FR2535204A1 - PROCESS FOR THE PREPARATION OF A VACCINE ADJUSTED, INACTIVE, AGAINST COCAR PLESTER - Google Patents

Abstract

PROCESS FOR THE PREPARATION OF AN INACTIVE VACCINE FOR THE CONTROL AGAINST COCAR PLAGUE, WHICH IS AN EPIZOOTIA AND WHICH MAINLY AFFECTS PIGS. THIS PROCESS CONSISTS OF CULTIVATING A VIRULENT STRAIN OF COCAR PLAGUE VIRUS "PHYLAXIA" DEPOSITED AT THE HUNGARIAN NATIONAL INSTITUTE OF PUBLIC HEALTH (OKI) UNDER NUMBER 0023682, ON A CELL CULTURE PREPARED IN A KNOWN MANNER. EVEN, THEN TO DISABLE THE CULTURE AND MIX THE INACTIVE ANTIGEN WITH AN INCOMPLETE OILY ADDITIVE AND TO HOMOGENIZE. THE INACTIVE VACCINE OBTAINED IS AS ACTIVE AS LIVE COMMERCIAL VACCINES, WHILE DOING NOT PRESENT THE SERIOUS DISADVANTAGES OF THESE LIVE VACCINES.

Description

The present invention relates to a process for the preparation of a

  adjuvanted vaccine, inactivated, against

cocar plague.

  The plague of cocar which owes its name to the one who described it first, the Hungarian ALADAR AUJESZKY (Zbl Bakt.

  Orig 1903 I, 32, 353-357) is a disease of domestic animals

  which has been known for a long time and which causes heavy losses It is mainly the pig herd, and

  especially young animals, which is affected.

  As the age of piglets increases, life-threatening diseases become rarer, it appears

  However, losses in the profitability of animal

  because of diseases of the respiratory system,

  central nervous system as well as their complications.

  Animals affected by the virus can carry the pathogen for a long time (1-2 years) and evacuate the virus under stress (eg from birth) and spread the disease ( J Am Vet Med Ass 176, 998-1000 and 1345-1347, 1980) Infected sows frequently lose embryos by abortion (Acta Vet Acad Sci Hung 12, 17-23, 1962) Infected pigs also play an important role in the transmission of cocar plague

  ruminants (beef, sheep) (Acta Vet Acad Sci Hung.

12, 53-58, 1962).

  The procedures for combating cocar plague are based on the fact that the animals most susceptible to the disease, namely piglets that are still breastfed, must be protected during the first weeks of life.

  their lives by specific antibodies.

  In Hungary, a technology has been developed to

  production of a preventive hyperimmune serum for the protection

  against the disease (Acta Vet Acad Sci Hung 7, 423-

  427, 1957) The hyperimmune serum obtained from pigs, reduces, when used at the desired moment, the losses, however its drawbacks are important: the preparation of the serum is very expensive, and moreover it is eliminated from the organism within a very short time (in 10 to 14 days) Its use in young animals and animals that have completed their growth, does not enter into question because of the high price of serum. The next step of protection against the plague of

  cocar consisted of the introduction of active immunization.

  Strains of virus have been attenuated in variable measures and in different ways and have been used for the production of vaccines In Hungary, a live vaccine

  which contains the living virus with a marked virulence

  born, has been on the market since 1961 This vaccine is used

  for the active immunization of pigs, ruminants and

  dogs (MAL 16, 42-45, 1961) The economic losses caused by

  Since the beginning of the vaccination program (Cah Med Vet 43, 240-247, 1974), the use of live viral vaccines has some disadvantages: 1) After immunization of pregnant sows , the

  antibody content of colostrum and milk ensures an immunity

  passive group of piglets, which persists for 4 to 6 weeks

  During this time, it is not possible to carry out the immunization with a live viral vaccine, because even the low level of colostral immunizing substances included in the disappearance is sufficient to inhibit the

  multiplication of the virus to the extent necessary for the

active immunity.

  (2) For these reasons, it may be found in the flock of animals in which vaccination has failed and in an infected flock those individuals whose immunity has

  failed ensure the perpetuation of virulent virus.

  3) The use of live vaccines is prohibited in some countries and in some cases this prohibition extends to the introduction of meat from animals

  immunized with live vaccines This limits the possibilities

  3 - export balances of the countries in which

  immunization with live vaccines.

  4) Although the attenuated vaccine made in Hungary

  has so far proven to be harmless and genetic-

  other countries report cases in which the viral strains used to produce the attenuated vaccine

  virulence (The Vet Bull 43, 465-480, 1973, Dtsch Tierarzt.

  Wschr 85, 381-420), which resulted in vaccine reactions

  too violent in animals treated with the vaccine.

  ) The attenuated vaccine used in Hungary is not completely safe in dogs and mink and

does not immunize cats at all.

  Due to difficulties in using the live vaccine, attempts have been made to produce an inactivated vaccine Formalin, ultraviolet radiation, epropiolactone, saponin, Crystallized Violet, and the like have been inactivated by inactivation. ethanol, and we used

  adjuvant, aluminum hydroxide gels (Acta Microbiol.

