CS252389B1 - Inactivated adsorbate vaccine against equine influenza and method of its preparation - Google Patents
Inactivated adsorbate vaccine against equine influenza and method of its preparation Download PDFInfo
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- CS252389B1 CS252389B1 CS858238A CS823885A CS252389B1 CS 252389 B1 CS252389 B1 CS 252389B1 CS 858238 A CS858238 A CS 858238A CS 823885 A CS823885 A CS 823885A CS 252389 B1 CS252389 B1 CS 252389B1
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- 241000283073 Equus caballus Species 0.000 title claims description 24
- 229960005486 vaccine Drugs 0.000 title claims description 22
- 206010022000 influenza Diseases 0.000 title claims description 13
- 238000000034 method Methods 0.000 title claims description 10
- 239000002156 adsorbate Substances 0.000 title claims description 7
- 238000002360 preparation method Methods 0.000 title description 4
- 230000035931 haemagglutination Effects 0.000 claims description 22
- 241000700605 Viruses Species 0.000 claims description 21
- 239000012530 fluid Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 241000712461 unidentified influenza virus Species 0.000 claims description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 6
- 239000011707 mineral Substances 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 229940024546 aluminum hydroxide gel Drugs 0.000 claims description 5
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical group O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- 239000008098 formaldehyde solution Substances 0.000 claims description 3
- 230000002779 inactivation Effects 0.000 claims description 3
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- 230000000391 smoking effect Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 9
- 235000013601 eggs Nutrition 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 241000283086 Equidae Species 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
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- 239000000463 material Substances 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
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- 102000002322 Egg Proteins Human genes 0.000 description 3
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- 101710154606 Hemagglutinin Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 3
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 3
- 101710176177 Protein A56 Proteins 0.000 description 3
- 239000000185 hemagglutinin Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000712431 Influenza A virus Species 0.000 description 2
- 108010093857 Viral Hemagglutinins Proteins 0.000 description 2
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- 230000000890 antigenic effect Effects 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
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- 230000000241 respiratory effect Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241001295925 Gegenes Species 0.000 description 1
- 241001500351 Influenzavirus A Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical compound [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229940024545 aluminum hydroxide Drugs 0.000 description 1
- 229940009859 aluminum phosphate Drugs 0.000 description 1
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
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- 235000011010 calcium phosphates Nutrition 0.000 description 1
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- 230000001492 haemagglutinating effect Effects 0.000 description 1
- 230000003067 hemagglutinative effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
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- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
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- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
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- 229960000380 propiolactone Drugs 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
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- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
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- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229960004906 thiomersal Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
Vynález sa týká inaktivovanej adscrbátovej vakcíny proti chrípke koní určenej na profyliaxiu respiratórnebo ochorenia vyvdaného vírusom chřipky koní a sposobu jej výroby. Inaktivovaná adsorbátová vakcína proti chrípke koní je určená na špecifickú profylax'u respiratórnebo ochorenia koní všetkých věkových kategorií.The invention relates to an inactivated equine influenza adsorbate vaccine for the prophylaxis of respiratory or disease caused by the equine influenza virus and to a method for producing the same. The inactivated equine influenza adsorbate vaccine is intended for specific prophylaxis of respiratory or equine diseases of all ages.
