NO122031B - - Google Patents
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- NO122031B NO122031B NO164282A NO16428266A NO122031B NO 122031 B NO122031 B NO 122031B NO 164282 A NO164282 A NO 164282A NO 16428266 A NO16428266 A NO 16428266A NO 122031 B NO122031 B NO 122031B
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- virus
- chicken
- mumps
- fetal
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- 241000700605 Viruses Species 0.000 claims description 23
- 241000287828 Gallus gallus Species 0.000 claims description 16
- 241000711386 Mumps virus Species 0.000 claims description 14
- 229960005486 vaccine Drugs 0.000 claims description 10
- 235000013601 eggs Nutrition 0.000 claims description 9
- 210000003754 fetus Anatomy 0.000 claims description 9
- 230000001605 fetal effect Effects 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 5
- 239000002609 medium Substances 0.000 description 8
- 208000005647 Mumps Diseases 0.000 description 7
- 210000003837 chick embryo Anatomy 0.000 description 6
- 208000010805 mumps infectious disease Diseases 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000002238 attenuated effect Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 230000006978 adaptation Effects 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 210000000991 chicken egg Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000004789 organ system Anatomy 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- 201000005505 Measles Diseases 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 229940095293 mumps vaccine Drugs 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229960001322 trypsin Drugs 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 208000006740 Aseptic Meningitis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 206010027201 Meningitis aseptic Diseases 0.000 description 1
- 206010029240 Neuritis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 206010039705 Scleritis Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000012568 clinical material Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000003027 ear inner Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 210000000256 facial nerve Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000003670 sublingual gland Anatomy 0.000 description 1
- 210000001913 submandibular gland Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
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
- A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
- A61K39/165—Mumps or measles 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
- C12N2760/18011—Paramyxoviridae
- C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
- C12N2760/18734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Communicable Diseases (AREA)
- Pulmonology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
o o
Fremgangsmåte ved fremstilling av Procedure in the manufacture of
levende kusmavirusvaksine. live mumps virus vaccine.
Foreliggende oppfinnelse angår en fremgangsmåte ved fremstil- The present invention relates to a method for producing
ling av en vaksine inneholdende et svekket, levende kusmavirus. ling a vaccine containing a weakened, live mumps virus.
Som folge av dets svekkede natur forårsaker viruset liten eller ingen klinisk reaksjon. Det befordrer imidlertid dannelse av en be- .r tydelig mengde motstoff mot viruset hos mennesker, og dette motstoff er vesentlig for å oppnå beskyttelse mot infeksjon av viruset og mot sykdom forårsaket av dette. As a result of its attenuated nature, the virus causes little or no clinical reaction. However, it promotes the formation of a significant amount of antibody against the virus in humans, and this antibody is essential for achieving protection against infection by the virus and against disease caused by it.
Kusma ansees på samme måte som meslinger som en av de mindre alvorlige barnesykdommer. Ved vanlig klinisk infeksjon er dette riktig. I spesielle tilfeller kan imidlertid de kliniske folger av virusinfeksjonen være alvorlige, og ettersykdommene kan være like odeleggende som ved meslinger. Mumps is considered, in the same way as measles, to be one of the less serious childhood diseases. In the case of common clinical infection, this is correct. In special cases, however, the clinical consequences of the viral infection can be serious, and the after-effects can be as devastating as with measles.
I det vanlige tilfelle av kusma inntrer den akutte febertilstand etter en inkubasjonstid på 18 - 21 dager. De viktigste kliniske symptomer er feber med ledsagende opphovning av spyttkjertlene på den ene side eller på begge sider, av og til også under medvirkning av de submaxillære og sublinguale kjertler. In the usual case of mumps, the acute fever occurs after an incubation period of 18 - 21 days. The most important clinical symptoms are fever with accompanying swelling of the salivary glands on one or both sides, occasionally also involving the submaxillary and sublingual glands.
