CS199365B1 - Adjuvant inactivated tissue vaccine against aujeszky disease and method of producing the same - Google Patents

Description

The subject of the invention is an adjuvanted inactivated tissue vaccine against Aujeezky's disease (AL) which induces resistance of immunized animals or their offspring with passively transmitted antibody and a method for producing the vaccine.

Aujeezky's Disease (AD) virus causes central nervous system and respiratory tract diseases in susceptible animals with fatal consequences especially in young individuals · The occurrence of this disease, mainly in large-scale animals, leads to mass losses and causes considerable economic losses, especially in piglets and mink ·

Hesistenoe against viral infections, in addition to factors causing natural resistance, is due to the presence of antibodies neutralizing the morbidity of the virus to induce an infectious process and cell-mediated immunity. Individual resistance factors against a given infectious agent are applied to different animal species to varying degrees depending on the genetic properties of the host reflecting in cell surface composition and metabolism, presence of non-specific inhibitors and other non-specific mechanisms, and pathogenesis of the relevant infectious disease ·

Viral infections presuming the activity of the virus to the target blood organ are successfully influenced by circulating antibodies, in other cases the participation of immune

199 365

199 385 mechanisms summarized under the term cell mediated immunity ·

In active immunization against viral infections, the essential condition for the application of a certain minimal antigenic mania by an appropriate route that would induce a sufficient antibody response, with viral antigens having to contain a component which induces the synthesis of antibodies capable of neutralizing the development of infectious oaemoenéai is the adherence of the virus to the cell at the site of the corresponding reoeptor (BORLAND JJ. Enterovirus enhancement of lato speoifio host cell, and subsequent alterations of coli protein and nuclei acid synthesla · Bact. Sov · 1964, 28 * 3), ie virus neutralizing antibody must block viral number corresponding to tgmto reoeptors. In rare cases, the development of an infectious process can be effectively intervened by neutralizing the components also at another stage of the viral replication loop, such as immunization against neuraminidase mixoviruses.

In the active immunization it is possible to use the so-called "adjuvant effect" of some substances, which both allow the production of antibodies to the uterine titers and also stimulate the so-called cell-mediated immunity.

In the Aujeazky's Disease Prevention Process (AD), a number of results have been published looking for the potency of preventing AD by effective immunization *

Limitation of mortality in pigs infected with Aujeazky's disease virus (AV) by passively administered antibody in the form of immune serum (ΚΟΥΝΌΚ J., GRECZI Ei Serum Ajezaky with disease in suckling pigs. Acta vet · hug · 1957 »7 * 423-427)» or gamaglobulin (LUEASHEV II ·, NIKIZIKA VS, Prophylaotio and theropathis peepertios of gamma globulln in Aujezsky β dioeace Veterinaria (Moscow) 1958, 35/9 »48 to 50), showed in field conditions the possibility of influencing the course of the disease immunization · However at this time was not available active vaccine, neither applied nor alive, expressed (MANNINGER R, MOCZY J. * Traite dos maladies interaes de animals domestiquee. Paris * Vigot Preros 1959 »EP · 457-464) that active immunization against AD is not maybe·

Attempts to prepare vacoiay against AD have been targeted in several ways *

(a) the development of an effective vaccinin, optionally with adjuvants,

(b) elaborating live avlrulent vacoins solekol avirulentnioh mutants;

o) obtaining a natural, low virulent strain from natural outbreaks of AD infection,

(d) elaboration of vacoins using a virulent virus that would not spread in the organism immunized with the jedinoe would not result in disease.

Numerous attempts have been made to prepare vaccinated vacoins. A standardized vaccinia without great efficacy for pigs was prepared (KOCH A, abraham E. Immunizationsversuohe mlt dem Aujezakysohem Virus. Acta vet. Hung. 1952, 2 * 270-279), (ŽUPFA A · *)

Versuoh zur Immunisierung von Laborateriumstioren und Sohweinen mlttels Pormaldehyde - und - UV Strahlen - iaaktivierten Aujezsky-virus. Arch.exp. Vet. met.1963, 17 * 595 to 613) · Formalized vaccine induced protection of pigs against the fatal consequences of infection

