CS231514B1 - Inactivated oil vaccine against Aujeszky's disease and its production - Google Patents

Abstract

The inactivated oil vaccine against Aujeszky's disease is intended for the specific prophylaxis of Aujeszky's disease in susceptible species of livestock and farm animals. It is prepared from attenuated strains of the Aujeszky's disease virus, which are harmless to pigs of all age categories upon arbitrary application, with complete or partial pathogenicity for rabbits preserved. The virus suspension is prepared by propagation in cell cultures and subsequently inactivated by formaldehyde and saponin. It is emulsified with a mixture of adjuvant oils so that a double water-in-oil-in-water emulsion is formed.

Description

Inactivated oil vaccine against Aujeszky's disease is intended for specific prophylaxis of Aujeszky's disease in susceptible species of farm and commercial animals. It is prepared from attenuated strains of Aujeszky's disease virus, which are harmless to pigs of all ages when administered at will, while retaining full or partial pathogenicity for rabbits. The virus suspension is prepared by multiplication in cell cultures and subsequently inactivated by the action of formaldehyde and saponin. It is emulsified with a mixture of adjuvant oils to form a double emulsion water in oil in water.

The invention relates to an inactivated oil vaccine against Aujeszky's disease in susceptible species of farm and commercial animals and a method of its production.

Aujeszky's disease virus causes an infectious disease in several species of farm and commercial animals, i.e. pigs, cattle, sheep, dogs, cats and fur animals, which are characterized by high morbidity and mortality and cause high national economic losses. Protective vaccination against Aujeszky's disease has so far been carried out mainly using live vaccines, produced from attenuated or avirulent strains [Zuffa, A., Grunert, Z., 1974: Les vaccines utilisés en Tchecoslovaquie contre la maladie D'Aujeszky., Cah. Méd. Vét. 43, 334 — 337; Bártha, A., 1961: Kísérletek az Aujeszky-féle virus virulenciájának szelídítésére., Mágy. állatorv. Lapja 16 (2), 42 — 45], Live vaccines from avirulent strains are harmless and their application in an appropriate dose induces protection against clinical disease in vaccinated animals upon their contact with the infection [Žuffa, O., 1976: The significance of specific prophylaxis for control of Aujeszky’s disease., Proc. 4-th Int. Pig Vet. Soc. Congr. Ames USA, G 10 (En]; Szabó, A., Grunert,

Z., 1971: Persistence of virulent Pseudorabies Virus in Herdis of Vaccinated and Nonvaccinated Pigs., Acta Virologica, 15, 87 to 94], Objections are raised against the use of live vaccines, on the grounds that they introduce infectious agents into the pig herd and population, which can persist in vaccinated animals and mediate the transmission of infection when vaccinated animals are moved to healthy herds [Škoda, R., 1976: The control of Aujeszky's disease in large swine units by use of attenuated live vaccines. Proc. 4-th Inst. Pig Vet. Soc. Congr., Ames. USA, G (6), Baskerville, A., Ferran, J. B., Dow, C., 1973: Aujeszky's disease in pigs., Vet. Bull. 43, (9), 465 — 480]. These objections to the use of live vaccines became justified especially after it was proven that the Aujeszky's disease virus, like other herpes viruses of humans and animals, can induce a persistent latent infection after a primary infection accompanied by clinical disease or proceeding inapparently [Sabó, A., Rajčáni, J. 1976: Latent pseudorabies virus infection in pigs., Acta virologica, 20, 208 — 214],

The use of live vaccines is therefore indicated in areas enzootically infected with Aujeszky's virus, while in areas and countries with sporadic occurrence of Aujeszky's disease, the use of inactivated vaccines is recommended.

An effective inactivated vaccine against Aujeszky's disease will be of particular importance and application in the protective vaccination of cattle, sheep and carnivores. These animal species are very susceptible to infection with Aujeszky's virus, and the disease is fatal in all cases. Live vaccines, harmless to the lowest age categories of the above-mentioned animal species, must therefore be produced from avirulent strains with limited ability to reproduce in the body of the vaccinated. However, this means that only a small amount of the inoculated vaccine virus is used during antigenic stimulation, which is manifested by a relatively weak immune response. With an inactivated vaccine, a relatively large amount of viral material can be concentrated into the application dose, the antigenicity of which can be further potentiated by binding to a suitable adjuvant.

