RU2129442C1 - Method of preparing vaccine against carnivores plague - Google Patents

Abstract

FIELD: microbiology, vaccines. SUBSTANCE: invention relates to preparing live culture vaccines against carnivores plague. For vaccine preparing a new attenuated strain "VNIIVVim-88" (registration number is "VNIIVVim Dep" in VGNKI collection) is used. Highly sensitive subline CV-1 of an African green monkey transplanted kidney cells is used as a cellular system for virus culturing and for drying medium - a medium of the corresponding composition based on protein products hydrolysis. Monolayer cell culture of subline CV-1 is maintained by serial passage and virus culturing in transplanted cell culture is carried out under roller conditions. Collection of virus-containing material is carried out at virus titer up to 3.5-4.5 lg TCD 50/cu. cm. Material is mixed with protective medium at volume ratio 3:2. Value pH is corrected at the range 6.4-6.8 and material is lyophilized. Method exhibits high productivity, simplified procedure, availability and easily realized in industrial scale. Vaccine produced by new method shows the enhanced effectiveness by immunogenicity, safety, stability at storage, harmless and low cost. EFFECT: improved method of preparing, high quality and properties of vaccine. 1 tbl, 3 ex

Description

 The invention relates to veterinary virology and biotechnology, and in particular to methods for producing live culture vaccines against carnivorous plague, and can be used in enterprises of the biological industry.

 A known method of producing live culture vaccines against carnivore plague (PE), including the adaptation of attenuated virus strains to primary cell cultures from tissues of organs of carnivores from canine and mustache families, African green monkey kidney tissue and chicken and quail embryo tissues, the cultivation of vaccine in these cultures virus and its stabilization by subsequent lyophilization in a suitable medium (US Pat. Cl. 424-89 N 2965544, 1960; N 3080291, 1963; CL 435-237 N 4224412, 1980. German Patents CL 30h6 N 1138888, 1963; N 1235505, 1967. Author St. S CP cl C 12 N 7/08 N 527 072, 1977;. Cl A 61 K 39/12 N 1287592, 1984)..

 There is also known a method for producing culture virus vaccines against PE, characterized in that as the cell system for growing the virus, lines of transformed, non-oncogenic transplanted cells from the honorary tissue of animals are used: green monkey (Vero N 4647), rhesus monkey (LLC-MK2), dogs and Syrian hamster (BHK-21S, BHK-21 13S) (US Pat. CL 424-89 N 3836626, 1974: N 4004974, 1977; N 4351827, 1982; N 5000951, 1991. England patent CL A 61 K 39/175 N 2053679, 1981. CPP patent, class A 61 K 39/175 N 82518, 1983. Grachev V.L. et al. // Virusol. - 1990. - N 1).

 The second method has undoubted advantages over the first, since in the production of vaccines there is no need to use expensive, non-standard and unsafe primary cell cultures. The main disadvantage of the second method is the low immunogenicity of the obtained vaccines, their vaccination dose varies from 1 thousand to 1 million TCD 50 (PFU 50). Another disadvantage of this method is that the reproduction of the virus of emergency in a number of transplantable cultures (LLC-MK2, BHK) proceeds within 3 to 7 (sometimes 10) days and is not accompanied by the development of a specific cytopathogenic effect (CPD), which in turn makes it difficult to evaluate the results cultivation, complicates and increases the cost of the manufacturing process and control of the vaccine (US Pat. CL 424-89, N 3836626, 1974; N 4351827, 1982. England patent CL A 61 K 39/175 N 2053679, 1981).

 Closest to the proposed method of obtaining a dry culture virus vaccine containing an attenuated strain of EPM virus PE, grown in a primary culture of tissue cells of Japanese quail embryos (EP), and drying stabilizers - sorbitol and gelatose (US Patent. CL 424-89 N 4224412, 1980) .

