CS253709B2 - Process for preparing inactive vaccine against "aujezsky" illness - Google Patents

Abstract

A vaccine against Aujeszky disease is produced by inoculating a cell culture, preferably of primary chicken embryo cells or primary calf testicle monolayer cells, with a virulent strain of the virus deposited in the Hungarian Institute of Public Health under the number 00236/82. The virus used to inoculate the cell culture is preferably obtained from a suspension of the brain of a rabbit that contracted the said strain of Aujeszky disease. The virus propagated in the cell culture is then inactivated, preferably with ethylene imine, optionally obtained by cyclising bromoethyl amine immediately prior to use, and then mixed with an adjuvant, which is advantageously an oily adjuvant e.g. Phylaxia incompletely oily immunoadjuvant. The vaccine is effective to protect livestock and domestic animals from Aujeszky disease.

Description

The present invention relates to a process for the production of an inactivated vaccine comprising an additive intended for occlusion against Aujeszkyna disease.

Aujeszky's disease, which got its name after the first person who described it, Hungarian Aladár Auueszka (Zbl. Bakt. Orig. 1903 I, 32, pp. 353 to 357) has long been a known disease of domestic animals causing severe losses. Above all, pigs, especially young animals, are attacked.

With the possessing age of selitium, the impotent killing of death is less of a fortune, diseases of the respiratory organs, the central nervous system, and the sacrificial loss of animals in animals. Virus-infected animals can kill the pathogens for a long time (1-2 years) and release the viruses under the influence of etrlepes (for example, at birth), thus spreading the disease (J. Am. Vet. Med. Ass. pp. 998-1000, 1345-1347, 1980).

Infected sows often lose the embryo by preterm labor (Acta Vet. Acad. Sci. Hung. £ 2, pp. 17-23, 1962). Infected pigs also have a significant role in the transmission of Aujeszky's disease to ruminants (cattle and sheep) (Acta. Vet. Acad. Sci. Hung. 12, 53-58, 1962).

The method of combating the risk of disease is based on the need to protect epecific antibodies of the disease most frequently attacked, suckling piglets, in the first weeks of their lives.

In Hungary, a production buffer for preventive Urea Serum Serum (AAta Vet. Acad. Sci. Hung. 7: 423-427, 1957) has been developed to counteract this disease. Uyperia-derived swine serum decreases losses when administered at the right time, but the drawbacks are significant: serum production is very expensive and in addition to being eliminated from the body in a short time (within 10 to 14 days). At high prices, sera does not command use in young animals and buoyant animals in consideration.

The next step to defend against AujmszkyUn disease is to introduce active immunization. Viral strains are attenuated in various ways and to varying degrees and used to produce the vaccine. In Hungary, a live vaccine, which contains a living virus with a weakened virus, has been circumferentially available since 1961.

It is used for active scenting of cattle, hogs and dogs (MAL. 16, 42-45, 1961). Disease-induced Unspenders' losses have decreased following the introduction of the vaccination program (Cah. Med. Vet. £ 3, pp. 240-247, 1974), but there are several drawbacks to the live virus vaccine:

1) Po ^ шигас! In pregnant sows, cbsaU provides colostrum and milk antibodies for 4-6 weeks of passive piglets. At this time, it is not possible to carry out a live viral vaccine, since even a small amount of anti-viral substances is sufficient to prevent the aid of the virus to the extent necessary for the formation of active immunity. -you.

2) For these reasons, there may be animals in the herd in which the vaccine has been of no use, and in the infected state, these insufficiently immune individuals will present the persistence of the viral virus.

3) The use of a live vaccine is prohibited in many soils and in many cases this prohibition extends to the importation of live vaccine meat and animals. This limits the export ability of a country where a living vaccine is immunized.

4) Despite the fact that in Hungary, elaborated, attenuated vaccines have proved to be harmless and genetically stable, foreign publications in cases where viral strains of the breast vaccine have suddenly regained their virulence (TUe Vet. BuH. 43. p. 43). .

pp. 465-480, 1973; Dtsch. Tierarzt. Wschr. 85, pp. 381-420), which led to a violent response to vaccination of vaccinated animals.

5) In Hungary, the used and vaccinated vaccine is not completely free of danger to the pigs and mink, it does not confer immunity to cats.

Inactivated vaccine production trials have been carried out precisely because of the problems associated with living vaccine. Formalin, ultraviolet light irradiation, beta-prupiulatin, caponin, crystal violet, and ethanol were used as inactivators as an aluminum hydroxide gel adjuvant (Acta Microbiol. Hung., 1951, 151-160; Arch. Exp. Vet). Med 21: 501-507 (1967).

