GB2129684A - A process for the production of an inactivated adjuvated vaccine against Aujeszky disease - 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

SPECIFICATION A process for the production of an inactivated, adjuvated vaccine against Aujeszky disease This invention concerns a processforthe production of an inactivated, adjuvated vaccine against Aujeszky disease.

Aujeszky's disease has been known for a long time as a disease of domestic animals that causes heavy economic losses. It received its name from Aladsr Aujeszky, a Hungarian who first described the disease (Zbl. Bakt. Orig. 19031,32353-357). This disease causes great losses primarilywith pig stock, especially piglets. With age, the death rate due to Aujeszky disease decreases but the disease ofthe respiratory organs, ailments ofthe nervous system and the resulting complications significantly reduce the eco nomyofanimal husbandry. Animals which have been infected with the virus may carry the viral pathogen for a relatively long time (1-2 years) and under certain stress effects such as pregnancy the disease is spread by defecation (evacuation) of the virus (J. Am.Vet.

Med. Ass. 176,1980998-1000; 1345-1347). Often, infected sows miscarry or have stillborn pig lets. (Acta Vet. Acad. Sci. Hung., 72,1962, 17-23.) The infected pigs play a great role also in the spreading of Aujeszky disease in ruminating animals such as cattle and sheep (Acta. Vet. Acad. Sci. Hung., 12, 1962,53-58, J.

Am. Vet. Med. Ass. 176. 1980, 1001-1003). The infected sows may evacuate via the milk both the virus and the colostral antibodies (Acta. Vet. Acad. Sci. Hung., 7, 1957,273-276, Acta. Vet. Sci. Hung., 13, 1963, 111-118).

The protectivetreatments against Aujeszky disease are basically directed at protecting the highly susceptible sucking piglets with specific antibodies againstthe disease in that most disease-prone period, thefirstweeks oftheir lives.

In Hungary as a first step of protection againstthe disease a technology has been worked out forthe production of a hyperimmune serum (vaccine) with the aim of a prophylactic treatment of sucking piglets (Acta. Vet. Acad. Sci. Hung., 7,1957,423-427). The hyperimmune serum is produced from pigs and can reduce the losses if used at an appropriately early time but its disadvantages are considerable: the production oftheserum is very expensive and the serum is rapidly evacuated from the organism, typically in 10-14 days. The high cost ofthe serum makes it impossible or uneconomic to use it for young and full grown (mature) animals.

The next step in the protection against Aujeszky disease was the introduction of an active immunisation process against the disease, in the course of which variouslyweakened virusstrainswere produced for the purposes of manufacturing vaccines. In Hungary since 1961 a live virus vaccine of reduced virulence has been marketed for use in the active immunisation of pigs, ruminants and dogs (may. 16, 1961,42-45). The amount of damage and economic loss caused by the disease has been greatly decreased in Hungary since the introduction ofawidespread vaccination programme (Cah.Med. Vet 43,1974, 240-247), but nevertheless the utilisation of a live virus vaccine has a number of disadvantages: 1. After immunising pregnant sows the antibody content of the colostrum and of the milk provides a passive immunity for a time which varies between 4-6 weeks with individual animals. During this time it is not possible successfully to immunise by using a live virus vaccination because the level of colostral antibodies evacuated is low and the antibodies are capable of preventing the achievement of that degree of proliferation ofthe vaccine virus as is required for active immunisation.

2. Forthe above reasons the stock may contain animals in which the vaccination was unsuccessful and these individuals of defective immunity assure the survival ofthe virulent virus in the infected stock.

3. In some countries the utilisation of live vaccine is officially prohibited and in certain cases this prohibition is extended to the meat originating from animals that have been immunised with a live virus.

Thus the possibilities of meat exportfrom countries using live vaccines are restricted.

