CS201802B1 - Inactivated adjuvant vaccine against the aujeszky disease and method of preparation thereof - Google Patents

Description

The invention relates to an inactivated adjuvant vaccine against Aujeszky's disease of susceptible species of farm and utility animals and to a process for its preparation.

Aujeszky's disease virus causes contagious diseases in a wide variety of farm and utility animals, ie pigs, cattle, ovines, dogs, cats and fur animals, which are characterized by high morbidity and mortality and cause high economic losses. So far, protective vaccination against Aujeszky's disease has been carried out predominantly with live vaccinia, produced from the attenuated and / or avirulent strains / frog, A., Grunert, Z., 1974. Med. Ver. 4 ^ »334-337} Bártha, A., 1961» Kérérletek az Aujeszky-féle virus virulenoiájának szelídítésére., Magi. állatorv. lapja 16/2 /, 42-45 / · live vaccines from avirulent strains are harmless and their application at an appropriate dose induces protection against clinical disease in vaccinated animals upon contact with the infection / Žuffa, A ·, 1976 »The signifikanoe of spscifio prophylaxis for control of Aujeszky * s disease. 4-th lnt. Pig Vet. Sec. Congr., Ames USA, G 10 / En /} Szabo, A., Grunert, Z., 1971 «Persistence of Virulent Pssudorabies Virus in Herda of Vaooinatsd and Monvaoolnated Pigs ·, Acta Virologica, 15, 87-94 / · Against Use The live vacoins are challenged to bring infectious agents into the breeding and population of pigs that may be present in the vaccinated animals and mediate the transmission of infection when moving vaccinated animals to healthy animals / Skoda, R ·, 1976: The

201 802

201 802 control of Aujeszky's disease in large ewine unity by using attenuated live vaooines. Proo 4-th Iqt. Pig Vet. SOC. Congr., Ames USA, Q 6 /, Baskerville, A., Perran, J.B., Dow, C., 1973, "Aujeszky and disease in pigs," Vet. Bull. £ 2, (9), 465-480]. These objections to the use of live vaccines became justified only after that, and has shown that Aujeezky's disease virus, like other herpesviruses in humans and animals, possibly after a primary infection !, accompanied by clinical disease or ongoing inapparently, induced a persistent / latent infection. And, Bajaji, <J «1976: latent paeudorabies virus infection in pigs. Acta virologica, 20, 208-214].

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

Of particular importance and application will find an effective inactivated vaccine against Aujeszky's disease in the protective vaccination of cattle and sheep. These species are very susceptible to Aujeszky virus infection, and the disease is lethal in all cases. The live vaccines, harmless to the lowest age categories of the animal species mentioned, must therefore be produced from avirulent strains and limited reproductive capacity in the vaccine organism. This means, however, that only a small amount of inoculated vaccinia virus is used in antigenic assimilation, as shown by a relatively weak immune response. For an inactivated vaccine, relatively large amounts of viral material may be concentrated into the application dose, whose antigenicity may be potentiated by binding to suitable adjuvants.

Inactivated vacoins, produced from organs of experimentally infected animals, have been shown to be ineffective. Initial attempts to prepare an effective inactivated vacoin against Aujeazky virus from virus grown in cell culture and inactivated by foxmaldehyde / Koch, A., Abraham, £ 1952, Xmmunlaationsversuohe mit dem Aujeszkiachen Virus, Acta vat. Hung. 11 (4), 270-279. Suffa, A. 1963 »Verauch zur Xmmunialrung von Laboratoriumatere und Sohweinen mittela Pormaldehyde- und UV / Strahlen inaktieviertem Aujeazky-Virua ·, Aroh.f. Exptl. Vet.-med. 17/3 /, 593-613), or by ultraviolet rays (previously cited 2uffa, Béládi, 1 ·, Ivánovica, D ·, 1954). Acta Mlorobiol. Aoad. Sol. Hung. 2 (1-2), 151-160], and using an aluminum hydroxide gel as an adjuvant, anda proved unsuccessful. Nor was the excitability of the Aujeazky virus virus grown on cell cultures with nitric acid to produce an effective inactivated vacoin. In Vaccines Prepared from Viruses Inactivated by Aaponin / Barbinsohi, C., Papadopol, M., 1966 »Gereetari prime immunity in the boil of the Aujeszky virus in a preparation of a virus dea cultura adaorbit and aaponinate. Fluoro. Inat Pasteur 111 (1-1964), 71-84], or crystal violet / ledjelkov, S. et al., 1968 »Verauohe zur Heratellung von Vakzine gagen Aujeszkyeohe Krankheit aua inaktiviertem Virus. 1. Mitt. Arch. exp. Vet. Med. 22, 499-507), the presence of an infectious virus (cited previously by Baskerville et al.) Cannot be excluded. 1973 /. A more interactive aa has been shown by a Haralambiev-prepared vacoin and apol. (Haralambiev, H., Mermeraki, K., Slmeonov, S., 1967.) «Unequalization according to the Amino Acid inactivlerton Vaccine gene according to Aujeezkysche Krankheit., Aroh. exp. Vet Med. 21/2 /, 501-507 /