However, the vaccine produced in this way was not sufficiently effective. Modern methods used to inactivate ethylethylenimine as an additive of D4A-ldetane (1973), DEAE, but dextran causes allergic reactions.

It is an object of the present invention to provide an inactivated vaccine for Aujeszky's oocyte,

(1) which is as effective or more effective than the attenuated Aujeszky vaccine,

(2) it would be harmless to an animal infected with Aujezsky's disease, irrespective of its species and age, and would provide adequate immunity,

3) would be suitable for i ^ m ^ r ^ n ^ ^ c ^ i piglets protected by ^ Ιρ ^^ Ιο! imurity.

The principle of the method of producing a vaccine of Aueczkyhu cannot be grown by Aujeszky nemmoi virus strain on cell culture, harvesting and inactivation of the virus and mixing and homogeneizing the virus-infected virus with the olefinic lynx according to the invention, characterized in that it is diseases stored in midirském Provincial Institute of health (Országos Kozegészségůgyi Intézet) under Cups in 00237/82 ικ ^^ νί least 10 ⁷ TKID _5Q / ml.

According to the invention, the Aujeszky Phhlaxia strain deposited from the various Aujeszky strains on the basis of their immunizing ability and selected under the number 00236/82 in OKI is inoculated into a primary fibroMasso cell culture of the chicken embryo and propagated. The virus-thickening liquid obtained is injected with a known inhibitor, preferably ethyleneimine, and then mixed with a known adjuvant, preferably with an incomplete oleic adjuvant.

The fact that the vaccine produced by the method of the invention is immunizable results from several facts. From a domestic epidemic affecting a virulent Phylixii strain, it is caused by the following biological olstices: it can successfully migrate both in tic cell cultures as well as in Hossitess animals, has a high infectious titer, and has good immunization ability.

A gentle process also contributes to the efficacy of the efficacy, during which the parameters (temperature, concentration of the composition, time) of the protective antigens are maintained. The inactivating agent is in the form of a dilute aqueous solution before use. The immunity of the gentle antigen is further enhanced by the incomplete Phhyaxii solution.

The vaccine prepared in the following examples is used for pigs, cattle and dogs, with immunization preferably being repeated.

The invention is illustrated by the following examples.

Example 1

Preparation of vaccine virus

Rabbits of 2 to 2.5 kg hmoonooti are infected intracerebrally with a strain of Auueszky's Phylaxia virus deposited at the Hungarian Regional Institute of Health under number 00236/82, isolated in an acute epidemic. Characteristic symptoms of Aujeszky's disease are observed in animals, death occurs after 72 to 96 hours. Under sterile conditions, the brain is removed and a 10% suspension is prepared therefrom using a sodium chloride solution. It is used for mass propagation of the virus.

Example 2

Production of virus suspension

According to Example 1, the virulent vaccine virus obtained is propagated to a primary on a chicken embryo fibroblast monolayer cell culture or a similar monolayer cell culture in a 2000 ml Roux flask (or in a rotating cradle flask).

Hanks' solution or MEM-H solution is used as the cell culture growth solution, Earl's solution or MEM-E solution is used to maintain and multiply the virus. The composition of these solutions is given in the following references: J. Nat. Cancer Inst. 31, 1974; Why. Soc. exp. . Biol. Copper. 21, 196 (1949); Science 139: 1959; Bakacs, Farkas: Orvosi virologia, -eddcina, Bnldaess).

Hanks' solution or MEM-H solution used to grow monolayer cell culture is enriched in 10% calf serum, whereas the solution used for virus multiplication does not contain any calf serum. After formation of the monolayer cell culture, the growth solution is decanted.

Pour the virus containing the maintenance solution onto the culture. The cultures are incubated at 37 ° C until complete disruption of the cells (after about 48 to 72 hours). The resulting virus suspension is then suitable for the manufacture of a vaccine čnaktivované if sterile and infectious titer measured in cult uněčné ^b ^ e is aksjorn ^10? TCCDDg / ml.

Example 3

Inactivation and formulation

The virus suspension obtained by the method of Example 2 is inactivated for 6 hours at 37 [deg.] C. with an ethyleneimine solution having a concentration of 200 [mu] g / ml. The solution is prepared immediately before use by cyclization of bromocithylamine. The ethyleneimine is then quenched with sodium sulfate and the pH of the activated suspension is adjusted to 7.2-7.4.

To check whether the suspension is truly inactive, the cell culture seeded as described is inoculated with the activated suspension and further inoculated with rabbits of 2 to 2.5 kg and 25 to 3.0 kg of sheep.

The viral material is then mixed with an equal volume of Phylaxia oil adjuvant. (Phylaxia oil adjuvant is new and consists of 73% -arcol 52 oil and 27% surfactant, which is a mixture of known TREATMENTS 80, TREATMENT 20 and TWEEN 60. The term "-acol 52" is used in all immunological literature and in the production of a vaccine for oil produced by Esso.)

