CS199865B1 - Combined vaccine against aujeszky disease and g-intestinal pathogenic microbes and method of producing the same - Google Patents

Description

(54) Combined vaccine against Aujeezky's disease and G-intestinal pathogenic microbes and method for its manufacture

The present invention provides a combined adjuvanted inactivated vaccine against Aujeszky's disease (AD) and G-intestinal pathogenic microbes that confer resistance to immunized animals, or their offspring, with a passively transmitted antibody, and a method of making the vaccine.

The Aujeezky's Disease (AV) virus causes central nervous system and respiratory-tract diseases in fatal susceptible animals with fatal consequences, particularly in young individuals. The occurrence of this disease, mainly in large-scale animals, especially in piglets and mink. Of the G-microorganisms that cause gastrointestinal diseases in pigs, the most common cause of the disease is enteropathogenic E coli, which, in addition to gastrointestinal infections, can lead to a general septic condition, often resulting in death.

Some serotypes of Escherichia microbes are more frequently associated with infectious diseases, due to significant virulence factors such as enterotoxin production. The surface structure - the wall - of these bacteria is both a biologically active component of the microbe, allowing adhesion to the intestinal mucosa and, secondly, a prominent antigen. Among the biological properties, the ability of the lipopolysaccharide complex, the so-called endotoxin, to non-specifically potentiate the antigenic stimulus, is significant. The multiplicity and variability of biological properties, including vlrulation, and antigenic structure assume the use of combined antigens to successfully prevent diseases caused by microbes of the genus Eecherichia. Separate immunization with individual antigen types is not feasible in the field.

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Resistance to infections, in addition to factors contributing to natural resistance, is due to the presence of antibodies neutralizing the ability to induce an infectious process _in cell-mediated immunity. Individual resistance factors for a given infectious agent are applied to different species in varying degrees depending on the genetic characteristics of the host reflected in the cell surface composition and metabolic pathways, the presence of non-specific inhibitors and their non-specific mechanisms and the pathogenesis of the respective infectious disease.

In active immunization against infections, the essential condition for the application of a certain minimal antigenic mass by an appropriate route that would produce a sufficient antibody response, while the microbial antigen must retain a component conducive to induction of antibody synthesis capable of neutralizing the development of infectious disease. For viral infections, it is believed that the condition for inducing the infection is the adherence of the virus to the cell at a site corresponding to the HOLLAND J.J. Enterovirus enhances specific host cells, and subseguent alterations of cell protein and nucleis acid synthesis, bact. Roar. 1964, 28 (3), a virus-neutralizing antibody must block the surface determinants of the viral portion corresponding to these receptors.In rare cases, it is possible to effectively intervene in the development of the infection process by neutralizing components involved in other stages of the viral replication cycle, such as immunization against myxovirus neuraminidesis.

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

From a review of the world literature on the possibility of using an inactivated vaccine to prevent and control AD, some generalizations can be attempted:

1) A prerequisite for the efficacy of the viral antigen is a gentle inactivation procedure, leaving the virion's surface structures free from damage BORGEN H.C., BSPENSEN L.: Xmmunizing effect of photolnaotlvated virus Zbl. Bakt. Parasite where l.Orig. 1970,

214 with 13 to 16.

2) The vaccine must present a sufficient antigenic pulse, given by the concentration of Borgen H.C. virus, Espensen L. 1970, ŠKODA R., WITTMANN G.: Immunization of Schweinen with vaccine and Aujeszky virus Zbl.Vet. copper. 1973, Heft 2 (127-138).

3) The selected immunization scheme of ŠKODA R., JAKUBÍK J. and Immunization of Schweinen with Aujeszky-virus-Vakzinen, hergestellt aus zwel Plaguenvarianten des Virus Zbl.Vet decides on the result of the immunization. copper. 1974, B 21, 234-241.

The inactivated vaccine prepared in the light of the foregoing assumptions can be matched in its efficacy, as measured by three neutralizing antibodies, to vaccines attenuated by BORGEN H.C., ESPENSEN L. Immunizing effect of photoinactivated pseudorabies virus Zbl. Bakt.Parasit where 1. Orlg.1970, 214 and 13 to 16, no risk for newborn piglets and high pregnant sows BOYADZHIEV S., GENEV Kh., GENOV I., VACHEN B., STOYANOV V., KALCHEV I.