Viremia ledsager ofte kusmavirusinfeksjon, og en rekke organer eller organsystemer kan være innblandet. Disse kan innbefatte bite-stikkelen, blærehalskjertelen, eggstokken, leveren, bukspyttkjertelen, milten, skjoldbruskkjertelen, nyrene, labyrinten, oynene, brisselen, myocardium, brystkjertlene og sentralnervesystemet. De viktigste syk-dommer er encephalitis, encephalomyelitis, neuritis i ansikts-nervene, i trigeminus eller i synsnervene, aseptisk meningitis (0,5-10 % av tilfellene), : scleritis, pancreatitis, som enkelte ganger forer til sukkersyre; og orchitis eller ovaritis, som av og til forer til atrophia og sterilitet. Alvorlige komplikasjoner er minst hyppige blant barn. De mer alvorlige tilfeller inntrer ved infeksjon av voksne mennesker som har unngått sykdommen som barn. Viremia often accompanies mumps virus infection, and a number of organs or organ systems may be involved. These may include the epididymis, prostate gland, ovary, liver, pancreas, spleen, thyroid gland, kidneys, labyrinth, eyes, thymus, myocardium, mammary glands and central nervous system. The most important illnesses are encephalitis, encephalomyelitis, neuritis in the facial nerves, in the trigeminus or in the optic nerves, aseptic meningitis (0.5-10% of cases), : scleritis, pancreatitis, which sometimes leads to sugar acid; and orchitis or ovaritis, which occasionally leads to atrophy and sterility. Serious complications are least frequent among children. The more serious cases occur when adults are infected who have avoided the disease as children.
o o
Kusma uten komplikasjoner hos barn er avkreftende, anstrengende for foreldrene og resulterer i. betydelig fravær fra skolen. Kusma med komplikasjoner, enten det gjelder barn eller voksne, kan variere fra en mild sykdom med tilsynelatende fullstendig helbredelse til ødeleggelse eller nedsatt kapasitet av viktige organsystemer som kan gjore pasienten til kropling'for livet eller være dbdelig. Mumps without complications in children is debilitating, stressful for the parents and results in significant absences from school. Mumps with complications, whether in children or adults, can vary from a mild disease with apparently complete healing to destruction or reduced capacity of important organ systems that can cripple the patient for life or be fatal.
Det var kjent for denne oppfinnelse ble gjort - jfr. en artikkel av J.E. Prier i Basic medical virology, s. 259-26<*>+ (Baltimore 1966) - at kusmavirus kan tilpasses'til vekst i fosterholdig honseegg med suksessive passeringer gjennom dette medium, og at meget godt til-passet virus kan dyrkes i kyllingfosterkultur. Samme kilde viser likeledes at det var kjent å fremstille en levende kusmavaksine ut fra virus dyrket i fosterholdig honseegg , Det var imidlertid ikke kjent at man ved på nærmere bestemt måte å kombinere passeringer gjennom fosterholdig honseegg med dyrkning i kyllingfostervevkultur kan fremstille en sikker men effektiv levende kusmavaksine. ;Ved hjelp av oppfinnelsen tilveiebringes der således nu en fremgangsmåte ved fremstilling av levende kusmavirusvaksine ut fra virus som er dyrket suksessivt i fosterholdig honseegg, hvilken fremgangsmåte utmerker seg ved at man anvender kusmavirus som har passert minst ti suksessive ganger gjennom fosterholdig hSnseegg og deretter er blitt dyrket i kyllingfostervevkultur inneholdende vev fra 9 til ;11 dager gamle kyllingfostere ved 30 - 38°C. ;Fremgangsmåten omfatter de folgende trinn: (A) isolering av det livskraftige virus i fosterholdig honseegg og tilpasning av dette til kyllingfostervevkultur, (B) utvikling av det svekkede, levende virus ved hjelp av dyrkninger i kyllingfostervevkultur og (C) fremstilling av vaksinen ut fra det erholdte svekkede, levende virus. Disse trinn skal beskrives hvert for seg. ;A. Isolering og tilpasning av livskraftig virus ;Kusmaviruset skaffes fra klinisk materiale, nemlig fra personer som vites å ha kusma. Isolering og tilpasning av kusmaviruset full-fores i