Chřipka koní je akútne, virusové vysoko infekčné ochorenie, ktoré s rožnou intenzitou postihuje koně všetkých věkových kategorií. Povodcem je Myxovirus influenzae equi, ktorý sa vyskytuje v dvoch antigénnych subtypoch, a to ako subtyp A equi 1 a A equi 2. Ochorenie je známe od polovice minulého· storočia, ale póvcdca bol izolovaný až poměrně nedávno. Heller a spol. vyslovili r. 1955 na základe serologických vyšetření domienku, že póvodicom chřipky korní sú virusy, které sú subtypmi chřipkového· virusu A (Bryans, J. T. a spol. Epizootologic features of disease caused by myxovirus influenze A equine, Am. J. vet. Res. 28, 1967, 122, s. 9—17, Jukic, B. Iníluenca konja: Serolcški nalaz u postepiaootskom rozdoblju, Vet Arh. 43, 1973, 1—2, s, 48—53). Ich předpoklad sa potvrdil izolováním chřipkového virusu v priebehu akutného respiratórneho ochorenia koní v CSSR (Sovinová, O. 1958, Acta virol., Praque, 2, 52).Equine influenza is an acute, viral, highly infectious disease that affects horses of all ages with great intensity. The causative agent is Myxovirus influenzae equi, which occurs in two antigenic subtypes, both subtype A equi 1 and A equi 2. The disease has been known since the middle of the last century, but the pathogen has been isolated relatively recently. Heller et al. pronili r. 1955, based on serological examinations of the suggestion that the root influenza virus is a virus that is a subtype of influenza A virus (Bryans, JT et al.). , 122, pp. 9-17, Jukic, B. Iníluenca konja: Serolcški nalaz u postepiaootskom rozdoblju, Vet Arh. 43, 1973, 1-2, pp. 48–53). Their assumption was confirmed by the isolation of influenza virus during acute respiratory disease of horses in CSSR (Sovinová, O. 1958, Acta virol., Praque, 2, 52).
vakcína prati chrípke koní a sposob jejthe vaccine to wash and treat equine influenza
Izolovaný kmeň s označením 138(A)Equi 1( Praha) 56, sa stal prototypovým kmeňom subtypu A equi 1 chřipkového· virusu koní (Blaškovič, D. Základny lekárskej virológie, Bratislava 1978). Kmeň izolovaný v roku 1963 v Miami na Floridě v USA, označený 274(A)Equi 2 (Miami) 63, sa stal prototypovým kmeňom subtypu A equi 2 virusu chřipky koní (Blaškovič, D. Základy lekárskej virológie, Veda Bratislava 1978).The isolated strain designated 138 (A) Equi 1 (Prague) 56, has become a prototype strain of equine subtype A equi 1 of influenza virus (Blaškovič, D. of Medical Virology, Bratislava 1978). The strain isolated in 1963 in Miami, Florida, USA, designated 274 (A) Equi 2 (Miami) 63, became a prototype strain of the equine 2 subtype of equine influenza virus (Blaškovič, D. Basics of Medical Virology, Science Bratislava 1978).
Izolováním povodců a jeho identifikováním sa otvorila cesta pre studium epidemiologie, patogenézy a imunogenézy chřipky koní.The isolation of the pest and its identification opened the way for the study of equine epidemiology, pathogenesis and immunogenesis.
Pokusy o aktívnu imunizáciu koní proti chrípke sa zakladajú na poznatku, že překonáme infekcie vyvolává chránenosť proti ochoreniu a praktická imunizácia na poznatku, že štruktúry virusu zodpovědné za vyvolanie protilátek a imunity predstavujú virusový hemaglutinin.Attempts to actively immunize horses against influenza are based on the knowledge that we will overcome infections give rise to disease protection, and practical immunization to recognize that the virus structures responsible for eliciting antibodies and immunity are viral hemagglutinin.