Inactivated vaccines, produced from the organs of experimentally infected animals, have proven ineffective. The first attempts to prepare an effective inactivated vaccine against Aujeszky's disease from the virus grown in cell cultures and inactivated with formaldehyde (Koch, A., Abraham, E., 1952: Immunisationsversuche mit dem Aujeszkyschen Virus., Acta vet. Hung. 11/4/, 270 — 279; Žuffa, A., 1963: Versuch zur Immunisierung von Laboratoriumstiere und Schweinen mittels Formaldehyde- und UV/ /Strahlen inaktieviertem Aujeszky-Virus., Arch. f. Exptl. Vet.-med. 17/3/, 593 — 613], or ultraviolet rays [previously cited Žuffa, A., Beládi, I., Ivánovics, D., 1954: Immunisierung von Laboratoriumstiere mit dem Virus der Aujeszkyschen Krankheit nach Dessen Inaktivierung mit Ultraviolettstrahlen, Acta Microbiol. Acad. Sci. Hung. 2(1-2), 151 — 160], using aluminum hydroxide gel as an adjuvant, proved unsuccessful. Nor did the disruption of the infectivity of Aujeszky virus grown in cell cultures with nitrous acid lead to the preparation of an effective inactivated vaccine. In vaccines prepared from viruses inactivated with saponin [Barbinschl, C., Papadopol, M., 1966: Cercetari privind imuzares in boala lui Aujeszky cu vacciu preparát din virus des cultura absorbit si saponinatat. Lucr. Inst. Pasteur III (1-1964), 71 — 84], or crystal violet [Nedjalkov, S. and spol,, 1968: Versuche zur Herstellung von Vakzine gegen Aujeszkysche Krankheit sus inaktiviertem Virus.

1. Mitt. Arch. exp. Vet. Med. 22, 499 — 507], the presence of an infectious virus cannot be ruled out [previously cited by Baskerville et al. 1973], The vaccine prepared by Haralambiev et al. proved to be effective. [Haralambiev, H„ Mermerski, K., Simeonov, S„ 1967: Untersuchungen uber die Immunogenitat der mit Eethanol inaktivierten Vakzine gegen die Aujeszkysche Krankheit., Arch. exp. Vet. Med., 21(2), 501 — 507] from tissue virus inactivated by ethanol with the addition of paraffin oil as an adjuvant. Since it is a simple mixture of water and oil, the vaccine emulsion separates into an aqueous and an oily phase after a short period of standing. Simeonov et al. [Si231514 meonov, S., Vasilev, V., Jotov, M., 1973: Sravnitelno izpitvanie na saponinovi vaksyny protiv bolesta na Aujeszky., Vet. Med., Nauki 10(2), 83 — 88] replaced the oil adjuvant saponinor. They compared the effectiveness of three inactivated vaccines prepared using formaldehyde, crystal violet and saponin. Borgen, H. C., Expensen, L., 1970: Immunizing effect of photoinactivated pseudorabies virus., Zhl. Bakt. I. Orig., 214(1), 13 — 16, presented in the above publication evidence that exposure of Aujeszky's virus to ultraviolet rays leads to the destruction of its infectivity without damaging the surface structures of virions and thus to the preservation of antigenicity. Since testing of the immunizing abilities of the virus inactivated by ultraviolet rays was done by applying large doses that were not practical for practical purposes, and since the technical difficulties of inactivation and the technological problems of processing the inactivated virus into a vaccine remained unresolved, this method of inactivation of Aujeszky's virus did not find practical application in the production of a vaccine against Aujeszky's disease. A convincing immune response was induced by a vaccine representing a virus grown on a cell culture of stable of the roach line BHK-21, inactivated by ethylethylenimine and bound to diethylaminoethyl dextran [Škoda, R., Wittmann, G., 1973: Die Immunisierung von Schweinen mit Vakzinen aus Inaktiviertem Aujeszkv-Viirus. Zbl. Vet. Med. B(2Q), 127 — 138]. A single administration of the vaccine to pigs in appropriate doses induced active antibody production and protection against infection with a defined amount of virulent virus. However, the degree of immunity after application of lower doses of the vaccine, or in cattle only after double vaccination, as required for the practical use of the vaccine, is not given.

De Lagneau et al. [DeLagneau, J. F. et al. 1975: Immunisation contre la maladie D‘Aujeszky a 1‘aide d‘un nouveau vaccin huileux a virus inactivé, Rec. Méd. Vet. 151(151), 561 — 575] used the classical method, that is formaldehyde, for inactivating Aujeszky’s virus, propagated on the 1B-RS-2 cell line and a lipoid adjuvant for stimulating the antigenicity of the virus. Double administration of the vaccine induced the formation of antibodies in laboratory animals and pigs in titers of 1: 16 to 1: 128 and protection against infection with a defined number of lethal doses. An effective inactivated vaccine against Aujeszky’s disease was prepared by Genov et al. by inactivating the virus with 1,3-propiolactone and then binding it to an adjuvant representing an aluminum hydroxide gel [Genov. I., Řezačka, A., Genová, A., 1967: Polučavane inactivirana s betapropiolactone vakcinina srešču bolestta na Auskie s štam Ruse]. Nedostátkom tejto vagíny je užívat inactiváčný prostried, ktcurý bol vyřaděný zo· zoznamu látkových pre přípravu biopreparátov, nakotko sa provála jeho mutagenicity [Pasrish, B. J., Searle, C. D., 1966: Carcinogenicity of beta-propiolactone and 4-nitro quinoline N-Oxide for the skin of Guinea pig., Brit. ]. Gancer 2.3, 200 — 205 j. Žuffa a spol. prepared an effective inactivated vaccine against Aujeszky's disease from a mixture of virulent and attenuated Aujeszky's disease virus inactivated by alcohol, bound to an oil adjuvant [Zuffa, A., Brányik, A., Salaj, J., Žuffová, ]., Menšík, ]., Štěpánek, J., 1980: Inactivated adjuvant vaccine against Aujeszky's disease and its method preparations. Copyright certificate no. 201 802],