199 385 adsorbed aa aluminum hydroxide (AIRAEETIAN V.G., JHACHATRYAN A.B., CHBANYAN M.S.,

ABELYAM K.E · '»Iaaunologic peopertles of inactivalted vaccinia against Aujezsky's disease. Veterinaria (Moscow) 1969, 46:34 to 36), (SOLOMEIN P.S.: Rieview of the use of aluminum hydroxide · formolized withers against Aujezsky's disaase Veterinaria (Moscow)

1955 »32/12: 11 to 14) and formulated as adsorbed on aluminum hydroxyd and treated with saponin after adsorption (RRESCIRA T., PIOSRINI A: Gontrollo d officiada di un vaccine on pseudsrabia (Malattla di Aujezsky) Veterinaria Italiana 1971, 22 / 7-8: 374-393) According to the authors, this vaccine has already faded after a single im administration to seroconversion, prevented the virus from appearing in the farlng and blood and infection after i.m · challengy virulent AD virus ·

In Prague (AIRAPETYAN VG, KCHATRYAN AB, CHBANYAN MS, ABELYAN KE: Immunologic properties of inaotivated water against Aujezsky's diseaoe. Veterinaria t (Moscow) 1969, 46:34 to 36) and (ERESCHRA T., ERIORINI A · Controllo di effio-ada di »un vacclaa oontro la pseudorabia (Malattla Aujezsky) Veterinaria Italiana 1971, 22 / 7-8: 374 to 393), the doses of viral antigen expressed in TCID ^ q (ATRAPETYAN VG, KHACHATRYAN AB, CHBANYAN MS, ABELYAN KE: Immunological properties of iaaotlvatted vaelnes against Aujezsky's diseaoe (Veterinaria (Moscow) 1969, 46:34-36) used virus as an infectious medium from kidney prlmscultures of 3 to 13 day rabbits, at 224 passages (tltr 10 ⁶ '^ The immunization dose administered in a volume of 3 ml of the subcutaneous pig contained at least 10 * * ² ΤΟΙΒ ^ θ of virus.

(PRESGBRA T., EIORINI A: Controllo di effioaoia un vaccine oontro la pseudorabia (Malattla di Aujezsky) Veterinaria Italiana 1971, 22 / 7-8: 374-393) used fresh isolated virus in passage 6 on a stable line of porcine kidney cells (IK-15) having a titre of 10? * - * tcID? q / biI infectious media · The formol formulated by the inactivated aluminum hydroxide adsorbed vaccine was administered sc in graduated doses of 10, 5, 2.5) 1.25, ml ( maximum 10 ⁸ '-? TCID ^ q virus per dose).

Although at work (AIRAPETYAN VG, KHACHATRYAN AB, CHBANYAN MS, ABELYAN KE: Immunologic properties of inaotivated vaocines against Aujeszky's diseaoe · Veterinaria (Moscow) 1969 »46:34 to 36), the evaluation of antibody titers of animals after vacoiuaol from In both cases, inactivation of the formol virus does not alter the antigenic structure of AV and aluminum hydroxide is used as an effective adjuvant.

Aluminum Hydroxide-Adsorbed Virus Affected by Saponin (BERBINSCHI C., PAPADOPOL Maria: Valaur immunogene d 'un vacoin inaetivo confere la, aaadoe d'Aujeszky chez la beuf, le monfeoa, l in pore efe pe chien. Archives vet. 1967 »3 * 59 to 69) The use of this type of vaccine has led to miscarriages, fetal damage, munification and death of young piglets in immunized sows (POPOV A ·, TCHENTCHEV I ·: Utilization of vaccinations contra les maladies & virus das animaux en Bulgarle Bull · Off. · Epizoot, 1965, 64: 195-202), (TONEVA 7 ·: Les vaocins vlvant utiliaés en Bulgarle contre la maladie d 'Aujezsky, Bull · Off.int.Epizoot.