Východiskovým materiálem pre získanie virusového materiálu na přípravu vakcín sú infekčné alento-amniotické tekutiny, ktoré sa podrobujú rozdielnym perifikačným postupem. Ich cielom je odstránenie vaječných bielkovín a získanie poměrně čistého a koncentrovaného vírusovéhot materiálu. Na inaktiváciu virusu v infekčných vaječných tekutinách sa najčastejšie používajú formai252389 dehyd (McQueen, J. L. a spol. Imunology of equine influenza, J. Am. vet. med. Ass. 155, 1969, 2, s. 265—271], formaldehyd zmiešaný v rovnakom pomere s glycerínem (Onta, T. a spol. Distribution of antibodies against various influenza A viruses in animals, Jap. J. vet. Sci. 40, 1978, s. 451—454), alebo betapropiolakton (Gazzei, V. A. a spol. One day purification of influenza A and B vaccines using molecular filtnation and other physycal metods. Microbiologica, 4, 1981, s. 403—412). Najčastejším sposobom inaktivácie virusu fyzikálnou cestou je ožiarenle ultrafialovými lúčmi (Tood, J. D. Comments on the immunology of equine influenza, J. Am. vet. med. Ass. 1955, 1969, 2, s. 272 až 277).The starting material for obtaining the viral material for the preparation of vaccines is infectious alento-amniotic fluids, which are subjected to different peripheral procedures. Their goal is to remove egg proteins and obtain relatively pure and concentrated viral material. For the inactivation of the virus in infectious egg fluids, the most commonly used form225389 dehyd (McQueen, JL et al., Immunology of Equine Influenza, J. Am. Vet. Med. Ass. 155, 1969, 2, pp. 265-271), formaldehyde mixed in equally with glycerin (Onta, T. et al. Distribution of antibodies against various influenza A viruses in animals, Jap. J. vet. Sci. 40, 1978, pp. 451-454), or betapropiolactone (Gazzei, VA et al. One day purification of influenza A and B vaccines using molecular filtration and other physycal methods (Microbiologica, 4, 1981, pp. 403-412). The most common method of physically inactivating a virus is irradiated by ultraviolet rays (Tood, JD). equine influenza, J. Am. vet. ass. 1955, 1969, 2, pp. 272-277).
K postupom purifikácie virusu patria adsorbčné, zrážacie, elučné, centrifugačné a filtračně metody. Za účelom dosiahnutia požadované] čistoty virusového materiálu sa používajú dve i viac metod v rámci jedného purifikačného postupu. K zrážacím metodám patří predovšetkým precipltácia virusu vychladeným alkoholem. Precipitáciu pomocou 12,5 % síranu barnatého použil Mizutani 1963 a 5 % kyseliny trichloroctovej Weremowicz, S. Polypeptides of equine inflúenza virus A (Equi) 2 Warszawa(9)69, zbl. vet. Med. B-27, 1980, s. 549—558). Na otázku ziastúpenia jednotlivých kmeňov vo, vakcíne a obsahu hemaglutininu v aglutinačnej dávke sú rózne názory. Všeobecne sa požaduje, aby vakcína obsahovala obidva subtypy chřipkového· virusu (Burki, F. Ein neuer Impflan gegen Pferdinfluemza, Zbl. vet. Med. 22, 1975, 2, s. 3—17), a aby obsah virusového hemaglutininu představoval pri subtype A equi 1 najmenej 512 hemaglutinačných jednotiek a pri subtype A equi 2 najmenej 1024 hemaglutinačných jednotiek. Aj keď ako vakcínu možno použit samostatný virus purifikovaný do rózneho stupňa, najčastejšie sa k němu pridávajú rózne adjuvantné látky, ktoré potencujú jeho antigénny účinek. Imunogenita vakcíny je totiž závislá ako od obsahu virusových antigénov, tak aj od imunopotencujúceho účinku adjuvantnej látky na ktorú je antigén naviazaný. Pri adjuvantných látkách ako· sú fosforečnan vápenatý, fosforečnan hlinitý, hydroxid hlinitý a alginát sodný, používané v obsahu 1,0-1,5 objemových percentách. Zriedkavo sa použilo, aj olejové inkompletné adjuvans (Tood, J. D. Comments on the immunology of equine influenza. J. Am. vet. med. Ass. 155, 1969, 2, s. 272—277], pri ktorom vzniká riziko vývoja nežiadúcich lokálnych reakcií.Virus purification procedures include