kyllingfostervevkultur etter at det på forhånd er blitt formert i fosterholdig honseegg. Inkubasjon av infiserte kulturer kan utfores ved 30 - 3^-°C (optimalt 32°C) eller ved 35 - 38°C ;(optimalt 36°C) ;B. Fremstilling av svekket, levende kusmavirus ;Viruset som under A er påvist å være kusmavirus, anbringes i ;glassflasker inneholdende kyllingfostervevkulturer tilberedt av finhakkede og trypsinerte, omtrent ti dager gamle kyllingfostere. Dyrkningsmediet kan f.eks. være det kjente medium 199 til hvilket det er tilsatt kalveserum. Etter tilsetningen av serumet inkuberes de infiserte cellekulturer ved suksessive dyrkninger ved 30 - 38°C i varierende tidsrom, hvorunder viruset svekkes. De virusholdige væsker innsamles deretter og lagres i fryst tilstand eller ved annen lav temperatur for å opprettholde virusets infektivitet. ;De ovennevnte suksessive dyrkninger utfores under anvendelse av et fortynnet innpodningsmateriale, og flere^innhøstninger foretas med varierende mellomrom. Infektivitetstitreringer utfores i HeLa-kultur, grivetapenyrevevkultur eller kyllingfostervevkultur. ;C. Fremstilling av vaksine ;Kusmaviruset som ble hostet etter disse suksessive dyrkninger, viste seg ikke å fremkalle sykdom hos aper og gnagere, å fremkalle liten eller ingen klinisk reaksjon hos mennesker og å fore til dannelse av en betydelig mengde motstoff. Virusinfektiviteten stabi-liseres ved hjelp av et egnet stabiliseringsmiddel såsom sucrose menneskealbumin, glutamin, fosfat eller blandinger derav. Etter titrering for bestemmelse av dets styrke fylles viruspreparatet på ampuller. Produktet kan lagres og utdeles i fryst tilstand, men blir fortrinnsvis frysetorret og oppbevart fritt for fuktighet. ;Forsok på mennesker: Omtrent 150 barn som ikke tidligere hadde hatt kusma, ble parenteralt gitt en dose av en vaksine av svekket kusmavirus. Praktisk talt samtlige barn reagerte ved en betydelig mot-stofftiter innen en måned etter vaksineringen. Barna viste få eller ingen kliniske symptomer, alt etter stammen av kusmavirus og antall dyrkninger i fosterholdig honseegg og kyllingfostervevkulturer. ;Eksempel ;Innpodningsmaterialet er det som fåes ved den ovenfor beskrevne isolasjonsmetode etter 10 suksessive passeringer gjennom fosterholdig honseegg. ;Ni til elleve dager gamle kyllingfostere finhakkes under aseptiske betingelser etter at hode- og halepartier er fjernet, og det finhakkede vev vaskes flere ganger med Hanks' BSS. Det vaskede vev trypsineres ved 36°C under anvendelse av 0,25 % trypsin ("Difco" 1:250) i tris(hydroxymethyl)-aminomethan-puffer i 2 - 3 timer. Tryp-sincelleoppslemningen oppsamles gjennom to lag sterilt osteklede og sentrifugeres med en hastighet av 1500 omdr./min i 5 minutter. Sammenpakkede celler oppslemmes på ny i dyrkningsmedium for telling. Dyrkningsmediet er medium 199 inneholdende 2 % agamma-kalveserum (oppvarmet ved 56°C i 30 minutter) og 50 mcg/ml "Neomycin". Flaske-kulturer plantes i en konsentrasjon av 350.000 levedyktige celler pr. milliliter. Etter inkubering ved 36°C i et tidsrom fra *+8 til 72 timer kan flaskekulturene anvendes for suksessive dyrkninger eller for vaksinefremstilling. It was known before this invention was made - cf. an article by J.E. Prier in Basic medical virology, pp. 259-26<*>+ (Baltimore 1966) - that mumps virus can be adapted to growth in embryonated chicken eggs with successive passages through this medium, and that very well adapted virus can be grown in chick embryo culture. The same source also shows that it was known to produce a live mumps vaccine from virus grown in a fetal chicken egg. However, it was not known that by combining passages through a fetal chicken egg with cultivation in chicken fetal tissue culture in a more specific way, a safe but effective live virus can be produced mumps vaccine. With the help of the invention, there is thus now provided a method for the production of live mumps virus vaccine from viruses that have been grown successively in fetal chicken