> Studios on Aujszky's disease. I. Comparison of vaccines. Vet.med.Naukl (Sofia) 1967

4/9 «19 to 26, HASIČ M., PETROVld M. t Kann durch Kristalvioletvykzine gegen Sohweinepest die Aujeszkysche Krankheit iibertragen werden? Zbl.Bakt. Parasitkde. 1962, l.Orig.

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185 <145 to 154 and without limiting its application to outbreaks Infections.

In no case can one agree with the assertion of MANNINGER R., MOCSY J. I Traité des meladies internee des anlmaux doméstiguee Paris <Vigot Fréres 1959, pp. 457 to 464, that active immunization against AD is not possible. Nor does the opinion of KRETZSCHMARK C. t Untersuchungen zur Epizootologie der Ausjeszkyschen Krankheit beim Schwein und daraus sich ergebende Schlůesforgerungen fír Prophylaxie und Bekampfung. Mh.Vet.Med. <1964, 17 <248-257 and ZUFAFA A.: Versuch zur Immunisierung von Laboratoriumstieren und Schweinen mlttels Formaldehyde. und-UV Strahlen - Inactivierten Aujeszyk-Virus Arch.exp.Vet.med. 1963, 17 <595-613, that the infected vaccinia against AD is ineffective.

The tissue inactivated adjuvant vaccine against Aujeszky's disease, which has been developed in recent years, has significantly addressed the issue of the destruction and prevention of Aujeszky's disease, especially in large-scale farming. In spite of its effectiveness, it has increased the number of already numerous vaccinations. As a mono-vaccinous vaccine, it did not have the benefits of the potential potency of the antigenic ipulse to be used in combination vaccines.

The aforementioned disadvantages are overcome by the combination vaccine against Aujeszky's disease and G-intestinal pathogenic microbes, and the method according to the invention, which comprises 10 ⁸ 'to 10θ' tcID 4 q inactivated mutants of Aujeszky's disease virus in one mL,

H

CNCTC AO 5/78, and 10 to 10 inactivated bacterial G-individuals, for example, enteropathogenic Escherichia coli or ealmonella, in a lipoid adjuvant.

A method for producing a combination vaccine wherein Aujeszky's disease virus CNCTC AO 5/78 is cultured on tissue cultures, most preferably on cells of a stable pork kidney line (PK in a serum-free maintenance medium, at 36-37 ° C for 18-48 hours, depending on in complete disintegration of the infected cells, and then the separated cell debris is inactivated by beta propiolactone, homogenized at a temperature of up to 42 ° C and with a lipoid adjuvant and with 10 to

Q by inactivated G-individuals, in particular by enteropathogenic Escherichia coli or ealmonella. The adjuvant used in the vaccine consists of a resorbable lipid mixture which is mixed with the viral suspension most preferably at 30 to 37 ° C with the addition of a bacterial suspension of G-microbes, the potency of which has been enhanced by repeated agitation of its bodies by repeated freezing and thawing.

One advantage of the combination vaccine is the use of the AV mutant of the invention. This Aujeszky's disease mutant was obtained by triple cloning of the AVI laboratory strain from the SVU Prague - Lysolaje, by selecting plaques morphologically distinct after two replication cycles on PK cells grown under agar blanket at 36 ° C. The clone obtained is characterized with

(a) the preserved antigenic structure

(b) preserved virulena for pig, rabbit, guinea pig and mouse

o) ability of rapid formation of necrosis foci in PK cells (plaques within 24 hours)

Q

(d) ability to achieve high concentrations in the infectious medium (TCID titers ≥ 10 log 10 and higher per ml medium). The effective antigen stimulus is ensured by a very gentle inactivation procedure represented by the use of propiolactone in final concentrations of 0.05 to 0.25%. The immunization effect is substantially enhanced by the addition of a resorbable lipoid adjuvant which

In addition to potentiating the effect of vacoin, 199 865 has the advantage that it does not require the forced cutting of lots of slaughter animals where the vaccine has been applied and which consistently meets the technical requirements for mass application and the presence of the Lipopolieacharin complex in the vaccine of bacterial members present. In addition to stimulating humoral adjuvant antibodies and the Lipopoliaaccharide complex, it also positively affects cell-mediated immunity. The outcome of immunization is influenced by appropriately selected posology, dosage, and revaccination. The open immunization schedule demonstrated the advantages of intramuscular administration of the vaccine over the administration of strictly subcutaneous and subcutaneous fat, especially in the case of an alamnestic response. Vaccination with the combined AV vaccine and selected E. coli strains results in potentiation of the AV antigen pulse and concomitant generation of antibodies against bacterial strains, which are preferably used in colostral immunity.