4. Although the Hungarian attenuated vaccine has hitherto proved to be harmless and genetically stable, observers abroad have given accounts of unexpected increases in the virulence of various attenuated vaccine virus strains in certain cases (The Vet. Bull. 43, 1973,465-480, Dtsch. Tierarzt. Wschr., 85,1987, 381-420) manifesting themselves in a serious vaccine reaction in vaccinated animals.

5. The Hungarian attenuated vaccine is not completely harmless for dogs and minks; it does not immunise cats.

Because ofthe problems associated with live vaccine, several attempts have already been made to produce inactivated vaccine, utilising formaldehyde, ultraviolet irradiation, heat, betapropiolactone, saponine, gentian violet (crystal violet) or ethanol as an inactivating agent and aluminium hydroxide gel as adjuvant (Acta Microbiol. Hung., 2,151-160,1954, Arch. exp. Vet. Med. 17,595-613,1963, Arch. Vet. 5, 107-14,1968, Arch. Exp. Vet. Med. 21,501-507, 1967).

These inactivation processes and adjuvants have not, however, resulted in sufficiently effective vaccines.

Certain researchers have described a modern technology of producing a vaccine adjuvated with DEAE dextran which has been inactivated by ethyl-ethylene imine,thetechnologyutilising a modern alkylating inactivating agent that exerts its effect on the nucleic acid of the virus (Zbl. Vet. Med., B, 20,127-138,1973).

However, the DEAE dextran adjuvation triggered an allergic reaction.

Taking the above into consideration, an aim of the invention is to produce an inactivated vaccine against Aujeszky disease which: 1. as regards its efficaciousness, gives good results and preferably gives the same or improved results as live attenuated Aujeszky vaccine; 2. is relatively harmless for animals, preferably of any age and species, susceptible to the disease and is capable of establishing active immunity of an appropriate level; and 3. may also be used forthe active immunisation of pigs possessing colostral immunity and maythus continuously assure a uniformly high level of specific antibodies.

This task has been sought two be solved by choosing from all the Aujeszkyvirus strains availableto us a strain deposited in the Hungarian National Institute of Public Health on 25th June, 1982 under No.00236/82, the so-called 'Phylaxia' Aujeszky virulent virus strain, on the basis of its immunising ability, and propagating it by inoculating a cell culture, e.g. of primary chicken embryofibroblast cells, with it. The thus obtained viral liquid is inactivated with a known inactivating agent, expediently ethylene-imine, and then mixed with a known adjuvant, preferably with an incomplete oily adjuvant known as 'Phylaxia'.

Several factors play a significant role in the excellent immunising effect of this vaccine. The so-called Phylaxia virulentvirus strain used in the production of the basic material and originating from a Hungarian epidemic may be biologically characterised by its efficient ability to reproduce, by the ineffective titre which is uniformly high equally in cell culture as in susceptible animals, and by good immunisability in which latter a decisive role is played bytheinactivation process preserving the protective antigens. In the course of comparative inactivation-kinetic tests, from among the known materials an optimal choice was made as to the identity and concentration of the inactivating agent; and the temperature and time parameters of the inactivation process have also been optimised as to effectiveness.The ethylene imine expediently used for inactivation is, according to our process, a dilute, and thus a harmless, aqueous solution which before use is cyclised e.g. from bromoethylamine. the immunising effect ofthe gently inactivated antigen is enhanced by the 'Phylaxia' incomplete oily adjuvant, the composition ofwhich was elaborated by us.

The thus obtained vaccine was used in the manner described below for active immunisation of infectionprone pigs, cattle and dogs, advantageously in such a mannerthatthe vaccination was expediently repeated.

The invention is further described by way of the Examples below without, however, limiting its scope to the implementations described therein.

Example 1 Rabbits of 2-2.5 kg weight were infected intracelebrally by a so-called Phylaxia virulent virus strain deposited at the Hungarian National Institute of Public Health under No.00236/82, which strain had been isolated from an acute Aujeszky disease epidemic.