The disadvantage of the inactivated vaccines against Aujeszky's disease proposed so far is that virulent strains of Aujeszky's disease virus are used for their production, either individually or in any ratio mixed with strains with partially reduced virulence. Another disadvantage is the low authigenic stability of the Aujeszky's disease virus inactivated with ethanol before emulsification with an oil adjuvant. This fact does not allow the simultaneous production of inactivated and live avirulent vaccines, due to the serious risk of laboratory contamination and, in addition, makes it impossible to cooperate in production with countries whose regulations do not allow the use of virulent virus strains in production conditions.

The above-mentioned shortcomings are solved by an inactivated oil vaccine against Aujeszky's disease of susceptible species of farm and commercial animals, containing in ml 10 ⁷ to 10 ⁹ TKID50 of the inactivated attenuated strain Buk-TK-900/IVa listed in the Czechoslovak Collection of Microorganisms at the Research Institute of Veterinary Medicine in Brno, Hudcova 60, under the designation CCM V-94, or 10' to 10 ⁹ TKID50 of the inactivated attenuated strain Buk-TK/650 listed in the Czechoslovak Collection of Microorganisms at the Research Institute of Veterinary Medicine in Brno, Hudcova 60, under the designation CAMP V-265, emulsified in an oil adjuvant and a method of its production from an attenuated strain of the Aujeszky's disease virus harmless to pigs of all age categories when applied topically, with preserved complete or partial pathogenicity for rabbits, with the advantage of using strains

Buk-TK-900/IVa, or Buk-TK/650, individually or in a mixture in a TubovoTny ratio, effective inactivation by formaldehyde and saponin while maintaining antigenicity and the ratio of inactivating substances to antigens of virus strains. The method of vaccine production is based on the fact that attenuated strains of Aujeszky's disease virus harmless to pigs of all age categories at any application, with preserved complete or partial pathogenicity for rabbits, are individually multiplied on primary cultures or subcultures of chicken or duck embryonic cells, grown on glass, in suspension or on microcarriers. For the multiplication of viruses, it is also possible to use susceptible stable cell lines with controlled biological properties. For the cultivation of cell cultures used for the propagation of attenuated strains of Aujeszky's virus, suitable nutrient media are used, especially those containing fragments of bovine milk albumin and the necessary amount of mammalian or avian serum [Portefield, JS, 1967: Animal Tissue Culture, in the collection edited by Maramorosh, K. and Koprowski, H., Acad. Press, New York]. The use of defined media containing substances affecting the metabolic activity of cells in layer or suspension cultures is not excluded. Cell cultures are infected with attenuated strains of Aujeszky's disease virus, at a multiplicity of infection of 0.1 to 1.0 TKID50 per cell. After 24 to 48 hours of incubation of the infected cultures at 37°C, the amplified virus is obtained in a volume of maintenance medium with an infectious titer of 10 ⁷ to 10 ⁹ TKID50 in 1 ml of suspension. The prepared virus suspension is freed from cell debris by filtration through gauze. Viral suspensions of attenuated strains of Aujeszky's disease virus with the specified infectious titer value are mixed individually or in a mixture in any ratio and subsequently inactivated by the action of formaldehyde in a final concentration of 0.05 to 0.20 weight percent at a temperature of 25 to 35 °C for 18 to 72 hours, by the action of saponin, which is also a component of the adjuvant, in a final concentration of 0.05 to 0.20 weight percent at a temperature of 25 to 35 °C for 18 to 72 hours, by the combined action of formaldehyde and saponin in one solution, or by subsequent addition of individual substances immediately one after the other, or until 72 hours have elapsed in the specified concentrations for 18 to 72 hours in the specified temperature range. After checking the inactivation of the virus, which is carried out by intramuscular application of the virus suspension to rabbits in doses of 2 to 10 ml, the antigens are emulsified with known oils in a defined ratio of water phase to oil. The mixture of adjuvant oils contains mineral oil with a viscosity of 20.10® to 120.10 ⁶ m ² .s ^-1 and emulsifiers in such a ratio as to form a double emulsion water in oil in water, the presence of which in the vaccine represents 13 to 52 weight percent, preferably 17.4 weight percent.