Revaccination three to five weeks after primary vaccination is appropriate for antibody levels. Revaccination of fertilized females, eg 7 to 14 days before farrowing, leads to the protection of colostrum piglets for the beer weeks after farrowing, at the time of the highest susceptibility of the animal to AV and anteropathogenic coli. A major advantage of applying the combination vaccine of the present invention against live AV vaccines is that they can be used to prevent AD, particularly in areas where they occur, without the risk of introducing live virus into the herd. Administration of the inactivated vaccine results in a 3 to 5 days post-immunization cessation of infection. This rapid effect can be explained by the booster effect of the vaccine in the soaked stock. Individuals not responding to vaccine administration are an exception. The vaccine does not induce a local or general reaction and can be used without risk in high-breeding animals. These advantages also apply to the bacterial strains included in the combination vaccine. Antibodies against these strains protect newborns in the risk period from enteropathogenic microbes of the corresponding serotypes. Vaccine efficacy is guaranteed for at least 6 months when stored at 3 to 7 ° C.

The vaccine for efficacy has been verified in pigs, guinea pigs, rabbits and results showing the general ability of this vaccine to induce antibody production. Production of the vaccine assumes the use of conventional ingredients and the manufacturing process itself can be carried out without the more complex instrumentation. Essentially, the equipment of a tissue culture laboratory and, in mass production, include the possibility of sterile replenishment of the product.

A mixture of atheropathogenic E. coli potentiates the efficacy of infected vacoins against AD using other adjuvants to the invention.

Example of production method of combined vaccine - prescription for 1000 ml.

Earl solution with 0.5% lactalbumin hydrolysate, pH 7.6, rinsed cultures of PK kidney cell line PK are infected with a volume of 1 ml of the high-titre Aujeszky's virus 8 CNNCC AO 5/78 virus having a titre of at least 10 * / ml and allowed to absorb to the cells at 36 ° C for 20 minutes. After adsorption, the maintenance medium was added in a volume of 1200 per 1200 mL Roux flask - Earl's solution with 0.5% lactalbumin hydrolysate pH 7.6 after treatment with 7.5% NaHCO 3 - and the cultures were allowed to incubate at 36-37 ° C until their complete disintegration, which usually occurs between 20 and 48 hours after infection.

At the time of complete citopathic damage to the cultures by viral replication, the medium was individualized

199,865 infected cultures were mixed, centrifuged at 6,000 rpm for 10 minutes using an angled head in a T 23 Janetzki centrifuge, and the supernatants were mixed again.

The viral centrifuged suspension is tempered to 37 ° C and inactivated by the addition of a 10%, refrigerated temperature of pre-cooled betapropiolactone to a final concentration of 0.15%. The inactivation takes place at a temperature of 36-37 ° C for 30 minutes, during which the suspension is mixed at least twice and the pH is adjusted as necessary, which usually drops after the addition of betapropiolactone. After 30 minutes of inactivation, the pH is checked, optionally adjusted to 7.6, and 1/5 volume of gram-negative intestinal bars propagated in Earl's solution with 0.5% lactalbumin hydrolysate are added to the virus suspension for 20 hours at 36 ° C, containing 1 ml 10 bacterial subjects that were subjected to a double freeze / thaw cycle and inactivated with formol at a final concentration of 0.4% for 48 hours at 36 ° C. The vaccine vaccine for sows generally comprises equal volumes of strains 157, 147, 64, 149, 141 typed according to the 0 antigen, at least two of which have LT enterotoxin present and the fimbrial K 88 antigen, Salmonella choleraesuis.

The inactivated viral suspension with a mixture of inactivated bacterial strains preheated to 37 ° C is mixed with 85 ml of warmed Aguasorb while stirring vigorously in a mixer or using a mixer, first adding an adjuvant with 250 ml of mixture, allowing the suspension to bind to the adjuvant. thickening of the mixture after stirring for approximately one minute and further, the remainder of the suspension is slowly added while stirring continuously. At the same time, the antibiotics penicillin and streptomyoin are added to the final concentration of aa 500 µl and 500 µl, respectively. gamma / ml vaccine, and fungizone 5 µg / ml. The total mixing time sufficient for perfect homogenization does not exceed 5 minutes, usually 4 minutes. The adjuvanted vaccine is sterile filled into vials and sealed with rubber stoppers and metal liners. Store at 4 to 6 ° C until use.