The cultivation of the EPM strain is performed in a roller unit with a multiplicity of infection of 0.0001 - 0.001 TCD 50 / cell suspension of EP cells with a seed concentration of 0.8 - 1.0 million per 1 cm3. As the growth medium, a 0.5% lactalbumin hydrolyzate in Hanks solution or medium 199 with 10% vol. serum of cattle (cattle), and as a support medium - environment 199. The cultivation of the virus is carried out for 11-14 days at a temperature of 36.5 - 37.5 ^o C and the speed of rotation of the vessels 12 - 25 about. in hour. Before lyophilization, the virus is mixed in a 1: 1 volume ratio with a protective medium containing equal volumes of a 20% sorbitol solution and a 6% gelatose solution. The titer of the vaccine virus in a dry preparation is from 2.8 to 3.8 1g TCD 50 / cm3 (Technical Specification TU 10.19. 87-89 for the vaccine against culture dry carnivore plague from the strain EPM. - Temporary instructions for the manufacture and control of the vaccine, associated against viral enteritis, botulism, pseudomonosis and carnivore plague, approved 08.08.92).

 The main disadvantage of the closest analogue is the use of EP as a cell substrate in the cultivation of the virus, which, as you know, are not free from foreign microorganisms (endogenous viruses and microplasmas). The elimination of this drawback is possible by creating specialized, constantly monitored poultry farms with a healthy livestock, but this extremely increases the cost of producing live vaccines and controlling the contamination of quail herds, eggs and biological product. Moreover, the indications used cannot guarantee the complete absence of contaminants in the objects of control.

 The second disadvantage of the known method consists in the non-standard nature of the used cellular substrate, and therefore, the non-standard of the virus and vaccine, which is due to the inability to obtain large batches of cell cultures homogeneous in their properties from the tissue homogenate and requires additional costs for monitoring the suitability of each tissue source. The yield of cells from one EP is low, usually not more than 40%, which also leads to high costs for obtaining industrial series of cell culture and increases the cost of the vaccine.

Other disadvantages of the analogue include a long cultivation process (10-14 days), which complicates and increases the cost of production of the drug. The vaccine from the EPM strain creates intense and prolonged immunity after administration by the carnivores in a large range of doses: minks, sables, ferrets - from 120 to 120 TCD 50, foxes and Arctic foxes - from 240 to 2400 TCD 50, for dogs - from 630 to 6300 TCD 50 of the vaccine the virus
The technical result of this invention is to obtain a harmless, highly immunogenic, storage stable culture vaccine against PE.

 The essence of the invention lies in the fact that from the strains of the virus of emergency in the manufacture of the vaccine use the new attenuated strain "VNIIVViM-88", as a cell system - highly sensitive subline CV-1 transplantable kidney cells of the African green monkey, from the drying media - an environment of the appropriate composition based on protein hydrolysis products.

 The proposed method for producing a live culture vaccine against PE includes: maintaining a monolayer culture of CV-1 subline cells by serial passaging, growing the virus in an inoculated cell culture under roller conditions, collecting virus-containing material when the virus titer reaches 3.5 - 4.5 1g TCD 50 / cm3, mixing it with a protective medium in a volume ratio of 3: 2, adjusting the pH of the mixture in the range of 6.4 - 6.8 and lyophilization.

The new vaccine strain, called “VNIIVViM-88” (registration number “VNIIViM-88 Dep” in the VGNKI collection), was isolated from the EPM strain by adapting it to the CV-1 subline through a series of intermittent passages and subsequent cloning using the limiting dilution method. The new strain is characterized by the following properties:
1) The strain has morphological properties characteristic of the state of emergency virus of the genus Morbillivirus of the family Paramyxoviridae.

 2) The strain propagates in a transplanted CV-1 subline cell culture with the development of a pronounced CPD in the form of symplasts and subsequent cell destruction, the virus titer in this culture reaches 3.5 - 4.5 1g TCD 50 / cm3. Without adaptation, the virus multiplies in a culture of cells from the tissues of chicken and quail embryos, causing a specific CPD and accumulating in titers up to 3.0 - 4.0 1g TCD 50 / cm3.