adsorption, precipitation, elution, centrifugation and filtration methods. Two or more methods are used in a single purification procedure to achieve the desired purity of the viral material. Precipitating methods include, in particular, precipltion of the virus with cold alcohol. Precipitation with 12.5% barium sulfate was used by Mizutani 1963 and 5% trichloroacetic acid. Weremowicz, S. Polypeptides of Equine Influenza Virus A (Equi) 2 Warszawa (9) 69, Coll. vet. Med. B-27, 1980, p. 549-558). There are different opinions on the question of the distribution of individual strains in the vaccine and the hemagglutinin content in the agglutination dose. Generally, the vaccine is required to contain both subtypes of influenza virus (Burki, F. Ein neuer Impflan gegen Pferdinfluemza, Zb. V. Med. 22, 1975, 2, pp. 3-17), and the viral hemagglutinin content is subtype A equi 1 of at least 512 haemagglutination units and, for subtype A equi 2, of at least 1024 haemagglutination units. Although a single virus purified to a different degree can be used as a vaccine, most often it is added with various adjuvants which potentiate its antigenic effect. Indeed, the immunogenicity of the vaccine is dependent both on the content of the viral antigens and on the immunopotentiating effect of the adjuvant to which the antigen is bound. For adjuvants such as calcium phosphate, aluminum phosphate, aluminum hydroxide and sodium alginate, used at 1.0-1.5% by volume. Oil incomplete adjuvants have also been rarely used (Tood, JD Comments on the immunology of equine influenza. J. Am. Vet. Med. Ass. 155, 1969, 2, pp. 272-277), which risks developing undesirable local effects. reactions.
Proti chrípke koní boli v posledných rokoch vyvinuté účinné vakcíny, ktoré sa s úspechom používajú na účely špecifickej profylaxie tohto virusového, ochorenia (EQUIFLUR — Anglicko, FLUVACR — USA, PREVACUN — NSR, RESEQUIN — NDR}. Spósob technologie přípravy uvedených vakcín sa ziakladá na použití špičkových tech4 nických, finančně velmi nákladných zariadení.In recent years, effective vaccines have been developed against equine influenza which have been used successfully for the specific prophylaxis of this viral disease (EQUIFLU R - England, FLUVAC R - USA, PREVACUN - NSR, RESEQUIN - GDR). it is based on the use of state-of-the-art technical, very expensive equipment.
Přednos! navrhovaného riešenia spočívá v tom, že bol vypracovaný postup izolácie a purifikácie virusového materiálu z injekčných vaječných tekutin kombinovaným použitím fyzíkálnochemických metod s minimálnymi nárokmi na 1'ahko dostupné technologické zariadenia, dodržaním ktorého sa připraví vakcína o vysokých kvaTtatívmych parametrech.Prefer! The proposed solution consists in the development of a process for isolation and purification of viral material from injectable egg fluids by the combined use of physicochemical methods with minimum requirements for readily available technological equipment, by which a vaccine of high quality parameters is prepared.
Táto úloha bola vyriešená následovně:This task was solved as follows:
Bola připravená inaktivovaná adsorbátová vakcína proti chrípke koní s minerálnym nosičom, ktorá obsahuje v 1 ml vakcíny inaktivované kmene virusu chřipky koní 138(AjEqui 1(Praha)56 CAPM V-122, o hemaglutinačnom titre 1280 hemaglutinačných jednotiek, 274(A]Equl 2(Miami)63 CAPM V123 a 245(A)Equi 2(Ditroit)63 CAPM V-304 oba o hemaglutinačnom titre 2560 hemaglutinačných jednotiek.An inactivated equine influenza adsorbate vaccine with mineral carrier was prepared and contained in 1 ml vaccine inactivated equine influenza virus strains 138 (AjEqui 1 (Prague) 56 CAPM V-122, with a haemagglutination titre of 1280 haemagglutination units, 274 (A) Equl 2 ( Miami) 63 CAPM V123 and 245 (A) Equi 2 (Ditroit) 63 CAPM V-304 both with a haemagglutination titre of 2560 haemagglutination units.