The given example does not limit or exhaust the subject matter of the invention in any way.

Example of execution

Primary cultures from chicken or duck embryos, their subcultures, or susceptible stable cell lines with controlled biological properties, grown on glass, in suspension or on microcarriers, are stripped of the growth medium consisting of Earle's solution with the addition of 7% by weight of calf serum and 0.3 to 0.5% by weight of lactalbumin hydrolysate. The cell cultures are rinsed 1 to 2 times with phosphate-buffered saline and infected with the attenuated strain Buk-TK-900/IVa or Buk-TK/650 of Aujeszky's disease virus, as specified in the description, at a multiplicity of infection of 0.1 to 1.0 TKID50 per cell. Infected cell cultures are poured with Earle's solution, enriched with 0.3 to 0.5 weight percent of lactalbumin hydrolysate and incubated for 24 to 48 hours at 37 °C. After specific degeneration of the cell culture, determined microscopically, the virus suspension is freed from cell debris by filtration through gauze into sterile neutral glass bottles. Formaldehyde is added to a final concentration of 0.2 weight percent and saponin to a final concentration of 0.1 weight percent in the virus suspension. Inactivation agents are added in a common solution, or by subsequent addition of individual substances immediately after each other. Inactivation takes place for 72 hours at a temperature of 30 °C. After checking the inactivation of the virus, which is carried out by intramuscular application of the inactivated virus suspension to rabbits in a dose of 5 ml, the inactivated virus suspension is mixed with 17.4 weight percent of the adjuvant oil mixture and the mixture is emulsified by homogenization in an appropriate device at room temperature.

The effectiveness of the inactivated oil vaccine against Aujeszky's disease, according to the invention, is confirmed by the results in the following table, which shows the titers of neutralizing antibodies in the blood serum of animals susceptible to infection with Aujeszky's disease virus depending on the amount of i. m. applied vaccine and the protection against the inducing infection defined by the amount of lethal doses.

NV-antibody titers in blood serum after two-time administration

--ml of vaccine

5

Table 1

Animal species

Protection against infection causing .. .LDjo in animals vaccinated with ... m,l vaccine

5

Carnivores , 4 — 16 8 — 32 N 10 ² 103 N Pigs 4 — 16 4 — 64 16 — 256 10 ³ IQ ⁴ 10 ⁴ Beef times- N 8 — 64 8 — 128 10 ⁴ IQ ⁶ 10® flow

SUBJECT

Claims (4)

g 19 231314 Table 1 Animal species NV-antibody titers in the bloodstream after two-stroke application - ml of vaccine 12 5 Protecting prod ucting infection LDo in animals vaccinated ... m vaccine 12 5 Carnivores 4-16 8 - 32 N 102 103 N Pigs 4 - 16 4 - 64 16 - 256 103 IQ4 104 Cattle N-N 8 - 64 8 - 128 104 IQ6 10® Flow SUBJECT An inactivated oil vaccine against Aujeszky's disease susceptible species of animal and commercial species, characterized in that 1 ml contains 107-109 TCID 50 of inactivated attenuated strains of Aujeszky's disease virus for pigs of all ages k-ate-gyries application, with complete or partial pathogenicity of the precursors, individually or in admixture in any ratio, emulsified in an oil adjuvant. 2. An inactivated oil vaccine according to claim 1, characterized in that it comprises an activated attenuated strain of Au-cyst virus virus Buk-TK-900 / IVa CCM V-94. 3. An inactivated oil vaccine according to claim 1, comprising an inactivated attenuated strain of Aujeszky's disease virus Buk-TK / 650 CAMP V-265. 4. A method of producing a vaccine as claimed in any one of claims 1 to 3, wherein the Aujeszky's disease virus-innocuous strain virus for all age categories in any application, which retains full or partial pathogenicity of precursors, is propagated to primary tumors, or subcultures of smokers or chimeric embryonic cells, or intrinsic stable cell lines with controlled biological properties, spread on glass, in suspension, or on micro-carriers, are mixed individually and subsequently inactivated by application formaldehyde in a final concentration of 0.05 to 0.20 weight percent at 25 to 35 ° C for 18 to 72 hours, by the effect of saponin at a final concentration of 0.05 to 0.20 weight per cent at at a temperature of 25 to 35 ° C for 18 to 72 hours or a combined effect of formaldehyde and saponin in one solution in said concentrations ch ratios priteplote 25-3 5 ° C for 18-72 hours anásledné is emulsified viruses suspenzias 13 to 52 weight percent zmesiadjuvantnýoh oils which contains oil and emulsifiers 1'ahkýminerálny.