 3) The strain is cultured in a monolayer of transplantable cells of the CV-1 subline under stationary and roller conditions; under optimal conditions, the maximum biological activity is reached after 44 - 52 hours.

 4) Irregularly forms plaques on HAO chicken embryos.

 5) Not contaminated by bacteria, fungi, mycoplasmas and extraneous viruses.

 6) Harmless to laboratory and susceptible animals, non-contagious, irreversible; non-pathogenic to humans.

 7) Induces the formation of intense and prolonged immunity against plague in vaccinated fur animals (minks, thorzofretoks, arctic foxes, foxes, raccoons) and dogs.

 8) Causes the formation of virus-neutralizing antibodies in titers 1: 8 - 1:32 and above; does not possess hemagglutinating properties.

 9) Genetically stable for 10 consecutive passages in CV-1 subline cell culture.

 In contrast to the known method, the virus is grown in a transplanted culture of cell subline obtained in VNIIVViM from the original culture of transplanted CV-1 cells from an African green monkey kidney (number 43.2 in the catalog of the VNIIVViM cell collection). In this case, the expensive, non-standard, low-tech and contaminated primary culture of EP cells is excluded from the technological process of manufacturing the vaccine.

 To grow the virus, a monolayer cell culture of the CV-1 subline is used, obtained by serial passaging with a transfer coefficient of 1: 3 - 1: 5 within 50 consecutive passages. To ensure standard production of the vaccine, a cryobank of working cells of the CV-1 subline was created, containing cells of passage 17 in the amount of 165 million, characterized by the stability of morphological, growth and karyological indicators for the absence of tumorigenicity and contamination by bacteria, fungi, mycoplasmas and extraneous viruses.

The new strain and used transplanted cells are easily cultured under roller conditions on Igla-MEM non-deficient nutrient medium with cattle blood serum (4–5 and 9–10% vol., Respectively), do not lose their properties after low-temperature storage and “refreshment” - restoration . At a temperature of 36.5 - 37.5 ^o C, the formation time of the monolayer of CV-1 subline cells and the duration of the virus cultivation are 44 - 52 hours (each), which is extremely beneficial for production.

 The use of a rapidly propagating strain and a highly productive cell line can increase manufacturability and productivity, reduce economic costs and reduce the cost of a new vaccine, and therefore provides its commercial advantage.

 The harmlessness, areactogenicity, high antigenic and immunogenic activity of the vaccine manufactured according to the new method are guaranteed by the conditions of adaptation and maintenance of the strain "VNIIVViM-88", in which stability of its attenuated properties, genetic and immunobiological characteristics is achieved. At elevated doses (up to 2000 TCD 50 / cm3), the vaccine from the VNIIVViM-88 strain is harmless and does not cause any deviations from the physiological norm in the behavior and food intake of the immunized fecivorous animals, signs of plague disease and death.

 The grafting dose of the vaccine from the VNIIVViM-88 strain contains: for minks and thorzofret — not less than 100 TCD 50 / cm cubic meter, for foxes, arctic foxes and raccoons — not less than 200 TCD 50 / cm cubic meter, for dogs — not less than 500 TCD 50 / cm3 When administered intramuscularly in a vaccination dose, the new vaccine does not cause post-vaccination reactions and complications. Immunity in fur animals occurs on the 14th - 21st day after a single vaccination, in adult dogs - on the 10-14th day after a single injection, and in puppies of dogs - after the second vaccination at the same time. Revaccination of adult carnivores is carried out annually in the same doses.

 The vaccine from the strain "VNIIVViM-88" creates 100% protection against plague among fur animals and dogs vaccinated from two months old. In vaccinated animals, virus-neutralizing (BH) antibodies to the PE virus are detected: in dogs and thorsofret - in titers 1:16 - 1: 128, Arctic foxes, foxes and raccoons - 1: 8 - 1:32. The duration of immunity in carnivores is at least 12 months.