Accompanied by symptoms characteristic of Aujeszky disease,the brains of rabbitsthatdied after an incubation period of 72-96 hours were removed under sterile conditions and were used for large-scale virus breeding byforming from them a 10% suspension prepared with physiological kitchen salt solution.

Example 2 The Phylaxia virulentAujeszky virus strain produced according to Example 1 was propogated in a primary chicken embryo fibroblast or primary calf testicle monolayer cell culture, in 2000 ml Roux bottles or in the rotary bottles of a rolling apparatus. The cell culture may take place in cell lines (BHK-21, or IB-RS-2 etc.) or in suspended or microcarriercultures also. As growth-stimulating liquid ofthe cell cult (colonies) Hanks solution or MEM-H solution may be used; as a preserving and virus-propagating liquid, Earle solution or MEM-E solution is used the composition of which is given in the following literature references: J.

Nat. Cancer Inst. 8: 83, (1974)-Proc. Soc. exp. Biol.

Med. 71,196, (1949) Science 139(1959), or Bakács- Farkas: Medical Virology, Medicina Publishers, Budapest (1965), pages 129-30. The Hanks or MEM-H solution used for cell culture are used with a supple ment of 10% young cattle salt and the virus propagating solutions do not contain any blood plasma. After the formation of a monolayer cell culture the nutrient or growth liquid is removed and then a viruscontaining preserving liquid is poured on the cultures.

The cultures are incubated at a temperature of 37"C until a complete cell-damaging effect sets in (after48 to 72 hours). The harvested virus suspension becomes usableforthe production of inactivated Aujeszky vaccine when it is sterile and its infective titre measured in the cell culture is at least 107TCID50/ml.

Example 3 The virus suspension produced according to Example 2 is inactivated. The inactivation is preferably performed with an ethylene imine solution of 200 uglml concentration, which has been cyclised before use from bromoethyl amine, at37 Cfor6 hours. The ethylene imine is neutralised with sodium thiosulphate and then the pH of the inactivated virus suspension has to be adjusted to 7.2-7.4. The process in which the live virus content becomes inactivated should be monitored by inoculation of the abovedescribed cell culture as well as by vaccination of rabbits of 2-2.5 kg and lambs of 25-30 kg weight.

In the interest of increasing the immunisability of the virus it is mixed togetherwith the same quantity of 'Phylaxia' incompletely oily adjuvant. The aqueous phase suspension and the oily adjuvant are transformed with the aid of a high pressure apparatus into a stable emulsion which does not separate out in the temperature range of 0 two +45"C. The inactivated and adjuvated vaccine is filled into sterile bottles and hermetically sealed.

Example 4 Rabbits, lambs, dogs, two-day old, three-week old and eight-week old piglets as well as sows in the last third of their pregnancy are intramuscularly and repeatedly inoculated with 2 ml doses of vaccine produced as described above. The rabbits and sheep inoculated with the experimental vaccine were kept under observation for 6 weeks whilethe pigs were keptunderobservationfor3 months during which time no vaccination reaction of any kind manifested itself.

Example 5 Two-day old, three-week old and eight-week old susceptible piglets were intramuscularly vaccinated twice at a three-week interval with 2 ml doses ofthe vaccine prepared according to Example 3. In the treated pigs specific antibodies formed which protected the vaccinated animals against a superimposed intranasal infection with a 2 x 106TCID50 virulent Aujeszky virus, while non-vaccinated susceptible pigs died as a result ofthe superimposed infection.

Example 6 Susceptible pregnant sows were vaccinated twice at a three-week interval in the lastthird of their pregnancywith 2 ml doses ofthe vaccine prepared according to Example 3 in such a way that the immunisation should be completed two weeks before the anticipated birth. In the colostrum ofthe immunised sows specific antibodies appeared as a result of which the virus neutralising titre ofthe blood plasma of the pigs, measured atthe age of seven days, was on average 1:16. This level of antibodies provides adequate protection against a superimposed infection with a virulentvirus of 2 x 06TClD50 titre.Control infections performed on the 30th, 60th and 90th days resulted in a death rate of 18,26 and 40%, respectively, for piglets of a decreasing level of colostral antibodies. With control piglets of the same age derived from non immunisedsowsthedeath rate was 100%.