199 385

1965, 64 (203-207). However, from the papers published (B3S8BIBSCHI C., PAPADOPOL Maria i Vale ur immunogene dun vaccin inactiv contre la maladle d 'Aujeszky ohez la bol, le monton, le porte et le fire. Archive vet. 1967, 5 * 59-69) the elimination of AT from contaminated pig holdings by systematic immunization and it is proposed in such holdings after inoculation to further immunize only breeding sows to interrupt the possibility of transferring the wind from dust to piglet by passively transmitted antibody (BSRBINSCHI 0, PAPADOPOL Maria, MARTA M) »Untersuohung Uber der psrophylatisohen ffert der inaktlvierten Aujeszky-vakalne in Sohweinesuchtbetriebeu Archive vet. 1968, 5 »1θ7 to 114) ·

The vaccine prepared (LTSBNKD IP, TSUÍBAL AN, KDLBACHNAJA MZ, Preparation and laboratory trilas of UHlEk Aujeszky ^f and dissassant virus vaccine, alone qnd ooabined with awine fever vaocin · Veterinary (Kijerv) 1965, is considered uninactivated) »38 to 43 in which the virus was inactivated with crystal violet and the efficacy of the vaccine was potentiated by aluminum hydroxydsm (BASKSSVILLE A, MoPERRAN JIBI, DOW O »Aujeszky * s dieease in piga The Teterinary Bulletin 1975, 45/9 * 465 * Ž 480) does not consider the inactivation of AT crystalviolete to be probable, since (UaSiC M «, PETROVC Mt Mt. Kanu durch Kristalvioletvakzlne gegeh Sobveinepest dis Aujeszkysohe Krankenheit Hbertragen werdea 7 Zbl.Bakt.Parasitkde. crystal violets ·

The crystalvioleate adaorbate vaccine has been prepared and verified (NBJDALKOT S., STOITSCHBV S, RIBAROV B., STIWVA N., Tersuche zur Herstelluttg von Aujeszky sche Krankheit aus Inaktlvierten Virus · I.Mitteilung. 1968, 22J.499 to 507), (NBJDALKOV S, STOILOVA B., RIBAROT B, TABAKOV B. i Z Ur Immunnitzt von Sangererkel na Impfung mit Adsorbatkristallviolettvakzifie gegen die Aujeszkysohe Krankheit Arch. F. , 2 ^ / 2 * 257-262) · Vaccine-Induced Antibodies Paradoxically, piglets of 10 to 20 days of age had higher titers in individuals with pre-existing titers, whereas there was no production in individuals without detectable antibodies. · According to experience (ARNADDOV H .: Immunity and antibody dynamics of pigs inoculated with Aujeszky's ethanol vaccine I.Influence of naturally occurring antilbodies (Veterinarnomeditains of Nauki 1975, 10/5 * 85 to 87) Prescribes colostral antibodies to prevent as well as viral antigen and pro-drug response are generally not present.

t

AH inactivated HHOg has been shown to be capable of revo a i.v. sowed with a dose of about 10 ® TCZD 50 AT to induce rabbit production of antibodies at titers of 52-128 that protected animals from challenging 500 TGID 50 homologous virus (ITASISOTA S., SKODA R., MATES T., SOKOL.) »Inactivacion of Aujeszky s Acta Tirologica 1965, 7 »7 to 15) · Subcutaneous administration of the virus to rabbits, with no exception, has no effect on antibody production · Efficacy on preaeate has not been verified and it is difficult to assume that an immunization schedule requiring numerous revacinains would be practicable.

199 395

The vaccine prepared by CH by inactivation of AV has been shown to be effective (BELIEVES I, IVANOVIGS G 'XBuuuniaiexrung the Laboratoriiuestioren mit Dam Virus Aujeszkyschen Krankheit nach deseen Inaktiviernng nit UT Strahlen · Acta Microbiol.Hung.19560, 2 to 15160, 2).

In a laboratory experiment in guinea pigs and rabbits and three sows, the vaccine against AB prepared photoinactivatives AT was propagated on primocultures of porcine kidney cells by methylene blue followed by exposure to light (BORGEN HC, ESEENSEN 1.1 Immunising efeot of photoinactivated virus Pseudorabie. as it where I.Orig.