201 802 from ethanol-inactivated tissue virus with the addition of paraffin oil as an adjuvant · Because of the simple mixing of water and oil, the vaccine emulsion is separated into a water and oil phase after a short period of time. Simeonov et al. (Simeonov, S., Vaeilev, V., Jotov, M, 1973). "Sravnitelno izpitvanie na saponinovi vakoviny protiv boleetta na Aujeszki." Med. Naukl 10 (2), 83-88] replaced the oil adjuvant with saponin. They compared the efficacy of three inactivated vaccines prepared with formaldehyde, crystal violet and saponin. Borgen, H.C., Expensen, L., 1970 »Immunized Effect of Photoinaotivated Pseudorabies Virus., Zbl. Bektas. I. Orig., 214 (1), 13-16, have shown in that publication that exposure of Aujeszky virus to ultraviolet rays leads to disruption of its infectivity without disrupting the surface structures of the virions and hence healing of the antigen. While testing of the immunizing capabilities of the ultraviolet ray inactivated virus was accomplished by applying large, unusable doses for practical purposes, and since the technical difficulty of inactivation and the technological problems of inactivating virus to vaccinin remained unresolved, this method of inactivating Aujeszky's virus was not found. A proven immune response has been induced by a vaccine representing a virus not grown on a stable hamster cell line BHK-21, inactivated by ethylethylenimine and bound to diethylaminoethyl dextran / Skoda, R., Wittmann, 6, 1973. Zbl. Vet. Med. B 20, 127-138]. Single administration of the vaccine to pigs at appropriate doses induced active antibody production and protection against infection! a defined amount of virulent virus. However, the degree of immunity after administration of lower doses of the vaccine, respectively, is not reported. in cattle only after double vaccination, as required for the practical use of the vaccine.

DeLagneau et al. / De Lagneau, J.E. »and apol. 1975: Immunization contre la maladie D * Aujeszky and l * aide dun nouveau vaccin huileux and virus inactive, Reo. Med. Vet. 151 (10), 561-575] used a classical method, i.e. formaldehyde, to stimulate the antigenicity of the virus and to stimulate viral antigenicity by lipoid adjuvant to inactivate Aujeszky virus propagated on the cell line IB-RS-2. Two-fold administration of vaccine induced antibody production in titers of 1 »16 to 1» 128 and protection against infection in laboratory animals and pigs! defined amount of lethal doses. Effective inactivated vaxcin against Aujeszky's disease was prepared by Genov et al. so that the virus inactivated 1,3-propiolactone and subsequently bound it to the adjuvant image of aluminum hydroxide gel / Genov, 1 », Cutter, A ·, Gene, A ·, 1967» inactivated with betapropiolactone vaccine sreche boleetta on Aueski with four Ruse / · Inactivity of this vaccine is an inactivation agent that has been removed from the list of substances useful for the preparation of biopreparations as it has been shown to be mutagenic / Eaerish, BJ, Searle, CD, 1966 »Carcinogenicity of beta-propiolactone -oxide for the skin of guinea pig., Brit. J. Cancer 20, 200-205].