Virulentně virusové kmene boli pomnožené oddelene na 10 dňových kuřácích zárodkoch po dobu 48 hodin pri teplote 35 až 37 °C. Odobraté virusové tekutiny boli inaktivované 7% roztokom formaldehydu pri teplote 2 až 8 CC po dobu 8 až 10 dní. Inaktivované virusové tekutiny sa precipitovali etanolom po· dobu 24 až 48 hodin ia po odstředění sa resuspendovali vo fosfátevom pufri tak, aby sa dosiahol požadovaný hemaglutimačný titer 1280 hemaglutinačných jednotiek u kmeňa 138(A)Equi 1(Praha)56 CAPM V-122 a 2560 hemaglutinačných jednotiek pri kmeňoch 274(A)Equi 2(Miami)63 CAPM V-123 a 245(A)Equi 2(Ditroit)63 CAPM V-304. Po odstránení nežiadúcich látok sa s výhodou zmiešali v rovnakom objemovom pomere a naviazali na nrnerálny nosič, ktorý představuje gél hydroxidu hlinitého vo výslednej koncentrácii 1 až 2 hmotnostných percent hliníka.Virulent virus strains were propagated separately on 10 day smoking germs for 48 hours at 35-37 ° C. The collected viral fluids were inactivated with a 7% formaldehyde solution at 2-8 ° C for 8-10 days. Inactivated virus fluids were ethanol precipitated for 24 to 48 hours and resuspended in phosphate buffer after centrifugation to achieve the desired haemagglutimation titer of 1280 haemagglutination units in strain 138 (A) Equi 1 (Prague) 56 CAPM V-122 and 2560 hemagglutination units for strains 274 (A) Equi 2 (Miami) 63 CAPM V-123 and 245 (A) Equi 2 (Ditroit) 63 CAPM V-304. After removal of the undesirable substances, they are preferably mixed in the same volume ratio and bound to a nrneral carrier which is an aluminum hydroxide gel in a final concentration of 1 to 2% by weight of aluminum.
Vakcína vykazuje následovně imunobiologické vlastnosti:The vaccine exhibits the following immunobiological properties:
a. je neškodná pre zvieratá všetkých věkových kategoriía. is harmless to animals of all ages
b. navodí včasnú a dlhotrvajúcu chránenosť očkovaných zvierat proti ochoreniu a infekcii jednotlivými kmeňmi subtypov A equi 1 a A equi 2.b. induce early and long-term protection of vaccinated animals against disease and infection by individual strains of subtypes A equi 1 and A equi 2.
c. je použitelná na profylaktické aj metafylaktické očkován:'ec. is applicable to both prophylactic and metaphylactic vaccines: e
d. cestou materinckých protilátek, očkovaných žrebných kobýl zabezpečí pasívnu chráněnosť žriebat až do obdobia, v ktorom móžu byť aktivně imunizované.d. by way of maternal antibodies, vaccinated stallions will ensure passive protection of the foals until they can be actively immunized.