Thus the specific antibodies received via the milk protect the suckling pigs against the disease at their most susceptible age.

Example 7 Piglets having colostral immunity derived from susceptible sows immunised according to Example 6 were vaccinated twice at the age of 7 and 28 days with 2 ml doses of the vaccine prepared according to Example 3. Specific antibodies were formed at a high titre (1 :44) in the piglets. These piglets were intranasally infected at the age of 60 days with a virulent virus of 2 x 106 TCID50 titre. The piglets which had colostral basic immunity and were vaccinated twice survived the infection without any symptoms while 100% ofthe control pigs which were not vaccinated and which originated from non-vaccinated sows died. Thus sucking piglets having colostral immunity may be successfully immunised with the inactivated vaccine; the uniform high level ofthe specific antibodies assures the homogeneous immune state ofthe stock.

Example 8 The sows twice vaccinated according to Example 6 were vaccinated once in a subsequent pregnancy with a booster vaccination using a 2 ml does ofthevaccine prepared according to Example3. The effectofthe booster vaccination was that the level of specific antibodiesinthecolostrum ofthesowswasagain high and thus when their piglets were given a control superimposed infection with a virulent virus of 2 x 106 TCID50the same results as described in Example 6 were obtained.The piglets of sows which did not receive a booster vaccination during their second pregnancy showed a higher percentage of death rate as a result of a superimposed infection (35% at the age of 6 weeks) and therefore in the interests of sufficient colostral protection sows having a basic immunity should be given a booster vaccination during each pregnancy.

Example 9 Lambs aged one month, three months and six months were immunised with a 2 ml dose of vaccine prepared according to Example 3 and then infected on the 15th day after the vaccination with a virulent Aujeszkyvirus of 1000 TCID50.100% ofthe control unvaccinated lambs died as a result ofthe infection while the immunised lambs survived the superimposed infection without any symptoms.

Example 10 One-month old and six-month old puppy dogs were immunised twice at a three-weekly interval with a 2 ml dose of the vaccine prepared according to Example 3. On the 15th day afterthe second vaccination the vaccinated puppies as well as the unvaccinated control dogs were infected with a virulent virus of 106 TCID50. The specific antibody level of the vaccinated dogs was high (1 :32) and they survived the infection without any symptoms while the unvaccinated control died showing symptoms characteristic ofthe disease.

Claims (10)

1. Aprocessforthe production of an inactivated vaccine against Aujeszky disease which process comprises propagating the Aujeszy virus strain deposited at the Hungarian National Institute of Public Health underthe number 00236/82 in a cell culture that is susceptible to the virus, inactivating the virus and homogenising the inactivated antigen with an adjuvantto prod uce the vaccine

2. A process as claimed in claim 1, wherein the adjuvant is an oily adjuvant.

3. A process as claimed in claim 2, wherein the oily adjuvant is Phylaxia incompletely oily immunoadjuvant.

4. A process as claimed in any one of claims 1 to 3, wherein the virus strain is inoculated into the cell culture in the form of a suspension of a brain of a rabbit that contracted the said strain of Aujeszky disease.

5. Aprocessasclaimed in claim 4,whereinthefirst to third passages ofthe rabbit brain are used to inoculate the cell culture.

6. A process as claimed in any one of claims 1 to 5, wherein the cell culture is a culture of primary chicken embryofibroblastcells or of primary calf testicle monolayer cells.

7. A process as claimed in any one of claims 1 to 6, wherein the virus is inactivated by ethylene imine.

8. A process as claimed in claim 7, wherein the ethylene imine was obtained bycyclising bromoethyl amine priorto use.

9. A process substantially as hereinbefore described in Examples 1 to 3.

10. A vaccine produced by the process claimed in any one ofclaims 1 to 9.