Inactivated virus was administered sc at a dose of 50 ml for dosages containing 5x10 TOID 50 virus. Erimovaccine resulted in an increase in antibodies from 0.5-1 and ₂ ΝΒ ^ θ to 0.9, respectively. · 1.2 in two immunized sows, 14 days after revacoination at 4,9 and 6,2-log.g ΝΒ ^ θ (which corresponds approximately to titers 32 and 64) · Ohallenge dose LD 10 LD50 rabbit (200 TOID 50) virulent virus led to an anamnesic reaction with an increase in antibody titers to 9.9 and 6.2 -log · 2 »®> ijq (00a 1024 and 64) without signs of clinical disease · Colostral antibodies protected pigs from immunized sows from the same ohallengine agent AT 1 both the control sow and her piglets had clinically manifest signs of disease to which the piglets succumbed ·

The striking effect of photoinactivated AT manifested by antibody and pig titers shows at least two facts at the same time as compared to other inactivated in action (1). Potoinactivaoe is a very gentle inactivation procedure leaving the viral surface unchanged · 2) The utility dose of virus five times ten to eight for dosing subcutaneously is a sufficiently effective anti-genic stimulus ·

The drawback of this vacoin is dosing (50 ml e.o · for dosi and a certain difficulty of vacoin preparation in large-scale operating conditions (1000 doses with 50 thousand.ml ·) ·

Etanulene inactivated AB virus mixed with paraffin oil (HARALAMBIET H., MERMERSKI K., STOET I., SIMEONOT Sj IOTOT M .: Investigating the immuno-inactivated Aujeszky virus * and the virus on sheep · Cr Aoed · bulg. Sol · 1966 , 19'441-444) was used for immunization of sheep, cattle and pigs. The experience with this vaccine was summarized after a three-year trial period (HARALAMBIET H., MERMERSKI K., SIEMONOT S., IOTOT M., STOET I ·, DIMITROTK ·, NIKOLOT 1 * * Untersusohungen Immunogenitfit der mit Fithanol inaktivierten Takzino gegen Aujazkysche Krankheit · Tet · med · 1967, 21 '501 to 507) · Vakoina was based on more effective than formalized vaccine according to (SOLOMKIN PS: Review of the use of aluminum hydroxide formolized vaooine against Aujeszky's disease · Teteriaarija (Moscow) 1955 »32 / 12'U to 14)., (BOIADZHIET S., GENET Kh., GEHOT I., TAOHET B. STOIANOT V. , KALCHEV I · studios on Aujeszky 'b desease · I · Com Tet · med · Nauki (Sofia) 1967 »4/9 * 19 to 26) considers the alcohol-inactivated vaccine against AB to be the best of the three inactivated vaccine types - alcohol-inactivated, formol-adsorbate and saponinadsorbate - compared (SIMEONOT S ., TASILET V., IOTOT M., MERMERSKI K. * Improvement of ethanol vaooine against Aujeszky * s disease I. Production of atanol199 395

Naukl 1971, 8/7 * 23-29), attempted to replace paraffinated adjuvant saponin in ethanol-inactivated vaccine. Neutralizing antibody titers in pigs immunized with this vaccine at two doses reach values of 8 days after revaccination with a persistence of 4 months, 6 months 4 or 2 (SIMEONOV S., YOTOV 11 «, VASILEV V., MERMBRSEI K. and Attemps to lmprove the ethanol vaccinated against Aujeszky 's disease · II · Storage and duration of immunity Veterinarskomeditsinski naukl (Sofia) 1972, 9/5 * 69 to 73) ·

Efficacy of ethanol vaccine with paraffin oil as adjuvant (HARAIAMBIEV H., MERMERSKI K., SIMEONOV S., YOTOV M., STOEV I., DIMITRO K ·, NIKOMV I. t. Arch · exp · Vet · med · 1967, 21 (501-507), tested on piglets with low titreohabs present, is difficult to evaluate because it cannot be ruled out that vaccination has been used as a secondary stimulus * and different antibody responses of piglets of different ages with low antibody titers · ¹ (GENOV I ·, RBZASHKA A ·, GENOVÁ A · * Product of propiolaotone - a vaccine against Aujeszky * s disease from the Russian vaocinal strain. Vet.med.'nauki ( Sofia (1967, 4/10 * 35 to 40) attempted to prepare an absorbent vaccine with sparingly inactivated AD beta - propiolactone virus. Emphasis was placed on the appropriate minimum effective dose of inactivating agent (0.05%) · The vaccine efficacy was evaluated in sheep and pigs ·