Tri kmene virusu chřipky koní subtypov A equi 1 a A equi 2 s označením 138(A)Equi 1(Praha)56, 274(A)Equi 2(Miami)63 a 245(A)Equi 2(Ditroit)63 nám poskytla Česko252389 slovenská chřipková centrála pri Ústave epidemiologie a mikrobiologie — Šrobárova 48, Praha 10. Kmene 138(A)Equi l(Praha)56 s označením CAPM V-122, 274(A)Equi 2(Miami}63 s označením CAPM V-123 a 245(AjEqui 2-(Ditroitj63 s označením CAPM V-304 sú tiež uložené v Československej zbierke mikroorganizmov na Výskumnom ústave veterinárneho lekárstva v Brně, Hudcova 60. Uvedené virusové kmene sa pomnožujú na 10 až 11 dňových kuřácích zárotíkoch po· infekcii do· alantoickej alebo· amniovej dutiny·. Infekčně alanto-amniové tekutiny odobraté z vychladených vajec sa klarifikujú odstředováním pri nízkých obrátkách a vyšetrujú na obsah hemaglutininu a infekčných jednotiek.Three equine influenza virus strains of subtypes A equi 1 and A equi 2 designated 138 (A) Equi 1 (Prague) 56, 274 (A) Equi 2 (Miami) 63 and 245 (A) Equi 2 (Ditroit) 63 provided by Czech Republic252389 Slovak flu center at the Institute of Epidemiology and Microbiology - Šrobárova 48, Prague 10. Strains 138 (A) Equi l (Prague) 56 designated CAPM V-122, 274 (A) Equi 2 (Miami} 63 designated CAPM V-123 and 245 (AjEqui 2- (Ditroitj63 with the designation CAPM V-304) are also deposited in the Czechoslovak Collection of Microorganisms at the Veterinary Research Institute in Brno, Hudcova 60. The above mentioned virus strains reproduce in 10 to 11 days old smokers after infection into allantoic or Infectious allanto-amniotic fluids collected from chilled eggs are clarified by low-speed centrifugation and examined for haemagglutinin and infectious unit content.
Infekčnosf viruse v vo vaječných tekutinách sa inaktivuje přidáním def novaného množstva formaldehydu, a virus z alantoamniových tekutin vyzráža pomocou etylalkoholu za chladu, získaný precipitát predstavujúci virusové částice a vaječné bielkoviny sa resuspenduje v takom objeme fosfátového· pufru definovaného zloženia a pH, aby výsledný hemaglutinačný titer pur fikátu bol pri kmeni 138(A)Equi 1 (Praha)56 CAPM V-122 1280 hemaglutinačných jednctiek a pri kmeňoch 274(A)Equi 2(M.'ami)63 •CAPM V-123 a 245(A)Equi 2 (Ditroit) 63 •CAPM V-304 2560 hemaglutjnačnóých jednotiek. Příprava jednotlivých purifikátov sa ukončí dokonalou elúciou virusu do fosfátového pufru pri zvýšenej teplote a vyčírením získanej vírusovej suspenzie odstředěním pri nízkých obrátkách.The viral infectivity in egg fluids is inactivated by the addition of a defined amount of formaldehyde, and the virus is precipitated from the allantoamnium fluids with ethyl alcohol in the cold, the precipitate obtained representing virus particles and egg proteins is resuspended in a volume of phosphate buffer of defined composition and pH to the resulting hemagglutin the purity of ficate was haemagglutinating units in strain 138 (A) Equi 1 (Prague) 56 CAPM V-122 and in strains 274 (A) Equi 2 (M.'ami) 63 • CAPM V-123 and 245 (A) Equi 2 (Ditroit) 63 • CAPM V-304 2560 hemagglutinating units. The preparation of the individual purified compounds is terminated by complete elution of the virus into phosphate buffer at elevated temperature and clarification of the obtained viral suspension by centrifugation at low speed.
Vakcína sa připraví zmiešaním purif kátov připevněných z jednotlivých kmeňov v rovnakom objemovom pomere a přidáním takého množstva hydroixidu, hlinitého A1(OH)3, aby jeho výsledná koncentrácia představovala 1,5 objemových percent. Přidáváme minerálneho nosiča k zmesi purifikátov sa robí postupné a v menších cbjemoch, aby nastalo dokonalé naviazanie virusových partlkúl.The vaccine is prepared by mixing the purified strains attached from the individual strains in the same volume ratio and adding an amount of Al (OH) 3 aluminum hydride to give a final concentration of 1.5 volume percent. Adding the mineral carrier to the mixture of purities is done gradually and in smaller volumes to allow perfect binding of the virus particles.