  Hung 2, 151-160, 1954; Arch Vet Vet Med 21, 501-507, 1967) Vaccines prepared in this manner, however, are insufficiently active. More modern methods use ethylethyleneimine for inactivation, and DEAE-dextran (Zbl Vet Med B 20, 127) as adjuvant. -138, 1973),

  however DEAE-dextran has triggered

  cal. The object of the present invention is to produce an inactive, cocar plague-active vaccine which: 1) is as active or more active as vaccines

  attenuated against the plague of cocar.

  2) is safe for all susceptible animals, regardless of breed and age, and provides adequate immunity; 3) is also suitable for active immunization of

  piglets still benefiting from colostral immunity.

  This object is achieved according to the present invention in that the strain of cocar plague virus called "Phylaxia" deposited at the Hungarian National Institute of Public Health (Orszagos K 5 zegészség Ugyi Intézet) under the number 00236 / 82, which was selected from the different strains of cocar plague virus because of its immunizing properties, is inoculated and cultured on a primary culture of fibroblastic cells

  The viral solution obtained is inactivated

  by a known inactivating agent, such as ethylene

  imine preferably, then mixed with a known adjuvant,

  preferably the incomplete oily adjuvant "Phylaxia".

  The excellent immunizing action of prepa-

  according to the present invention is due to many factors. The virulent "Phylaxia" strain which

  national epidemic is characterized by the biological properties

  following: it can be successfully grown too

  in cell cultures than in animals receiving

  it has a high infective titre and is

  good immunization ability The process of inactivation

  aided management contributes to the excellent activity, its

  meters (temperature, concentration of the inactivating agent,

  duration) allowing the protective antigens to be preserved.

  Ethylene imine, which is used as an inactivating agent, is cyclized before being used in the form of a dilute aqueous solution. The immunizing action

  inactivated antigens in a controlled manner is further

  incomplete adjuvant "Phylaxia".

  The vaccine prepared in the manner to be described hereinafter in the Examples is used for the immunization of pigs, cattle and dogs, and the immunization is

repeated preference.

  The invention will be better understood by means of the

  description which will follow, which refers to

  examples of implementation of the method object of the present invention. It should be understood, however, that these examples of implementation are given solely by way of illustration of the subject of the invention, of which they

  in no way constitute a limitation.

Example 1

PREPARATION OF THE VACCINANT VIRUS

  Rabbits weighing from 2 to 2.5 kg, intracerebrally, of a strain of cocar 'Phylaxia' plague virus isolated from a case of acute epidemic, are deposited at the Hungarian National Institute of Public health

  under number 00236/82. The characteristic symptoms are

  The brain is extracted under sterile conditions and is used to prepare a 10% suspension with a physiological saline solution. This suspension is used for the

multiplication of the virus en masse.

Example 2

PREPARATION OF THE VIRAL SUSPENSION

  The virulent vaccinating virus obtained according to Example 1 is cultured on a primary culture of cells in a single layer of fibroblasts of chicken embryos or testes of calves, in boxes of Roux of a volume.

  2000 ml (or in the bottles in rotation of a

  "Rolling" solution). A growth solution for cell culture, a Hanks solution or a

  MEM-H solution for maintenance and multiplication

  viruses, an Earle solution or MEM-E solution is used. The composition of these solutions can be

  found in the following publications: J Nat Cancer Inst.

  8, 83, 1974; Proc Soc. Exp. Biol Med 71, 196, 1949; Science 139, 1959; Bakacs, Farkas: Orvosi virologia, Medicina, Budapest) The solution of Hanks or the MEM-H solution used for the growth of the cell culture in a single layer is supplemented with 10% of calf serum, while solution used for the multiplication of the virus does not contain calf serum After the cell culture in a single layer has been contituted, ie after confluence of the cells, the growth solution is removed. cultures of the holding solution containing virus The cultures are incubated at 37 C until the total destruction of the cells (after about 48 to 72 hours) The virus suspension obtained is suitable for the production of the inactivated vaccine, if it is sterile and if its infective titre, as measured in

  a cell culture, is at least 107 TCID 50 / ml.

Example 3

INACTIVATION AND FORMULATION

  The virus suspension obtained in accordance with Example 2 is inactivated with a solution of ethyleneimine, the concentration of which is 200 g / ml, at 37 ° C. for 6 hours. The ethyleneimine solution is prepared just

  before use, by cyclization of bromethylamine.

  The ethylene imine is then decomposed with sodium thiosulfate and the pH of the inactivated suspension is adjusted to 7.2-7.4. To control whether the suspension is effectively inactivated, a cell culture prepared as described above is seeded. top, with this suspension, then vaccinate with this suspension rabbits weighing 2-2.5 kg

  and sheep weighing 25-30 kg.

  The viral material is then mixed with an equal volume of incomplete oil adjuvant "Phylaxia" The aqueous virus suspension and the oily adjuvant are treated in a high pressure emulsifier, to give a stable emulsion which does not decompose in the temperature range is between 0 and + 45 C. The finished vaccine is introduced sterilely into ampoules which are

closed away from the air.