Although the amount of virus antigen in vakeině was small (maximum 10 ⁵ TCID 50 for dose), the authors provide evidence after primovakcinaoi sheep and pigs identical titers 16, 32 · after revkacinaoi

The importance of a sufficiently large antigen pulse and the potential effect of an adjuvant is outlined in the work (SKODA R., WITTMANN G., Die Immunizierung von Schwlaen to have Vakzinen and Aujeszky-Virus inactivlerten · Zbl.Vet.med.1973, Heft 2> 127 to 138) · The authors reported poor efficacy of previously inactivated vaccines. They attempted to concentrate the virus obtained from tissue cultures of BHK 21 cells, so that the final amount of viral angigen in the vaccine corresponded to a dose of 5 of TCθθ TCID ^ q in the volume of the competitive vaccine. Two doses of virus, 5x10 6 and 10 6 TOID 6, which were administered i.a. or with diethylamino-ethyldextrans (DBAE'-D) and further compared to efficacy with virus inactivated by the same procedure applied without adjuvant and with Preude's incomplete adjuvant. Experiments have shown that ΞΕΙ inactivated virus indicates antibodies that reach neutralization titers of 2 and 4 after 4 weeks. The incomplete adjuvant did not potentiate the antigenic properties of the virus used. DEAE -E compared to virus alone in virus adjuvanted anti-virus response resulted in earlier and higher antibody responses (8 to 32). · After priming, titers of 1 to 4 are maintained for up to 15 weeks, revacinating increasing titers to 9 to 32, however, antibodies decrease as fast as after the primary response, so that 80 to 105 · day resistenoe on infectious burden was detectable in 13 · vacoinated piglets out of 20.

189 368

The authors conclude that revaccination did not lead to an increase in immunity. At the same time, it is noteworthy that in animals vaccinated with this type of vaccine, contact infection with virulent virus secreted by the upper respiratory tract of artificially infected animals did not result in a typical secondary response (aoamnestic reaction) and that antibody titers begin to rise after 4 to 7 days. JAKUBÍK J. «Immumleslerung von Schweinwn mit AujeszkyvirusVakzinen, hergestelit aze vol Plawttenvariantea Des Virus. Zhl.Vet.laed. 1974, B 21,

234-241) the authors relate the time interval of revaccination from priming to the persistence of antibodies in the discussion.

From the literature review on the possibility of using inactivated vaccine to prevent and control AD, some generalizations may be attempted.

1. A prerequisite for the efficacy of the viral antigen is a gentle inactivation procedure, leaving the surface atrophy of the virion intact (BGRGEN H.H., ESPENSEN L. t Immunizing effeot of photoinaotivated pseudorabies virus. Zbl. Bact. Parasitkde 1. Orig. 1970,

214 (13-16).

2. The vaccine must present a sufficient antigenic pulse given by the concentration of the virus.

3 · The immunization scheme chosen also determines the outcome of the immunization (SKODA R., JAKUBIK J. t Immunlaierung von Schweinen mit Aujeszkyvirus-Vakzinen, hergestellt aus zwei Plaquenvarianten der Virus. Zbl.med.med. 1974, B * 21, 234-241) .