Vynález bližšie objasňuje nasledujúci příklad praktického prevedenia:The invention is illustrated in more detail by the following example:
Příklad dnové kuracie zárodky sa očkujú do alantoickej dutiny dávkou 103 TKIDjo/v 0,1 až 0,2 ml inokula a zárodky inkubujú 48 hodin pri teplote 35 °C. Vajcia s prežívajlicími zárodkami sa po ukončení inkubácie schladia pri -j-2 až -j~8°C a po· 24 h.dinách sa z nich odoberajú alanto-amniotické tekutiny. Infkečné vaječné tekutiny cdobraté a egalizované pri každom virusovém kmeni samostatné s požadovaným hemaglutinačným titrcm sa inaktivujú přidáním 0,1 objemových percent 7 % roztoku formaldehydu pri teplote 2 až 8 °C počas 10 dní. Kontrola účinnosti inaktivácia sa robí očkováním 10 dňových kuřácích zárodkov vaječnými tekutinami odobratými po ukončení hemaglutiinácie na prítomnosť hemaglutininu. Inaktivované virusové tekutiny každého kmeňa sa ponžijú na přípravu purifikátov. Spracovanie virusových materiálov je založené na principe precipitácie virusových preinov pomocou etylalkoholu. K aliantoamniotickej tekutině udržiavanej pri -j-4 stupňoch Celsia sa přidává 96 % etylalkohol vychladený na teplotu —38 CC do výsledné j koncentrácie 30 až 40 %. Po 48 hodinovom státi pri chladničkovej teplote sa obsah fiiaš premieša a odstreďuje pri nízkých otáčkách počas 60 min. Supernatant sa kontroluje na obsah hemaglutininu a pri zistení hemaglutinačných titrov 1 : 80 a viac, sa podrobí opakovanéj precipitácii. Sediment predstavujúci vyzrážané virusové partikule a vaječné bielkoviny, sa resuspenduje v takom objeme fosfátového pufru o pH 7,2, aby výsledný hemaglutinačný titer purifikátu bol 1280 hemaglutinačných jednotiek pri kmeni 138(A)Equi 1(Praha]56 CAPM V-122 a 2560 hemaglutinačných jednotiek pri kmeni 245(A)Equl 2(D tro.it)63 CAPM V-304 a 274(A)Equi 2(Miami)63 CAPM V-123. Stupeň nariadenia závisí preto· od východiskového· hemaglutinačného titru egalizovaných alantoických tekutin, a nie od množstva získaného sedimentu. Virus resuspendovaný. vo· fosfátovom pufre sa pretrepáva počas 2 hodin pri 37 °C, čím sa dosiahne elúci virusu z nariedeného sedimentu. Následnou 60 min. centrifugáciou pri nízkých obrátkách 700 g sa odstránia nežiadúce částice a prímeesi vyzrážaných bielkovín. Ako· ménerálny nosič sa použije gél hydroxidu hlinitého (výrobok Biovety, n. p., Nitraj. Purifikáty kmeňov 138(A)Equi 1(Praha)56 CAPM V-122, 245(A)Equi 2(Ditroit)63 CAPM V-304 a 274(A)Equi 2(Miami)63 CAPM V-123 sa zmieša jú v rovnakom objemovom množstve.An example of day-old chicken germs are inoculated into the allantoic cavity with a dose of 10 3 TKIDjo / in 0.1-0.2 ml inoculum and the germs are incubated at 35 ° C for 48 hours. Surviving eggs are chilled at -j-2 to -j-8 ° C after incubation and allanto-amniotic fluids are collected after 24 hours. Infectious egg fluids collected and leveled in each virus strain separately with the desired haemagglutination titre are inactivated by adding 0.1 volume% of 7% formaldehyde solution at 2-8 ° C for 10 days. Control of inactivation efficacy is done by inoculating 10 day-old smokers of embryos with egg fluids collected after hemagglutinination for hemagglutinin. The inactivated virus fluids of each strain are used to prepare the purified compounds. The processing of viral materials is based on the principle of precipitation of viral preins using ethyl alcohol. To the aliantoamniotic fluid maintained at j-4 degrees Celsius is added 96% ethyl alcohol cooled to -38 ° C to a final concentration of 30 to 40%. After standing for 48 hours at refrigerator temperature, the contents of the flasks are mixed and centrifuged at low speed for 60 min. The supernatant