CS250799B1 - Live atenurvated vaccine against distemper and its preparation - Google Patents

Abstract

The invention relates to a live] attenuated vaccine against distemper and a method of its preparation in cell cultures. Distemper is a viral disease of dogs and carnivorous fur animals, which is manifested by catarrhal inflammation of the mucous membranes of the respiratory and digestive systems and frequent changes in the central nervous system. The disease causes significant losses, as the mortality rate reaches up to 50% on average. Distemper is a dreaded disease, especially in larger dog breeding stations and multi-breeding carnivorous fur animals, because when it occurs, especially young ones die en masse.

Description

The disease elicits a ‘virus belonging to the group of non-hemagglutinating paramyxoviruses, which contains ribonucleic acid, is pH-labile, ether-sensitive, with helical symmetry and envelope. It is resistant to drying and freezing, withstands freeze-drying. Canine distemper virus is excreted from the infected organism by virtually all secretions, especially eye and nose, urine and faeces. The natural infection of the susceptible organism occurs through the upper respiratory tract, the oral cavity and the tonsils. Little susceptible to infection are infants that are protected by specific antibodies obtained from the mother.

Specific prophylaxis is based on passive immunization by administration of hyperimmune serum or immune serum globulins, but active immunization is most commonly used. Initially, these were inactivated vaccines prepared from a suspension of organs of experimentally infected dogs, the virus being inactivated by formaldehyde. To increase the effect, aluminum hydroxide gel was added to the inactivated antigen. Inactivated valkci induce only short-term immunity and require repeated inoculation, possibly using a live virulent virus (TAMOGLIA, TW: Inactivated canine diatomiper virus vaccine. J. Amer. Vet. Med. Assoc., 149, 1966, 5: 640 to 647). We have achieved the most in active immunization of vaccines containing live attenuated distemper virus. Attenuation was achieved by passage of flood virulent stones on ferrets (Green strain) or smoking embryos (Onder-stepoort strains, Lederle, Wiscomsim, etc.), which hurt late adapted to cell cultures from 1 ' of smokers emibryí. These vaccines are harmless, effective, relatively easy to prepare and well stored in a lyophilized state (CROGHAN, D.L .: Attenuated live-virus canine distemper ivaccines).

J. Amer. Vet. Med. Assoc., 149, 1966, 5: 622-665).

In the literature, the following authors have described the adaptation and cultivation of an attenuated distemper virus in cell cultures from smoking embryos (KARZON, DT - BUSSEL, RW: Cytopathic effect of canine distemp- er virus tissue culture. Science, 130, 1959, 1). 708-1709; JIRAN, E .: Cultivation of distemper virus on tissue cultures, Vet. Med., Prague, 10, 1965, 2: 87—92; PRYDIE, J .: Stu-dieis with an attenuated cante dite-mper vaccine -derived from -chick embryo cures.Res.Vet. S-ci., 9, 1968,: 443-452; GÓRSKA, C .: Namnaženia wirusa nasówki w hodowli komonek, Med. Wet., 24, 1968, 9: 565-569).

The procedures cited by the authors are suitable for the preparation of laboratory vaccine samples, as they require too many surgeries. For example, in infection! Washing, a-dsorption of the β-virus for a period of 1 to 2 hours, removing infectious taoculum is required to cultivate the virus suspensions with a relatively low amount of infectious virus.

However, large-scale technology requires at least a number of operations with high yields. This can be achieved by applying shorter cultivation methods using smoking embryos derived from specified pathogen-free (SPF) poultry farms, with the vaccines prepared having the following parameters:

1. In accordance with defined culture conditions, virus growth in dc cell cultures is carried out by characteristic cytopathic changes that allow quantitative determination of the virus content.

2. The virus content of the freeze-dried vaccine must not be less than IO ³ · ⁰ TKID 50. ml ^-1 .

3. The vaccine is harmless to all ages of dogs and carnivorous fur animals, whilst chicks without maternally derived antibodies can be successfully immunized from the age of six weeks.

4. The vaccine virus shall not be excluded from the organism of vaccinated animals and shall not be transmitted to co-housed susceptible animals.

5. Inoculum of susceptible animals induces partial protection against disease from day 4 to 5 and complete protection against cichorenl and infection at 2 to 3 weeks after vaccination.

6. Immunity induced by a single vaccination lasts for at least one year and is accompanied by the presence of serum antibodies.

-7. The offspring of immunized mothers are passive protected by maternal antibodies and for a period during which they may be actively immunized.

8. The vaccine virus also sufficiently immunizes in combination with other viral, possibly bacterial or parasitic antigens, thus permitting the preparation of multi-dose combined live and taactivated vaccines.

This task was solved as follows:

A live, distilled vaccine p-roti distemper, lyophilized, containing at least 10 ml of at least 10 ³ TKID 50 particles of distemper virus, strain CDV-F-BN 10/83 C-APM V-289 was prepared.