A disadvantage of the inactivated Aujeszky's disease vaccines hitherto proposed is that they are prepared or from virus not grown on stable cell line cultures, which affects the harmlessness of the vaccines due to the potential oncogenicity of such tissues, or on primary cultures prepared from kidneys of 1 to 3 months pigs.

201 802 costs · In addition. pri doposia! of the prepared inactivated Aujeazky's disease vaccines, no immunizing effect was declared for all species of susceptible and economically interesting animals by different doses of vacoin.

To solve the inactivated vaccine adjuvant Aujeazkyho disease susceptible species of animals, obeahujúoou in 1 mL 10® '1O ° and ^7, 5 Tkid ^ / ml of inactivated virus Aujeazkyho disease, strain HM-Raca-5, in the Collection of Microorganisms for the Seakoslovenakej The Executive of the Veterinary Institute in Brno Medlánky, Hudcova ul. 60 under the designation CCM V-95 and 10 ⁷ to 10 ⁸ TKID, ₁₀ / ml of inactivated Aujeazky's disease virus Buk-TK-200 deposited in the previously cited collection CCM V-93, emulsified in lipoid saponin adjuvant and method for its preparation, comprising selecting and appropriate ratios of immunogenic viral strains with the ability to grow on heterologous tissue relative to vaccines, effective inactivation with ethanol and saponin, while maintaining antigenicity and the ratio of inactivating agents to the altered antigen of selected virus strains.

The Aujeszky's disease virus, namely the strains BUK-TK-200 and Raca TK-5, are especially propagated in a known manner on primary cultures or subcultures of smokers, or duck embryonic cells, grown on glass, or in suspension! propagation of the aforementioned strains of Aujeszky virus will be carried out using appropriate nutrient media, in particular those containing bovine milk albumin fragments and the necessary amount of mammalian or avian animals / Portefleld, JS, 1967: Aniraal tissue culture, in an editorial by Maramoroah, K. and Koprowski The use of defined media containing substances affecting the metabolic and mitotic activity of cells in layered or suspension cultures is not excluded. Cell cultures are infected with both the BUK-TK-200 strain and the Raca TK-5 strain at a multiplicity of d 0.1 to 1.0 TKIDg ₀ per cell. After 24-48 h incubation of the infected cultures at 37 ° C and the virus is propagated in a volume of maintained handrail with infectious titers in the Baca TK-5 strain from 10 ° C to 10 ° C and in the BUK-TK-200 strain from 10 ^{7 ,} ° to ΙΟ ⁸ ' ⁵ ΤΚΠ> _5ο in 1 ml of suspension. Prepared virus suspensions of Aujeszky virus strains are freed of cellular detritus by sedimentation, centrifugation or filtration. Virus suspensions of the Bača TK 5 and BUK-TK-200 β strains are mixed together in any ratio, preferably 1: 2, preferably 1: 2 they are subsequently inactivated by treating ethanol in a final conententory of 15 to 25 mass per cent at 25 to 35 ° C for 18 to 72 hours, by the action of aaponin, which is currently adjuvanted, in a final conententric from 0.05 to 0.4 mass per cent at 20 to 35 ° C for 18 to 72 hours, the combined effect of ethanol and saponin in a single solution, or the subsequent addition of the individual substances immediately thereafter, or until 72 hours in said concentrators over a period of 14 to 72 hours within the temperature range indicated. Following viral inactivation control by intramuscular administration of rabbits at 2 to 10 ml doses, antigens are emulsified with known adjuvant oils at a defined aqueous phase to oil ratio. 25 ° 0 and subsequent

201 aa cooled to 2 to 10 ° C · Content of adjuvant oils containing mineral oils with a cinematic viscosity of 20.10 ^ to 120.10 ^ m ² · a ” ¹ , esters of higher fatty acids such as lanolin, arlaoel, cereol and tween emulsifiers in such a ratio as to form a water-in-oil or water-in-oil-in-water emulsion whose proportion in the vaccine is 12 to 60% by weight, preferably 16% by weight.