Example 4

  VERIFICATION OF VACCINATION REACTIONS

  The vaccine obtained in the manner just described is inoculated intramuscularly at a dose of 2 ml to the following experimental animals: rabbits, sheep, dogs, piglets at 2 days of age, 3 weeks and 8 weeks, sows in the last third of gestation.

  Pigs are monitored for 3 months and other animals

  for 6 weeks During this time, he did not

  no reaction to vaccination.

Example 5

PIGLET IMMUNIZATION

  Piglets of 2 days, 3 weeks and 8 weeks were vaccinated by the intramuscular route, each with 2 ml of vaccine. After 3 weeks,

  treatment was repeated. In treated animals, it appears that

  specific antibodies that protect piglets from intranasal infection with a dose of 2 xl D 6 TCID 50 of virulent cocar plague virus, while control animals, which do not had not been vaccinated, died when they were infected with the same

dose.

Example 6

PRODUCTION OF COLOSTRAL IMMUNITY

  Pregnant sows are vaccinated

  during the last third of their gestation, intramuscularly

  2 ml of the vaccine obtained according to Example 3 The treatment is repeated 3 weeks later, choosing the dates of vaccination so that the second vaccination takes place 2 months before the date

scheduled for calving.

  In the colostrum of treated sows, specific antibodies appear and, therefore, a mean neutralization titre of 1:16 can be measured on the seventh day in the blood serum of suckling pigs. protection against infection with the virulent virus at a dose of -8- 4 x 10 TCID 50 Control vaccinations at the 30th, the 90th and the 90th day (when the colostrum content of antibody decreases) have resulted in the treated group, mortalities of 18, 26 and 40% respectively, while piglets from non-immunized mothers all died. The specific antibodies contained in the milk thus protect young animals at the age at which they are present.

more susceptible to the disease.

Example 7

  VACCINATION OF PIGLETS DURING THE INTERVAL OF

  TIME O COLOSTRAL IMMUNITY EXISTS

  Piglets from immunized mothers

  according to Example 6, which benefit from colostral immunity, are vaccinated on the 7th and 28th days each with 2 ml of the vaccine prepared according to Example 3. Specific antibodies are formed with a high titre (1:44) At 60 days of age, piglets are infected intranasally with virulent virus at a dose of 2 x 10 TCID 50 Experimental animals show no symptoms while controls (this is unvaccinated piglets, from non-immunized mothers) died 100% As a result, it is possible to successfully immunize even piglets for breastfeeding, which benefit from

  colostral immunity, by the inactivated vaccine.

Example 8

FREQUENCY OF VACCINATION

  Breeding sows that were vaccinated twice during pregnancy, as described in Example 6, were re-vaccinated with 2 ml of vaccine (booster vaccination), whereas they were again pregnant. the antibody titer again increased sharply in the colostrum of treated sows, and their piglets were protected against control infection with 2 x 10 TCID 50. In piglets of unvaccinated sows during their second mortality 9 - was higher (35% at 6 weeks of age) This

  shows that in the interest of sufficient colostral protection

  health, sows must undergo a booster vaccination

during pregnancy.

Example 9

IMMUNIZATION OF SHEEP

  Sheep aged 3 and 6 months were immunized

  with 2 ml of the vaccine prepared according to Example 3.

  Fifteen days after vaccination, the animals were infected with virulent cocar plague virus at one dose

  1000 TCID 50 All animals in the unvaccinated control group

  were born, while the treated sheep did not

you have no symptoms.

Example 10

  One month old and six month old dogs were

  treated with the vaccine prepared according to Example 3.

  The treatment was repeated 3 weeks later Fifteen days after the second vaccination, the treated dogs were infected with the virulent virus, as was an untreated control group. The antibody titre in the blood of the immunized dogs is 1: 32, these dogs did not become ill, while in the control group, the dogs presented

  symptoms characteristic of the disease and are dead.

  As is apparent from the foregoing, the invention

  tion is not limited to those of its modes of implementation, of realization and of application which have just been

  described more explicitly; it embraces

  milk all variants that may come to the mind of the technician in the field, without departing from the frame or the

scope of the present invention.

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Claims (4)

  1) Process for the preparation of an inactivated vaccine for the control of cocar plague, characterized in that the strain of virulent cocar plague virus "Phylaxia" deposited at the Hungarian National Institute of Public Health is cultivated (OKI) under the number 00236/82, on a   cell culture prepared in a manner known in itself.   same, in that one inactivates this culture and in that one   mix the inactivated antigen with an oily adjuvant plet and homogenize.   2) Process according to claim 1, characterized in   what is used for seeding the cell culture, the first to third passages of the suspension   rabbit brain virus deposited.   3) Process according to claim 1, characterized in   what is inactivated the virus suspension by ethylene-   imine prepared immediately before use, by cycli- bromethylamine.   4) Process according to Claim 1, characterized in that the incomplete oily adjuvant is used,   the incomplete immunoadjuvant "Phylaxia".