Said viral strain was adapted to primary or secondary cell cultures prepared from smoking germs. As growth medium, the Parker medium used was enriched with bovine serum, in which the virus was propagated at 36-38 ° C for 24-48 hours, i.e. until a monolayer was formed to form a monolayer. the curing surface. Thereafter, the growth medium was exchanged for the maintenance medium, according to Parker, in which the vaccine virus was produced at 36-38 ° C for 5-9 days. After making clear cytopathic changes, the viral material was mixed with the lyophilization medium to contain at least 10 ⁴ TKID 50. ml “ ¹ .

Vaccine strain of distemper virus designated CDV-F-BN-10/83, listed in the Czechoslovak Collection of Keys-Micro-Organisms at the Institute of Veterinary Medicine in Brno, Hudcova 60 under the designation CAPM V-289 was bred from distemper virus, strain Onderstep-oort, attenuated by serial-passages -of chorioallantoic membranes of smoking germs (HAIG, 1956, -cit. ŽUFFA, A. - ŠKODA, R .: Preparation of avianized vaccine against distemper. Final report, Bio-veto, np, Nitra, 1959, p. 38), by adapting it to cell-cultures prepared from non-smoking embryos derived from specified pathogen-free pathogen-free (SPF) farms, followed by selection of a line characterized by high immunization efficacy and very good growth properties on said tissue type with the development of significant cytopathic changes accompanying viral m-foot.

The vaccine production method is based on the fact that a distemper vaccine strain harmless to all ages of dogs and carnivorous fur animals when applied on the reverse side is assisted in primary or secondary cultures of smokers of embryonate cells. mounted on glass, plastic, cultured in a stationary manner or in rolled bottles, or on microcarriers.

The non-smoking embryonic cell cultures are prepared exclusively from hatching egg embryos derived from a cure-free cure of specified pathogenic germs. Appropriate nutrient media, in particular those containing a defined amount of amino acids and vitamins enriched with a suitable culture serum, are used to cultivate the cells used to propagate the vaccine virus.

Cell cultures are infected with canine distemper vaccine strain at a multiplicity of infection of at least 0.005. They are infected for 24 to 48 hours. cells grown on the culture surface by replacing the paste medium with the maintenance medium to which an infectious inoculum is added.

The maintenance medium is of the same composition as the growth medium but does not contain serum and has a adjusted sodium bicarbonate content to 0.2 wt. %. Alternatively, the virus-cell mixture is allowed to incubate for 60 to 120 min. at 37 ° C. Thereafter, the cell suspension is diluted in growth medium to contain 400,000 to 800,000 cells per ml. Alternatively, infectious inoculum can be added to the cell suspension in the growth medium and poured into the culture vessel without prior incubation. In 24 to 48 hours the growth medium is exchanged, without rinsing the monolayer cells, for maintenance and incubation is continued under the same conditions.

Upon detection of the first cytopathic changes (usually in 72 to 96 hours), the medium is exchanged at 24 hour intervals into the ilyophilization medium and stored frozen until lyophilization, or the kittivation is allowed to continue until more than 50% of monolayer cells it is affected by virus-specific changes and the virus suspension is collected only once.

The homogenized virus material, with the required content of infectious units, free of contaminating microorganisms, is filled into ampoules or vials, lyophilized, sealed and stored at refrigerated temperature.

The following examples are not intended to limit or exhaust:

Claims (7)