The vaccine of the invention has the following composition:

8.5

Culture medium containing 10 µl of virus mutants in 1 ml

Aujeszky's disease-CNCTC Ao 5/78, inactivated 0.15% betapropiolactone 72 parts

A mixture of E.ooli having 0 antigens 157, 147.64, simultaneously β in the presence of LT enterotoxin and K 88 antigen. inactivated with 0.4% formol 20 parts

Aguasorb hydrophilic anhydrous 8 parts

Penicillin and streptomycin and 500 µl, respectively. gamma / ml Fungizon 5 µm / ml

2. Tissue culture medium containing 1 ml of 10 TCKD Aujeszky's Disease Virus CNCTC Ao 5/78, inactivated 0.35% betapropiolactone 73 parts

A mixture of E.coli having 0 antigens 149, 141,147 and Salmonella choleraesuia, wherein at least one of the E.coli strains (0149 »K 88 a, c,) produces LT enteroxin and has a K 88 antigen, inactivated 0.4% formolene 20 parts by parts

Aguasorp hydrophilic anhydrous

Penicillin and streptomyoin a and 1000 µl, respectively. gamma / ml Fungizon µg / ml

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The innocuousness of the vaccine is verified both during its manufacture and for the finished product. During manufacture, broth and blood agar sterility tests are checked under aerobic and anaerobic culture conditions for the sterility of the bacterial suspension for each strain used in the vaccine (verified by 1% of the volume of each strain). The safety of the combined adjuvant vaccine is verified by a three-rabbit test, by which 2 ml of the vaccine is administered intramuscularly and the clinical condition of the animal, including incl. temperature curves. The vaccinated rabbits also serve to verify vaccine efficacy by simultaneously evaluating titers of neutralizing antibodies at least 11,16 and if the test animals are protected from infectious load after revaccination (titers 128 to 256). Titers of agglutination antibodies are evaluated by direct agglutination reaction β by individual strains boiled or inactivated formol. A vaccine that results in titers of agglutinating antibodies of at least 1 and 80 against the individual strains present in the vaccine (calculated on the actual dilution of serum without the addition of antigen) is considered satisfactory, with the greatest emphasis being on reactions with formalized antigens.

The presence of LT enteroxin in the preparation is evaluated quantitatively before inactivation by titrating the bacterial filtrate to VERO in the cells. A formulation having a titre of at least 64 is considered suitable.

For the use of the combined vaccine against Aujeszky's disease virus and G-rods, which was applied during the disease of piglets and sows, the following loss of loss occurred:

1st quarter 1977 - 700 deaths 27,25% of births

IX Quarter 1977 - 259 to death 11.10% of births

III. Quarter 1977 - 162 deaths 6.03% of births

In the following period the infection was no longer occurring.

Claims (3)

SUBJECT OF THE INVENTION Combination vaccine against Aujeszky's disease and G-intestinal pathogenic microbes, characterized in that in 1 ml it contains 10 ⁸ ^ _and g 10 '' TCID inactivated mutants of the virus 5 8 ^5θ Aujeszky's disease CNCTC AO 5/78 and 10 ^? up to 10 inactivated bacterial G-individuals, e.g., enteropathogenic Escherichia coli and / or salmonella, in a lipiode adjuvant. 2. A method according to claim 1, characterized in that the Aujeszky's disease virus CNCTC AO 5/78 is cultured on tissue cultures, most preferably on cells of a stable pork kidney (PK) line in a serum-free maintenance medium at a temperature of 36-37 ° C. for 18 to 48 hours, depending on the complete disintegration of the infected cells and then, after separation of the cell debris, inactivated by betapropiolactone, homogenized at a temperature of up to 42 ° C with a lipoid adjuvant and 10? To 10? Inactivated G-individuals, in particular enteropathogenic Escherichia coli or salmonella. 3. A method according to claim 2, wherein the adjuvant used in the vaccine is a resorbable lipid mixture which is mixed with the viral suspension most preferably at 199 865 at a temperature of 30-37 ° C with the addition of a bacterial suspension of G-microbes whose antigenic potency was enhanced by repeated disruption of their bodies by freeze-thawing.