eggs, which method is characterized by using mumps virus that has passed at least ten successive times through fetal chicken eggs and has then been grown in chick embryo tissue culture containing tissue from 9 to 11 day old chick embryos at 30 - 38°C. The method includes the following steps: (A) isolation of the viable virus in embryonated chicken eggs and adaptation of this to chicken fetal tissue culture, (B) development of the weakened, live virus using cultures in chicken fetal tissue culture and (C) production of the vaccine from attenuated, live virus was obtained. These steps must be described separately. A. Isolation and adaptation of viable virus The mumps virus is obtained from clinical material, namely from people known to have mumps. Isolation and adaptation of the mumps virus is fully grown in chicken fetal tissue culture after it has previously been propagated in embryonated honse eggs. Incubation of infected cultures can be carried out at 30 - 3^-°C (optimally 32°C) or at 35 - 38°C ;(optimally 36°C) ;B. Preparation of attenuated, live mumps virus ;The virus which under A has been shown to be mumps virus is placed in ;glass bottles containing chick embryo tissue cultures prepared from minced and trypsinized approximately ten-day-old chick embryos. The cultivation medium can e.g. be the known medium 199 to which calf serum has been added. After the addition of the serum, the infected cell cultures are incubated in successive cultures at 30 - 38°C for varying periods of time, during which the virus weakens. The virus-containing fluids are then collected and stored in a frozen state or at another low temperature to maintain the infectivity of the virus. The above-mentioned successive cultivations are carried out using a diluted inoculum, and several harvests are made at varying intervals. Infectivity titrations are performed in HeLa culture, guinea pig kidney tissue culture or chicken fetal tissue culture. C. Preparation of vaccine The mumps virus coughed up after these successive cultures was shown not to cause disease in monkeys and rodents, to cause little or no clinical reaction in humans, and to produce a significant amount of antibody. The virus infectivity is stabilized by means of a suitable stabilizing agent such as sucrose human albumin, glutamine, phosphate or mixtures thereof. After titration to determine its potency, the virus preparation is filled into ampoules. The product can be stored and distributed in a frozen state, but is preferably freeze-dried and stored free of moisture. ;Experiments on humans: Approximately 150 children who had not previously had mumps were parenterally given a dose of an attenuated mumps virus vaccine. Practically all children reacted with a significant antibody titer within one month of vaccination. The children showed few or no clinical symptoms, depending on the strain of mumps virus and the number of cultures in fetal chicken eggs and chicken fetal tissue cultures. ;Example ;The inoculation material is that which is obtained by the isolation method described above after 10 successive passages through a fetal egg. Nine- to eleven-day-old chick embryos are minced under aseptic conditions after head and tail portions are removed, and the minced tissue is washed several times with Hanks' BSS. The washed tissue is trypsinized at 36°C using 0.25% trypsin ("Difco" 1:250) in tris(hydroxymethyl)aminomethane buffer for 2-3 hours. The tryp-sin cell slurry is collected through two layers of sterile cheesecloth and centrifuged at a speed of 1500 rpm for 5 minutes. Packed cells are resuspended in culture medium for counting. The culture medium is medium 199 containing 2% agamma calf serum (heated at 56°C for 30 minutes) and 50 mcg/ml "Neomycin". Bottle cultures are planted at a concentration of 350,000 viable cells per milliliters. After incubation at 36°C for a period of *+8 to 72 hours, the bottle cultures can be used for successive cultivations or for vaccine production.
