripravujú sa tkanivové kultúry z 11 dňových C/E kuřácích embryí, z ktorých sa odstránia oči a vnútornosti. Tkanivové kultúry sa kultivujú v bazálnom Eaglovom médiu (BEM) s prímesou 5 % fetálneho telacieho séra a antibiotik (penicilín a streptomycín). Po 24 hodinovéj kultivácii sa médium odstráni a tkanivové kultúry sa infikujú vírusom kliešťovej encefalitidy (kmen Skalica, ktorý je uložený v čs. zbierke arbovírusov Virologického ústavu SAV pod čislom Cl. gl. 53) so 7,5 log ΒΏ^θ/Ο,ΟΙ ml. Adsorbcia prebieha po dobu 90 min pri teplote 37 °C. Potom sa přidá čerstvé BEM s prímesou 5 % inaktivovaného séra a antibiotik. Na prvý den sa médium stiahne a vyčíri. Purifikácia virusu sa robí diferenciálnou centrifugáciou pri 25.000 ot. za min. po dobu 2 h. Odstráni sa supernatant a sediment sa resuspenduje do 100 krát menšieho objemu izotonického fyziologického roztoku o pH 7,2 s prímesou 50yug gentamycínu na 1 ml. Po vyčírení sa určuje titer virusu pomocou hemaglutinačného testu. Preparát sa za stálého miešania 3 krát štiepi éterom, 1 krát počas 5 min, druhý krát rovnakým dielom počas 18 h pri teplote 4' °C, 3 krát počas 5 min. Po vyčírení sa éter odsaje, zbytky éteru sa odstránia dusíkom. Vakcína sa lyofilizuje a skladuje pri teplote -20 °C. Skúšky neškodnosti vakcíny sa robia intracerebrálnouTissue cultures are prepared from 11 day C / E chicken embryos from which the eyes and viscera are removed. Tissue cultures are cultured in basal Eagle medium (BEM) with 5% fetal calf serum and antibiotics (penicillin and streptomycin). After 24 hours of cultivation, the medium is removed and tissue cultures are infected with tick-borne encephalitis virus (strain Skalica deposited in the Czechoslovak Arbovirus Collection of the Institute of Virology of the Slovak Academy of Sciences under number Cl. Gl. 53) with 7.5 log ΒΏ ^ θ / Ο, ΟΙ ml. The adsorption is carried out for 90 min at 37 ° C. Fresh BEM is then added with 5% inactivated serum and antibiotics. On the first day the medium is withdrawn and cleared. The virus is purified by differential centrifugation at 25,000 rpm. per min. for 2 h. The supernatant is discarded and the sediment is resuspended in 100 times smaller volume of isotonic saline pH 7.2 with 50 µg gentamycin per ml. After clarification, the virus titer is determined by a hemagglutination test. The preparation is cleaved 3 times with ether, 1 time for 5 min, the second time for 18 h at 4 ° C, 3 times for 5 min. After clarification, the ether is filtered off with suction, the ether residues are removed with nitrogen. The vaccine is lyophilized and stored at -20 ° C. The vaccine harmlessness tests are made intracerebral
- 3 ”- 3 ”
233 279 inokuláciou očkovacej látky po 0,1 ml úalšej skupině bielych myší. Počas pozorovacej doby 14 dní sa u žiadneho z pokusných zvierat neolfevilo poškodenie zdravotného stavu, ktoré by súviselo s aplikáciou skúšaného preparátu. Skúška na nepřítomnost živého virusu sa robí na 5 až 8 g bielych myšlach. Myšiam sa inokuluje po 0,03 ml intracerebrálne vakcíny neriedenej a Salšej skupině myší sa inokuluje po 0,03 ml vakcína riedená 1:10. Po 7 dňoch sa odoberie od poloviny myší mozog na Salšiu pasáž. Celkove sa robia 2 pasáže. Sledovanie tvorby protilátok sa robí na 15 g my šlach, kto:rým sa podá intraperitoneálne vakcína po 0,1 ml, dní po prvej dávke sa odoberie od myší krv a sérum sa vyšetřuje v hemaglutinačno-inhibičnom teste. Druhá dávka vakcíny sa podá po 1 mesiaci. 