1. Method of cultivation: Cell suspension prepared using proteolytic ferments from day germ-breeding germs derived exclusively from domestic pathogen-free pathogen-free (SPF) rearing in a growth medium containing a defined amount of 5 amino acids and vitamins. The sera intended for tissue cultures are cultured on glass, plastic, in a stationary manner or in roller bottles, optionally in suspension on microcarriers. 2. Method of iufection of cell cultures by vaccine virus: Vaccine virus inoculum in which the virus is stabilized by the addition of 10 wt. % dimethylsuilfoxide; it is kept frozen. The use of said stabilizer allows the virus inoculum to be stored in the frozen state even at temperatures above -70 ° C. The concentration of dimethylsulfoxide after mixing the inoculum with the paste medium is not toxic to cell cultures and therefore does not need to be removed. Cell cultures prepared and cultured in accordance with point 1 shall be infected with the vaccine virus at a multiplicity of infection of at least 0,605 in the following ways: a) Growth monolayer infection: Growth medium is removed from the monolayer of cells after 24-hour growth, and the cell culture is infected with inoculum added to the maintenance medium to replace the growth medium. IBJ cell suspension in growth medium at a density ^of about 16. ml ^-1 cells are mixed with the vaccine virus inoculum, and the mixture is incubated at 37 ° C for 30 min. After the adsorption is complete, the infected cell suspension is diluted in growth medium to contain 400,000 cells per ml and cultured at 37 ^and C for 24 hours, i.e. until complete monolayer cells are formed on the culture surface. This is followed by replacement of the growth medium with the maintenance medium. cj Vaccine virus inoculum is added to the cell suspension in growth medium at a density of 4100 600 per 1 ml. The suspension is immediately poured into a culture vessel and incubated at 37 ° C for 24 h. j. up to the formation of monolayer cells on the culture surface. This is followed by replacement of the growth medium with the maintenance medium. 3. Collection of vaccine virus: The vaccine virus is produced in a maintenance medium that is the same as its basic composition. maintenance, except that it does not contain serum and the sodium bicarbonate concentration is up to 0.30 wt. %. Virus from cell cultures grown according to point 1 and infected according to point 2 are obtained by the following methods: (a) The maintenance medium is left unchanged on the cultures until extensive cytopathic changes are determined microscopically. A suitable lyophilization medium is added to the virus maintenance medium in a proportion of 2 parts of the virus suspension and 1 part of the ilyophilization medium. The mixture is further stored frozen in culture containers until freeze-dried. b) After the appearance of the first symptoms of specific cytopathic changes, the maintenance medium is decanted and the same amount of maintenance medium is poured into the culture vessels. The procedure is repeated at 24 h. intervals until a large cytbpatic effect is produced throughout the monolayer cells, wherein the final treatment is carried out according to a). The lyophilization medium is immediately added to the viral suspension obtained by repeated changes of the maintenance medium and kept frozen. Homogenised viral material with the required content of infectious pathogens (at least 10 ⁴ TKID 50.ml ^-1 ), free from icontaminating microorganisms, is filled into ampoules or vials, lyophilized, airtight and refrigerated. The vaccine prepared according to the above procedure comprises at least ID ^1.0 TKID 50 ^/ ml vaccine virus. During this procedure, it is also possible to successfully cultivate many vaccine strains of distemper virus adapted to the cell cultures of the smoking embryos. 4. Preparation of the combination vaccine: Lyophilization medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other protein source and gelatin or dextran (BJOVARNICK, MR - MILLER, JC - SNYDER, JC: The effects of certain salts, aminoacids, sugars and proteins on the stability of rickettsiae.]. Bacteriol., 59, 1950,: 509-552) and is suitable for lyophilization of other types of vaccine viruses. Therefore, if an infectious suspension containing another vaccine virus type, e.g. strains of parvoviruses of dogs or mink, these suspensions can be mixed with each other in any radius and lyophilized. 1 'R E D' E T A live attenuated canine freeze-dried vaccine, characterized in that it contains in 1 ml at least the HP TKJ? M distemper virus strain, strain CDV-F-BN 10/8 1 CAPM V-289 adapted to cell cultures. 2. The live attenuated canine vaccine of claim 1 comprising a lyophilization medium in an amount of? 5 to 50% by weight. % based on the total weight of the vaccine. 3. A method of producing a podium 1 and 2 podium vaccine, wherein the attenuated strain of distemper virus adapted to cell cultures is propagated on primary or secondary cell cultures prepared from carrot smokers in a growth medium of the park enriched with bovine serum at mating from 36 to 38 ^? C for 24 to 48 hours until a monolayer is formed on the covalent surface, and then the vaccine virus is produced in Parker stage holding medium and at a temperature of 38 to 38 ° C for 5 to 9 days, until cytopathic changes are made, and then the virus the material is mixed with the lyophilization medium so as to contain at least ¹⁰ % TKfD 50. ml “ ¹ . 4. A method according to claim 3, wherein the virus inoculum is intended to infect cell cultures? is stabilized by the addition of 10 to 20 wt. % Climethylsulf oxide. 5. A method for producing a vaccine according to steps 3 and 4, characterized in that the infection of the cells EZU for virus addition is done by removing the medium from the lumped monolayer cells and adding the virus inoculum to the maintenance medium, mixing the cell suspension with a density of up to ^1% in the growth medium with the virus inoculum, incubating ⁿ 33 to 38 C for 60 to 120 min., then 10 to 2'0-fold diluted with growth medium c is cultured at the same temperature until the monolayer vytvoreni.a kulfivačnom surface, · the cell suspension in growth medium at a density of 400 000 up to 800,000 cells per mt of virus inoculum is added and introduced into a culture vessel under the indicated conditions. 6. A method of producing a vaccine according to any one of Claims 1 to 5, wherein the vaccine preparation virus is produced in the maintenance medium until a wide variety of cytooalic changes are made and then one-off, or ρο the first signs of specific changes appear. at one-day intervals until complete disruption of the monolayer cells. 7. A method according to claim 1, characterized in that a lyophilic mode comprising sucrose, phosphate buffer, sodium or potassium glulamate, bovine albumin or other protein source and gelatin or dsxtran is added to the viral suspeirdi immediately after it has been obtained. and the mixture is kept frozen until Jyophilization. Soverography, n. p., plant 7, Most Price 2,40 Kčs