An inactivated vaccine prepared with respect to the above assumptions may be equal in efficacy as measured by neutralizing antibody titer to attenuated vaccines (BORGEN H. 0., ESPBNSEN L. t Immunizing effect of ohotoinactivated pseudorabies virus. Zbl.baot. Parasitkde I. Orig. 1970 , 214-12-16) without risk of newborn piglets of high-breeding sows (BOYADZHIEV S., GENEV Kh., GENOV I ·, VACHEV B., STOYANOV V, KALCHEV I ·,: StUdies on Aujeszky 'a disease I. Comparison of Vet.med.nauki (Sofia) 1967, 4/9 * 19 to 26), (HASIČ M., PETROVIČ M. * Kann durch Kristalvioletvakzine gegen Schweinepe st according to Aujeszkysohe Krankheit Hbertragen werden, Zbl. Bact. Parasitasche · 1962, I. Orig ... 185 (t 145-154) and without limiting their application to outbreaks of infection. In any case, it is no longer possible to agree with the assertion (Meanlnger R., Moczyj * Traité des maladies internes des animavx domestigques. Papis »Vigot fréves 1959, ΡΡ · 457 to 464) Neither can we agree (Kretzschmar 0, Unter suchvagen zur Eplzotologie der Aujeszkyschen Krankheit heim Schweinund daraus sich ergebende Schluss forgerungeu fflr Prophylaxis und Bekamptung Mh.Vet.med. 1964, 19 * 248 to 257), Zuffa A. Versier Laboratoriumstieren und Schweinwn mittels Pormaldehyde - und - UV strains inactivated Aujeszky virus. Sheet. exp. (1983, 17: 595-613) that the inactivated AD vaccine is ineffective. This review of world literature shows that active immunization with an inactivated vaccine perfectly protecting the immunized animal is not resolved.

199 3BS

These deficiencies are remedied by the adjuvanted inactivated tissue vaccine against Aujestky's disease according to the invention, which consists of an Aujeszky's disease virus iaaktivevaua, which has ONOZO Ae 5/78 mutaaeta - achieving at least ΙΟθ '^ Ι ^ ΤΟΙΒ ^ ΤΟΙΒ ^ ΤΟΙΒ ^ ΤΟΙΒ ^ ΤΟΙΒ ^ ) in admixture with absorbable adjuvants at a concentration of 6 to 8 percent. A method for producing this vaccinia is characterized in that the infectious medium of susceptible tissue culture cells infected with at least a multiplicity of 1 Aujeszky's disease virus mutants - CNOTO Ao 5/78 - per cell and after infection stored at 26-37 ° C, takes place during the developed period. virus-induced tissue damage, preferably 20 hours after infection, the tissue holder and virus agglomerates are removed, the amount of virus is verified, the infectious medium is inactivated by adding 10 percent of the hetapropalator to a final konoentraoe of 0.05 to 0.025 percent under pH control, titration, adjust the viral antigen concentration (at least to 10 ^{8 (} TOH)), add antibiotics, adjuvant to a concentration of 6-8%, mix to form a viscous vaccine. The selected Aujeszky's disease virus-ONCTO Ao 5/78 mutant achieves titers in excess of 9 / 1θβ ^ θ TCID ^ q (ml) on porcine kidney cell cultures in the form of a primoculture or most suitably stable PK line. The inactivation takes place at 30 to 37 ° C for 15 to 30 minutes.

One advantage is the use of the Herpes virus suis AV mutant according to the invention, strain CNOTC Ao 5/78 MS. This type of Aujeszky's disease virus mutant was obtained by AU 1 from the SVI Prague - Iysólaje, the selection of salaries morphologically distinct after two replicative cycles on cells of the PK line cultivated under an agar blanket at 36 ° C. 0. The clone obtained is characterized by »

a) maintained autigenic structure,

(b) preserved virulence for pig, rabbit, guinea pig and mouse;

c) ability of rapid formation of necrotic lesions in cells of PK line with characteristic morphology,

(d) ability to achieve titers in excess of 10θ TCID ^ q in mu in the culture medium of susceptible cell tissue cultures.

The mutant AV achieves titers greater than 10.9 TOID ^ q / biI on porcine kidney cells, the possibility of eventual dilution of the infectious medium up to a minimum value of 10 ⁸ ' individuals underlined by the immunizer-scheme. An effective antigenic stimulus is provided by a very gentle inactivation procedure, represented by the use of betapropiolactone in final concentrations of 0.05 to 0.25%. for slaughter animals where the vaccine has been applied and whose consistency meets the requirements of the technical possibilities for nasal application. In addition to stimulating humural antibodies, the adjuvant positively affects cell-mediated immunity. The outcome of immunization is influenced by a suitably selected posologil, dosage and revaccination. A proven immunization schedule has shown the advantages of intramuscular administration of the vaccine over the administration of a strictly subcutaneous and subcutaneous fat, particularly in an anesthetic response. For the surface

