RU2129443C1 - Live vaccine "ks" against classic swine plague and method of prophylaxis of classic swine plague - Google Patents

Abstract

FIELD: veterinary science, agriculture, biotechnology. SUBSTANCE: invention relates to a method of preparing vaccines and a method of prophylaxis of classic swine plague. Vaccine has an attenuated strain "KS" of classic swine plague virus and dried milk as a stabilizing agent. Vaccine shows the enhanced activity against classic swine plague and its administration at high doses ensures to abolish diseases in unfortunate large swine breeding farms. EFFECT: enhanced effectiveness of vaccine, improved method of prophylaxis. 3 cl

Description

 The invention relates to biotechnology and for the production of vaccines for the prevention and treatment of classical swine fever (CSF). For the specific prevention of CSF, the LK-VNIIVViM vaccine is known containing the lapinized Chinese vaccine strain "C" obtained by adapting and propagating it in a testicular tissue culture of lambs (RU 2064302C, A 61 K 39/187, 07.27.96) (B. S. Romanova). A disadvantage of the known vaccine and the method of its use is the inability to eliminate CoES in dysfunctional industrial enterprises.

 The essence of the invention also lies in the fact that the vaccine is administered in large doses repeatedly. Repeated administration of the vaccine in large doses (hypervaccination) allows eliminating CSF in dysfunctional farms.

 The technical result of the invention is to significantly increase the activity of a live vaccine against CSF and the possibility of its use to eliminate the disease in large pig farms.

 The invention consists in the following. The vaccine contains an attenuated strain of "KC" virus CoES and a stabilizer (drying environment).

The KC vaccine is a white, porous, dry mass obtained by lyophilization of a mixture of virus-containing culture fluid with a drying medium. The mass fraction of moisture in the dry vaccine is in the range of 1-4%. Dry vaccine in vacuum bottles at 2-6 ^o C retains the initial activity for at least 12 months. With prophylactic vaccination, a single dose of the KC vaccine is 1000 ImD ₅₀ , and with the elimination of CoES, 100000-1000000 ImD ₅₀ .

Strain "KC" was obtained from the Chinese lapinized strain "C" of the CSF virus by prolonged passage and selection in a culture of cells of non-porcine origin. Phenotypically, the strain "KC" differs from the vaccine strain "C" in that it does not cause a hyperthermic reaction in rabbits when administered intravenously. It belongs to the genus of pestiviruses, the family of flaviviruses. The diameter of the virions is 40-60 nm; floating density 1.12-1.13 g / cm ³ ; sedimentation coefficient 140S. The genome consists of 12,300 nucleotides and encodes a polyprotein of 4,000 amino acids. The genome of the "KC" strain differs from the genome of other known vaccine strains of the CSF virus in that it has approximately 1000 nucleotide substitutions.

 In the shell of the virion 3 glycoprotein with a molecular weight of 23-57 kD. The nucleocapsid contains a protein with a molecular weight of 25 kD. For the synthesis of neutralizing antibodies and protective immunity, a glycoprotein with a molecular weight of 55 kD is responsible.

For safety and immunogenicity, the strain "KC" fully meets the requirements of OIE (OIE) and differs from similar vaccine strains ("C", "GP", "Tiverval" and others) only in a higher concentration of the virus obtained by reproduction in cell culture ( in 1 ml of at least 10 ⁸ doses infecting the cell culture - 10 ⁸ TCID _{50 / ml} ).

Strain "KC" has the following biological properties:
multiplies without a cytopathic effect;
detected by antibodies labeled with peroxidase (PLA) or polymerase chain reaction (PCR);
safe for pigs of any age, including pregnant sows and newborn piglets;
not reversible (6 consecutive passages of the vaccine virus on frost-free piglets);
does not cause persistence in vaccinated pigs and quickly disappears from the blood (after 7-10 days);
quickly (after 3-4 days) creates a long intense immunity (at least 2 years, observation period);
free from contamination with viruses, bacteria, fungi and mycoplasmas;
stored for a long time in dry and liquid form at different temperatures (+ 2-70 ^o C).