Kyllingfostervevkulturer fremstilles i glassflasker under anvendelse av medium 199'inneholdende 2 % inaktivert kalveserum som dyrkningsmedium. Tre eller fire dager etter plantingen tas dyrkningsmediet ut under aseptiske forhold, og flaskekulturene vaskes fire ganger med Hans' BSS, idet der benyttes 100 ml pr, vask, og podes med 2,5 ml fortynnet viruspreparat pr. flaske. 70 ml av medium 199 inneholdende 10 % av et egnet stabiliseringsmiddel for virusinfektiviteten tilsettes hver flaskekultur, og flaskene inkuberes ved 30 - 3^-°C. "Neomycin" i en konsentrasjon av 50 mcg/ml innlemmes i vekst-og vedlikeholdsmediet. Det foretas flere innhøstninger med 2 - •+ dagers mellomrom, og flaskene påfylles nytt vedlikeholdsmedium inneholdende det ovennevnte stabiliseringsmiddel. Ti suksessive dyrkninger utfores alle ved omtrent 32°C. Infektivitetstitreringer av hver innhostning utfores i HeLa-kultur eller i grivetapenyrekultur. Hver innhostning foretas aseptisk, idet der benyttes en steril be- holder. Det tas ut prover for bestemmelse av den mikrobielle sterilitet, og den resterende del skallfryses i et bad av torris og alko-hol. De virusholdige væsker lagres ved -70°C i en elektrisk drevet fryseenhet, for ett eller flere innhostede materialer velges ut for fremstilling av vaksinen. Chicken fetal tissue cultures are prepared in glass bottles using medium 199 containing 2% inactivated calf serum as culture medium. Three or four days after planting, the culture medium is taken out under aseptic conditions, and the bottle cultures are washed four times with Hans' BSS, using 100 ml per wash, and inoculated with 2.5 ml of diluted virus preparation per bottle. 70 ml of medium 199 containing 10% of a suitable stabilizer for virus infectivity is added to each flask culture, and the flasks are incubated at 30-3°C. "Neomycin" in a concentration of 50 mcg/ml is incorporated into the growth and maintenance medium. Several harvests are made at intervals of 2 - •+ days, and the bottles are filled with new maintenance medium containing the above-mentioned stabilizer. Ten successive cultures are all carried out at approximately 32°C. Infectivity titrations of each instillation are performed in HeLa culture or in liver tape kidney culture. Each inhalation is carried out aseptically, using a sterile container. Samples are taken to determine the microbial sterility, and the remaining shell is frozen in a bath of dry ice and alcohol. The virus-containing liquids are stored at -70°C in an electrically powered freezing unit, for one or more coughed-up materials are selected for the manufacture of the vaccine.
Ett eller flere egnede innhostede materialer velges ut etter at det er foretatt infektivitetstitreringer. Det utvalgte materiale tas ut fra fryseren og tines opp. En prove tas ut for kontroll og for å undersbke hvorvidt den er sikker. Den gjenværende væske klares, og en prove tas ut for å teste materialet på aper. Passende mengder ytter-ligere stabiliseringsmiddel tilsettes den gjenværende væske. Væsken fordeles på skåler og torres. Etter torring blir skålene lukket og forseglet og holdt i beredskap for tilberedelse av en vaksine ved tilsetning av sterilt vann. One or more suitable coughed-up materials are selected after infectivity titrations have been carried out. The selected material is taken out of the freezer and thawed. A sample is taken out for control and to check whether it is safe. The remaining liquid is clarified, and a sample is taken out to test the material on monkeys. Appropriate amounts of additional stabilizer are added to the remaining liquid. The liquid is distributed in bowls and dried. After drying, the bowls are closed and sealed and kept in readiness for the preparation of a vaccine by adding sterile water.