199 39S antibodies are preferable to revaccinao within three to five weeks after primovacoination in comparison and revacoineas after 14 days after vaccination. Revaccinae of fertilized females eg sows 7 to 14 days prior to childbirth, results in protection of piglets by colostrum for at least 50 days after childbirth, At the time of the animal's greatest susceptibility · The main advantage of the application of an effective inactivated vaccine against live vaccines is the possibility of its use in the prevention of AD, especially in areas where it is present without risk of introduction of live virus into the herd. 5 days after the immunization procedure · This rapid effect is explained by the booster effect ** of the vaccine in the infested brood. Individuals not responding to vaccine administration are a rare exception · Takciaa does not induce a local or aspen reaction and can be used without risk in high-breeding animals ·

Vaccine production assumes the use of common ingredients, and the manufacturing process itself avoids more demanding instrumentation · 7 essentially involves the equipment of tissue culture laboratories and, in the case of mass production, the possibility of sterile filling of the product ·

The efficacy of the vaccine is guaranteed for at least six months when stored at 3 to 0 ° C.

Immunity has been verified in pigs, guinea pigs, rabbits and mice with results showing the general ability of this vaccine to induce antibody production and protect immunized individuals from virulent AT infection.

Example:

The vaccine according to the invention has this composition of infectious tissue culture medium 95 parts aquasorb hydrophilic anhydrous 7 parts penicillin and streptomycin about 500 µl / gema / ml

Kuagizone 5 µg / ml

An example of a process for producing 1000 ml of a vaccine according to the invention:

Karl solution with 0.5% lactalbumin hydrolysate treated with 7.5% AnHCO3 solution to pH 7.6 rinsed large cultures of RK cells - stable pig kidney line in 1200 ml Roux bottles are infected with 1.0 ml virus strain - Aujeszky virus mutants diseases - ONCTC Ao 5/78 ·

The infected cultures are placed in a virus adsorption thermometer for 20 min and then overlaid with 100 ml of maintenance medium composed of Charles solution with 0.5% lectal-buminohydrolysate, pH 7.6 after treatment with 7.5% NaHCO 3 solution.

The infected cultures are incubated at 36 ° C. ° C in thermostat until cytophic changes are in the form of complete tissue destruction · Generally incubation does not exceed 48 hours ·

The infectious medium is decanted to form a single initial suspension and centrifuged at 5000-6000 rpm for 10 min. Using an angled head of a Jantzkl T 23 Supernatant1G centrifuge.

199 39S nl fluids are aspirated and inactivated by adding a ten-percent pre-cooled aqueous betapropiolactone solution to a final concentration of 0.05 to 0.25% t most preferably 0.15% a and 1000 ml of viral eentrifuged suspension 15 ml of 10% aqueous betapropiolactone solution with continuous stirring. with the viral suspension after the addition of betapropiolactone is stored in a water bath of 30 to 36 ° C and for 50 minutes, by adjusting the pH for 10 minutes to

7.6 by addition of bicarbonate · During the inactivation period, the viral suspension is mixed twice every 10 minutes · After completion of the inactivation after 50 minutes, it is repeatedly checked or adjusted to pHt with antibiotics penicillin and streptomyoin added to 500 µl, gamma and Fungs 120 N / ml * With continuous stirring, preferably in a mixer or high shear mixer, 85 ml of warmed Aquasorb is added to a volume of 250 µl of the virus suspension, allowing the suspension to bind to the adjuvant, resulting in thickening of the mass within about 1 minute from the adjuvant addition. gently adding the remainder of the virus suspension while stirring continuously · The total mixing time sufficient for perfect homogenization does not exceed 5 min, usually 4 minutes ·

The adjuvanted vaccine is sterile filled with dc medicaments and sealed with rubber stoppers and metal sockets. Until use, it is stored at a refrigerator temperature of 4 to 6 ° C.