The aqueous virus suspension and the oil adjuvant are processed in a high-pressure emulsifier to a stable suspension that does not disintegrate in the temperature range of 0 to +45 ° C. The finished vaccine is filled under sterile conditions into an ampoule and the vials are then sealed airtight.

ml were inoculated intramuscularly in these trials, three weeks and eight weeks, and sows for three months, other animals for a period of three months.

EXAMPLE 4

Vaccination test

The vaccines obtained as described above are dosed in rabbits, sheep, dogs, piglets, at the age of 2 in the last third of the pregnancy. The vepers will flourish after this time with no vaccine response.

six weeks. IN

Example

Immunization of piglets.

Piglets aged two days, three weeks and eight. The vaccines were vaccinated weekly for each ml of vaccine. After three weeks, the vaccination is repeated. Treated animals vunrkají speeífické roltil ^{p n} t ^{ky, k é} ter ^ CHR ^ For ^instance piglets e ^di ntranatální jLntetoí távtou ² x ¹⁰ ^ tiida virnSsntníhl the gone-kyho virus konnrolní the untreated piglets at the same dose die.

He said a 6

Generation of colostral immunity • In the last third of pregnancy, breeding sows are treated with the vaccine of Example 3 used in a quantity of 2 ml. The treatment is repeated after three weeks, but the treatment time is chosen such that the second vaccination is two weeks before the expected date of litter of piglets.

Specific defenses are produced in the colts of treated sows and can therefore be measured. on the seventh day in suckling piglets, the mean non-salivation titer 1:16. The viruses neeuralizační t r ^p ^ oc ^h oskytuje wound ^about and ^P ro VZI-ru ^ n ^ í virus infection ^can VCE ^ ² * K ^ ⁶ TCID.

animals in

Coonrous inoculations on days 30, 60 and 90 (when the content of preservatives in the coiostre decreases) of the group to a moortait of 18, 26 and 40%, while all piglets ossuned in the milk containing the special preservatives protect the youngest for the most acute disease.

leads to treated sows die. when they are

Example

Vaccination of calves during the period of colostral immunity is vaccinated with the substance

Piglets of immunized sows of Example 6 having a colostral immune system

On the 7th and 28th day, according to the method of Example 3, always 2 ml of the vaccine. SsPsiiiiCé protective forms in high titer (1:44). At the age of 60 days, the pigs are infected intaanatnlně skin can vi ^ ^ j ^ ue ^ r ^{^,} ^ nm vieem E ^and VCE ² x TCID ^ 10®.

The test animals do not show any symptoms, whereas the control animals from unimmunized sows and unvaccinated piglets die 100%. Thus, it is also possible to successfully immunize seeded piglets having colostral ΟυΓΐΟ by inactivated vaccine.

Example 8

Frequency of vaccination

Prepared according příkaadu 6 during its březoosi twice vaccinated breeding sows when new březoosi again očkuuí 2 mi vaccine (repeated očkooáinní. This in koiostru treated breeding sows titre preservatives again strongly increased and jejceh seiata are then protected by ^p rooi tornnroiní ^ fekci ² x 10® TCID ^.

In contrast, piglets show higher mortality during the second pregnancy of unvaccinated breeding sows (35% at 6 weeks of age). This shows the importance of the need for repeated vaccination to achieve sufficient colostral protection in breeding sows during their pregnancy.

Example 9

Immunization of sheep

Sheep at 3 and 6 months of age are immunized with 2 ml of the vaccine of Example 3. The animals are infected with the virulent Aujeszky's disease virus at a dose of 1000 ΤΚΙϋ dní days after vaccination. All animals from the unvaccinated control group die while treated sheep show no signs of disease.

Example 10

Dogs of one month and six months of age were treated with the vaccine of Example 3 in an amount of 2 ml. The treatment is repeated after three weeks. Dog treatment 15 days after vaccination and the untreated control group are infected with the virulent virus. The titre of preservatives in the blood of the immunized dogs is 1:32 and the animals do not get sick, while the control group perishes with the characteristic signs of the disease.

Claims (1)

OBJECT OF THE INVENTION A method of producing an inactivated Aujeszky's disease vaccine by growing a strain of Aujeszky's disease virus on cell culture, harvesting and inactivating the virus, and mixing and homogenizing the inactivated virus with an oil adjuvant, characterized in that the virus strain is a virulent, pathogenic Aujeses virus strain Phylaxia OKI No '. 00236/82 in quantities of at least ΙΟ ⁷ ΤΚΙϋ ^ θ / πιΙ.