7 dní po prvej vakcinácii sa vyšetřuje sérum myší na přítomnost hemaglutinačno-inhibičných protilátok. Ochranný test sa robí na dospělých bielych myšiach, ktoré sa imunizujú jednou dávkou vakcíny intraperitoneálne po 0,1 ml. Po 7 až 8 dňoch sa robí challenge živým vírusom. Myši sú chráněné proti 100 LD^q (myšie smrtelné dávky) virusu. Skúška sterility vakoíny sa robí podía Čel. 3.233 279 by inoculating the vaccine with 0.1 ml of another group of white mice. During the observation period of 14 days, none of the test animals suffered from any lesions related to the application of the test preparation. The test for the absence of live virus is performed on 5-8 g of white mice. Mice are inoculated with 0.03 ml of the intracerebral vaccine undiluted and a saler group of mice is inoculated with 0.03 ml of the 1:10 diluted vaccine. After 7 days, half of the mice are brain removed for the Saler Passage. A total of 2 passages are made. Monitoring of antibody production is performed on 15 g of my tendons, which is given an intraperitoneal vaccine of 0.1 ml, days after the first dose, blood is drawn from the mice and the serum is examined in a hemagglutination-inhibition test. The second dose of vaccine is given after 1 month. 7 days after the first vaccination, the serum of mice is examined for the presence of hemagglutination-inhibiting antibodies. The protective test is performed in adult white mice that are immunized with a single dose of 0.1 ml intraperitoneally. After 7 to 8 days, the challenge is a live virus. Mice are protected against 100 LD ^ q (murine lethal doses) virus. The vacoin sterility test is performed according to TLC. Third
Příklad 2Example 2
Postupuje sa ako v příklade 1 s tým rozdielom, že sa virus kultivuje prJL 37‘ °C počas 48 h.The procedure was as in Example 1 except that the virus was cultured at 37 ° C for 48 h.
Příklad 3Example 3
Postupuje sa ako v příklade 1 s tým rozdielom, že sa virusové partikule separujú v sacharózovom gradiente hustoty (10 až 30 % saúharóza/.The procedure is as in Example 1 except that the virus particles are separated in a sucrose density gradient (10-30% sucrose).
Příklad. 4Example. 4
233 271233 271
Postupuje aa ako v příklade 1 a tý» rozdielom, že flnálny preparát po oxtrakoil éterem aa opracuje formalínom v konečnom riedení 1:2 000, udržuje aa po dobu 3 dní pri teploto 4 °G, formalín aa odatránll tak, že k 100 ml vzorky ca přidala 1,7 ml 5 % pyrosiřičitana eodného. Po zmiežaní aa vzorka naplní de dia lyzačného sáčku a tento aa dialyzujo v 3. lltroch fyziologiekého roztoka pri teploto 4 °0 po dobu 20 hodin. Vakoína aa po dia lýze lyofilizujo·Proceed as aa as in Example 1, except that the final preparation after oxtrakoil ether aa is treated with formalin at a final dilution of 1: 2,000, and maintained for a period of 3 days at 4 ° C, formalin aa is removed to 100 ml sample ca added 1.7 ml of 5% sodium pyrosulphite. After mixing aa, the sample is filled with a de dia lysis bag and this aa dialyzed in 3 L of physiological saline at 4 ° C for 20 hours. Vaccine and freeze-dried after dia lysis ·
Vynález móže nájeť široké použitie v médieíne, ako vakoína proti víraau kliožťovoj encefalitidy.The invention can find widespread use in a medium, such as a virus vaccine and human encephalitis.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS836759A CS233279B1 (en) | 1983-09-16 | 1983-09-16 | Method of subunit vaccine preparation against tick encephalitis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS836759A CS233279B1 (en) | 1983-09-16 | 1983-09-16 | Method of subunit vaccine preparation against tick encephalitis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CS675983A1 CS675983A1 (en) | 1984-05-14 |
| CS233279B1 true CS233279B1 (en) | 1985-02-14 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS836759A CS233279B1 (en) | 1983-09-16 | 1983-09-16 | Method of subunit vaccine preparation against tick encephalitis |
Country Status (1)
| Country | Link |
|---|---|
| CS (1) | CS233279B1 (en) |
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1983
- 1983-09-16 CS CS836759A patent/CS233279B1/en unknown
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| Publication number | Publication date |
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| CS675983A1 (en) | 1984-05-14 |
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