 Commission tests have confirmed the full compliance of the strain "KC" with the requirements and were the basis for its deposit in the collection of microbial cultures VGNKI. The strain "KC" assigned registration number: strain "KC-DEPT".

 The invention is illustrated by the following examples.

 Example 1. To prepare the vaccine, use the strain "KC", which is propagated in cell culture. The culture virus is combined with the drying medium in the ratio necessary to obtain a vaccine with a given immunogenic activity. The resulting mixture was poured into 1 ml into 3 ml vials and lyophilized. Vials with a dry vaccine under vacuum are sealed with rubber stoppers and wrapped in aluminum caps.

Example 2. Under optimal cultivation conditions (method of growing cells and virus, multiplicity of infection, duration of incubation, treatment of infected culture), the accumulation of strain "KC" reaches a high level of 8.0-9.2 lg TKID _{50 / ml} , which is 100-1000 times the titer of all known vaccine strains used in the manufacture of live vaccines against CSF. A high degree of correlation of the biological activity of the KC strain was established during titration in PK-15 cell culture (PLA, TKID _{50 / ml} ) and during titration in pigs (ImD _{50 / ml} - an immunizing dose that protects 50% of vaccinated pigs). 1 IMD ₅₀ corresponds to 1-3 TKID ₅₀ .

Example 3. When optimizing the manufacturing conditions of the dry vaccine "KC", the effect of various stabilizing media and the lyophilization regimen is studied. As cryoprotectants, substances widely used for these purposes are used: sucrose, lactose, mannitol, gelatin, peptone, lactalbumin hydrolyzate, etc. The use of these substances in various combinations and concentrations allows the vaccine to be stabilized, although its infectious (immunogenic) activity decreased by 1 , 3-2.5 lg. The use of dried cow's milk as a cryoprotective medium was preferred. The decrease in the infectious activity of the vaccine does not exceed 0.7-1.0 lg. Drying of the KC vaccine with such a protective medium showed high reproducibility. So from 30 series of dry vaccines, 27 series had an activity of 7.2-7.7 lg TKID _{50 / ml} , the rest - 6.7-7.0 lg TKID _{50 / ml} . At 2-6 ^o C dry vaccine almost maintained its original activity for 12 months.

 Example 4. Epizootological activity of the vaccine "KC" was tested in two farms of industrial type (25000-27000 pigs; reproduction-fattening) unfavorable for CoES.

 In both farms, CoES proceeded similarly against the background of systematic prophylactic immunization with the LK-VNIIVViM vaccine according to the instructions and was characterized mainly by high morbidity (40-50%) and death (25-30%) of piglets especially in the post-weaning period. After the diagnosis of the disease (CSF) was established, the “KC” vaccine was used in the farms at an increased dosage and within the time period stipulated by the instructions for its use. As a result of hypervaccination for 8-10 months, the disease of CSF stopped. Thus, it was proved that in large dysfunctional farms, CoES can be eliminated only by hyper vaccination. This goal can be achieved if the dose of the vaccine is 100 or more times higher than the usual dose and if the vaccination is given on time.

Claims (3)

1. A vaccine against classical swine fever, containing an attenuated strain of swine fever virus and a stabilizer, characterized in that as an attenuated strain it contains a strain of VGNKI N "KS-DEP" of swine fever virus with an infectious activity titer of 7.0 lg TCID _{50 / ml} and more.  2. The vaccine according to claim 1, characterized in that it contains dried cow milk from the stabilizers. 3. A method for the prevention of classical swine fever, including the intramuscular administration of a vaccine, characterized in that the vaccine used is the vaccine according to claim 1 and is administered to pigs in dysfunctional farms many times in large doses of 100,000 - 1,000,000 IMD ₅₀ (hypervaccination).