The efficacy of the vaccine was verified in experiments in piglets, rabbits and guinea pigs using subcutaneous and iatramuscular administration. The highest efficacy was demonstrated in guinea pigs showing a high antibody response after primary vaccination (.256 tit) at the same dose per weight unit. lower titers were obtained in piglets (64 to 128), rabbits after orimovacoination only responded to dose increase. · Revaccination in all cases resulted in antibody responses to high titers (256 to 512 against 1000 TOID ^ q in tissue culture neutralization test) and animals at the same time was resistant to infection with 10? TCID? q infectious virus administered im The vaccine administration to rabbits was also a safety test as the rabbit corresponds to fatal diseases already infected with Aujeszky's virus units. · The inactivation procedure used in the rabbits proved to be fully sufficient to rid the Aujeszky's disease virus of infectivity while maintaining antigenicity.

Evidence of Efficacy Tests Three vaccines were tested, of which A - Adjuvant Inactivated Tissue Vaccine, B - Lyophilisate

C - another vaccine

The vaccines were administered i.m · in the following amounts, each to three rabbits:

Whole vaccine dose for pig

1/2 vaccine dose for pig

1/4 vaccine dose for pig

1/10 vaccine dose for pig

1/20 vaccine dose for pig

199 36S

At 11 dal pa vacclnaoi, all rabbits were revaccinated with the same doses and in the same way. For the next 10 days, rabbits, including three non-vaccinated controls, were infected with Aujeazky's disease virus (Bucvrest 200 passage strain) at a dose of 10. rabbits observed 14 days · The test was performed twice as I and II titraoo ·

I · titraoo

Vaccine Vaccine dose 1/20 1/10 1/4 1/2 1/1 AND 33 66 100 ALIGN! 100 ALIGN! 100 ALIGN! (B) 0 0 0 0 0 0 0 0 0 0 0 % survival of rabbits after challenge

II

Vaccine Vaccine dose - ............. 1/20 1/10 1/4 - 1/2. 1/1 AND 33 33 50 100 100 ALIGN! (B) 0 0 33 33 66 C 0 0 0 0 33 Survival of rabbits after challenge

100% protection is recorded for both whole and half doses of vaccine, while other B and C vaccines have by no means ensured Immunity of all vaccinated rabbits · 17 trials still 5% dose of vaccine A protected a third of vaccinated A · Control rabbit death after 4 and 5 day challenge ·

Proof of harmlessness!

Again, three vaccines A, B, 0 were used, which were administered to 5 rabbits weighing 2 kg at a dose equal to and a vaccine dose for pig and another 5 rabbits at half dose, ie:

Vaccine A 5 and 2.2

Vaccine B 2 cm @ -1 and 1. cm @ -1

Vaccine 0 2 cm cm and 1.0 cm ^

Observed 21 days · '

Harmless test results! Vaccine A 10 · one rabbit died 1 day after vaccination, the whole vaccine dose · A 'oh infection was excluded as the cause of mortality

Claims (4)

Adjuvant inactivated tissue vaccine against Aujeszky's disease, characterized in that it consists of an inactivated mutant of Aujesky's disease virus - CNOTO Ao 5/78 - achieving titers of at least 10 ® * TCID ^ q / ml in the tissue culture nutrient solution, with absorbable adjuvant in koneentraoi 6 to 8% · 2. A method for producing an adjuvanted inactivated vaccine against Aujoezkyhe disease according to item 1, characterized in that the infectious medium of susceptible tissue culture cells infected with at least a multicloneity of 1 Aujoszkyhe ohoroby virus - CNOTO Ao 5/78 per cell and polnfollol stored at 56-37 Harvesting at the time of the developed virus-induced tissue damage, preferably 20 hours after infection, the tissue debris and virus agglomerates are removed, the virus titer is verified, the infectious medium is inactivated by adding 10% of betapropiolactone to the final conoentra 0, 05 to 0.25% under pH control, titration results adjust viral antigen concentration, preferably less than 10 ΤΟΙϋ ^ θ / ηΙ, add antibiotics, adjuvant to conoentrao 6 to 8%, mix to form viscous vaccine · Production method according to claim 2, characterized in that the selected Aujeszkóhe ohoroby virus mutant - CNCTO Ao 5/78 - achieves titers in excess of 9 log ^ TCJD ^ Q / ml on porcine kidney cell cultures in the form of a primoculture or most suitably stable line - PK . 4. A process according to claim 2, wherein the inactivation takes place at 30-37 [deg.] C for 15-30 minutes.