Execution example

The primary cultures of the smoking or duck embryos or their subculture forming a continuous monolayer on the surface of the culture vessel glass are freed from the growth medium consisting of Earl's solution enriched with 7 weight percent calf serum and 0, 4 pereent lactalb mininol hydrolyzate, rinsed 2 to 3 times with saline and individually infect the strains BUK-TK-200 and Raca-TK-5 at the multiplicity of infections 0.1 to 1.0 TKIDg ₀ per cell » Infected cell cultures are overlaid with Earl's solution enriched with 0.3wt% lactalbumin hydrolyzate and incubated for 24-48 hours at 37 ° C. · After virus-specific tissue degeneration, microscopically, incubation is discontinued to obtain infectious liquid that is free of cell detritus by centrifugation or filtration through oez gauze. Two parts by volume of the virus suspension of the strain Buk-XK-200 and an infection titre of at least 10? TKID ^ ₀ / ml and one part by volume of the suspension of the strain Rača-TK-5 with an infection titre of at least 10? mixed virus suspension! aa add ethanol to a final concentration of 20% by weight and a solution of saponin at a concentration of 10% by weight in water to a concentration of 0.1% by weight in the virus suspension. The inactivation agents are added in a common solution or immediately after each other. · Inactivation takes place for 72 hours at 30 ° C.

Four parts by volume of the mixed inactivated viral suspension are mixed with 0.85 parts by weight of light mineral oils (e.g. Marcol), 0.15 parts by weight of higher fatty acid esters (e.g. Arlaoel) and 0.02 parts by volume of emulsifier (e.g. Tween 80) and the mixture is emulsified by stirring at high speed, or under pressure (for example Turmix or Ultrathurax) at room temperature for 60 seconds. The lipoid-saponin vaccine emulsion is stabilized by standing for 16 hours at room temperature and subsequently stored at 2-10 ° C. C ·

The efficacy of the inactivated adjuvanted vaccine against Aujeszky's disease according to the invention is confirmed by the results in the following table which shows the titres of virus-neutralizing antibodies in the blood sera of various animal species susceptible to Aujeszky virus infection depending on the amount of vaccine administered and the a lot of lethal benefits.

201 802

Table 1

Kind of animal Serum VN-antibody titers in blood serum after two-phase application ···. ml vaccine Protected against induced infection •• LDe. in vaccinated animals ·· ml vaccine 1 2 5 10 1 2 5 mink 2-4 4-16 4-32 N 10 ² 10 ³ N pigs 4-16 8-32 8-64 N 10 ³ 1O ⁴ 10 ⁴ bovine cattle 2-16 8-64 8-128 32-256 10 ⁴ 10 ⁶ 10 ⁶

SUBJECT MATTER Ν X LE Z U

Claims (3)

Inactivated adjuvant vaccine against Aujeszky's disease of susceptible species of farm and utility animals, characterized in that it contains 10 µl to 10 µl of TKIDg ₀ / ml inactivated Aujeszky's disease virus of the strain Raca-TK-5 CCM V-95 in 1 ml. and 10? ' ³ to 10 ⁸ »5 TKID, ₁₀ / ml of inactivated Aujeszky's disease virus Buk-TK-200 CGM V-93» emulsified in lipoid or lipoid-saponin adjuvant · 2. A vaccine preparation method according to claim 1, characterized in that the Aujeezky's virus strains are propagated on layered or suepensive primo-cultures or subcultures of smokers or duck embryonic cells and then inactivated by the action of ethanol at a concentrated concentration in medium of 10 to 25% by weight. at a temperature of 25 ° C to 35 ° 0 for 20 to 72 hours, or by saponin concentration in a medium concentration of from 0.1 to 0.33% by weight at 25 ° C to 35 ° C for 12 to 72 hours, or by combining the effects of ethanol and saponin in any order or in a common solution at the indicated concentration ratios at 12 ° C to 37 ° C for 12 to 72 hours and then emulsifying 80 to 420 ml of viral suspension in adjuvanted oils containing 60 to 85 ml light mineral oils and 3 to 13 ml fatty acid esters · 3. The method of preparation of the vaccine according to claim 2, characterized in that the emulsion formed and stabilizes at 12-25 ° C for 10-28 hours and is subsequently cooled to 2-8 ° C. Low: 14 ° C