Færre eller flere suksessive dyrkninger av viruset i kylling-fostervevkulturen kan foretas i den hensikt å svekke viruset. Eksempelvis ga tyve suksessive passeringer gjennom fosterholdig honseegg ved 30 - 38°C med to påfolgende passeringer gjennom kyllingfoster-vevkul tur ved 30 - 38°C et godt resultat. Fewer or more successive cultivations of the virus in the chicken-fetal tissue culture can be carried out with the intention of weakening the virus. For example, twenty successive passes through embryonated chicken egg at 30 - 38°C with two consecutive passes through chick embryo-tissue cool at 30 - 38°C gave a good result.
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Application Number | Priority Date | Filing Date | Title |
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US47965365A | 1965-08-13 | 1965-08-13 |
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NO122031B true NO122031B (en) | 1971-05-10 |
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NO164282A NO122031B (en) | 1965-08-13 | 1966-08-12 |
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JP (1) | JPS5238089B1 (en) |
BE (1) | BE685270A (en) |
BR (1) | BR6681974D0 (en) |
CH (1) | CH475355A (en) |
CS (1) | CS161688B2 (en) |
CY (1) | CY536A (en) |
DE (1) | DE1617621B1 (en) |
DK (1) | DK116677B (en) |
ES (1) | ES330274A1 (en) |
FI (1) | FI43094B (en) |
FR (2) | FR1509944A (en) |
GB (1) | GB1140766A (en) |
IL (1) | IL26244A (en) |
NO (1) | NO122031B (en) |
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Publication number | Priority date | Publication date | Assignee | Title |
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FR2076787A5 (en) * | 1970-01-28 | 1971-10-15 | Merieux Inst | Lyophilisation stabiliser - for viruses contg potassium phosphate lactose and albumin |
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1965
- 1965-08-01 IL IL26244A patent/IL26244A/en unknown
-
1966
- 1966-07-19 CS CS4877A patent/CS161688B2/cs unknown
- 1966-08-03 GB GB34791/66A patent/GB1140766A/en not_active Expired
- 1966-08-04 ES ES0330274A patent/ES330274A1/en not_active Expired
- 1966-08-08 CH CH1137766A patent/CH475355A/en not_active IP Right Cessation
- 1966-08-09 BE BE685270D patent/BE685270A/xx not_active IP Right Cessation
- 1966-08-09 BR BR181974/66A patent/BR6681974D0/en unknown
- 1966-08-10 DE DE1966M0070534 patent/DE1617621B1/en active Pending
- 1966-08-11 FI FI2099/66A patent/FI43094B/fi active
- 1966-08-12 FR FR73061A patent/FR1509944A/en not_active Expired
- 1966-08-12 DK DK414266AA patent/DK116677B/en unknown
- 1966-08-12 FR FR72964A patent/FR5807M/fr not_active Expired
- 1966-08-12 JP JP41052676A patent/JPS5238089B1/ja active Pending
- 1966-08-12 NO NO164282A patent/NO122031B/no unknown
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1970
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Also Published As
Publication number | Publication date |
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GB1140766A (en) | 1969-01-22 |
BE685270A (en) | 1967-02-09 |
FI43094B (en) | 1970-10-01 |
ES330274A1 (en) | 1967-06-16 |
IL26244A (en) | 1970-03-22 |
JPS5238089B1 (en) | 1977-09-27 |
FR5807M (en) | 1968-02-19 |
BR6681974D0 (en) | 1973-12-26 |
CS161688B2 (en) | 1975-06-10 |
DE1617621B1 (en) | 1971-03-04 |
DK116677B (en) | 1970-02-02 |
FR1509944A (en) | 1968-01-19 |
CH475355A (en) | 1969-07-15 |
CY536A (en) | 1970-04-10 |
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