is checked for hemagglutinin content and subjected to repeated precipitation upon detection of hemagglutination titers of 1:80 or more. The sediment representing the precipitated virus particles and egg proteins is resuspended in a volume of phosphate buffer at pH 7.2 such that the resulting haemagglutination titre of the purity is 1280 haemagglutination units for strain 138 (A) Equi 1 (Prague) 56 CAPM V-122 and 2560 haemagglutination units of strain 245 (A) Equl 2 (D tro.it) 63 CAPM V-304 and 274 (A) Equi 2 (Miami) 63 CAPM V-123 The degree of regulation therefore depends on the initial haemagglutination titre of the equalized allantoic fluids, The virus resuspended in phosphate buffer is shaken for 2 hours at 37 ° C to elute the virus from the diluted sediment, followed by 60 min centrifugation at low speed of 700 g to remove unwanted particles and admixtures precipitated. Aluminum hydroxide gel (product Biovety, np, Nitraj. Purified strains 138 (A) Equi 1 (Prague) 56 CAPM V-122, 245 (A) Equi 2 (Ditroit) 63 CAPM V-304 and 274 (A) Equi 2 (Miami) 63 CAPM V-123 are mixed in equal volume.
Zmes vírusov sa pre třepáním dokonale premieša a za stálého pomalého miešania sa po malých dávkách přidává také množstvo gélu hydroxidu hlinitého, aby výsledná koncentrácia hllníka Al vo· vakcíne bola 1,5 pere. Po přidaní 0,2 objemových percent 5°/o roztoku tiomerzalu je příprava vakcíny proti chrípke koní ukončená.The virus mixture is thoroughly mixed for agitation and the aluminum hydroxide gel is added in small portions in small portions with constant slow mixing so that the final concentration of Al aluminum in the vaccine is 1.5 pens. After the addition of 0.2 volume percent of a 5% thiomersal solution, the preparation of the equine influenza vaccine is complete.
Oč nnosť inaktivovanej adsorbátovej vakcíny proti chrípke koni, pripravenej podfa vynálezu potvrdzujú výsledky uvedené v nasledujúcej tabulke: Tabulka uvádza tilre nehemaglutinačne-inhibičných protilátek zisťované u žriebat a koní různého veku po očkovaní vakcínou připravenou podlá vynálezu.The results of the inactivated equine influenza adsorbate vaccine prepared according to the invention are confirmed by the results in the following table: The table shows the non-haemagglutination-inhibiting antibody filters found in foals and horses of different ages after vaccination with the vaccine prepared according to the invention.
HI-titre uvedené v logaHI-titers listed in the logo
3mesačné 6mesačné žriebátá žriebátá dospělé koně Vysoko žrebné Potomstvo kobyly od vakc. K kmeň 138 (A) 1 6,0 11,4 kmen 245(A)2 4,4 10,0 kmeň 274(A)2 6,0 10,33 months 6 months old foal foal adult horses Highly drawn offspring of mares. K strain 138 (A) 1 6.0 11.4 strain 245 (A) 2 4.4 10.0 strain 274 (A) 2 6.0 10.3
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| Application Number | Priority Date | Filing Date | Title |
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| CS858238A CS252389B1 (en) | 1985-11-15 | 1985-11-15 | Inactivated adsorbate vaccine against equine influenza and method of its preparation |
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| Application Number | Priority Date | Filing Date | Title |
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| CS858238A CS252389B1 (en) | 1985-11-15 | 1985-11-15 | Inactivated adsorbate vaccine against equine influenza and method of its preparation |
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| CS823885A1 CS823885A1 (en) | 1987-01-15 |
| CS252389B1 true CS252389B1 (en) | 1987-08-13 |
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1985
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