In contrast to analogues, a protective medium based on products of complete and incomplete hydrolysis of proteins containing enzymatic peptone or whey protein hydrolyzate or enzymatic muscle protein hydrolyzate, sorbitol or sucrose, hydrolyzed gelatin is proposed to stabilize the physical and immunobiological properties of the new vaccine. A positive effect is achieved by mixing the virus-containing material with a vaccine virus titer of 3.5 - 4.5 1g TCD 50 / cm3. and a protective environment in a volume ratio of 3: 2. The proposed environment is guaranteed to provide a stable eutectic zone of the drug in the range from minus 42 to minus 35 ^o C, minimal loss of the titer of the virus during lyophilization (not more than 0.5 lg), high shelf life of the vaccine.

The table below shows the physical and immunobiological properties of 5 series of dry culture vaccine against emergency prepared according to the new method both from long-stored (series 1) and freshly obtained virus-containing material (series 2 and 5), immediately after manufacture and after 12 - 13 month of storage of the drug at a temperature of 4 - 6 ^o C. The titer of the virus in a freshly prepared preparation is 3.0 - 4.2 lg TCD 50 / cm3. The shelf life of the vaccine is at least 12 months at a temperature of 4 - 6 ^o C, at a storage temperature from minus 50 to minus 40 ^o C it increases to 18 months.

 Thus, the proposed method provides a dry culture vaccine against PE from the VNIIVViM-88 strain, which is characterized by harmlessness, high immunogenicity, the absence of contamination by extraneous microorganisms (bacteria, fungi, mycoplasmas and pathogenic for carnivorous viruses), and high preservation of its useful properties. A new method of manufacturing a vaccine is simple to implement, affordable and can be implemented on an industrial scale.

 To confirm the advantages of the present invention provides examples of specific performance.

 Example 1. Obtaining virus-containing material.

To obtain a vaccine, the virus is grown in a monolayer culture of CV-1 subline cells on a roller unit with a productivity of 48 dm cube. material in one cycle. The transferred culture is obtained by serial passaging with a reseeding coefficient of 1: 4. As the growth medium used Igla-MEM with 9.5% vol. serum cattle. At an incubation temperature of 37 ^o C and a vessel rotation speed of 11 vol. per hour, a monolayer of cells is formed after 48 hours.

The cultivation of the virus is carried out for 48 hours at a temperature of 37 ^o C, regularly looking at the infected culture under a microscope. As a supporting medium use Igla-MEM with 4.5% vol. serum cattle. At the end of the process, the virus-containing material is monitored for contamination with bacteria, fungi, mycoplasmas and the titer of the virus in the CV-1 subline cell culture. In our experience, from one roller unit carrying 96 roller vessels, 48 dm cube was obtained. sterile virus-containing material with a virus titer of 4.25 lg TCD 50 / cm3, which is used to make a dry culture vaccine.

 Example 2. The manufacture of the vaccine.

 For immunization of dogs and fur animals, one series of vaccine is prepared in ampoules and in vials, respectively.

 Before lyophilization, the virus-containing material is mixed with a protective medium in a volume ratio of 3: 2. Using a 6% (by weight) aqueous succinic acid solution, the pH of the mixture is adjusted to 6.6.

 The vaccine is packaged in 1 cm cube. in glass ampoules, 5 cm3 each. - in glass bottles and freeze-dried. Ampoules with dry vaccine are sealed off, bottles are hermetically sealed.

 The resulting preparation is controlled in terms of appearance, color, solubility, moisture content, contamination by bacteria, fungi, microplasmas, biological activity (virus titer according to CPD in CV-1 subline cell culture), harmlessness (for thorzofretc at a dose of 3000 TCD 50), reactogenicity and immunogenicity (in experiments on thorsofret). The test results of the vaccine series 3 and 5, prepared respectively for immunization of dogs and fur animals, are presented in the table.

 Both series of prepared vaccines meet regulatory requirements and are suitable for use.

 From 48 dm cube 80,000 doses for dogs or 2 million doses of vaccine for immunization of fur-bearing animals can be made of the virus-containing material obtained on only one roller unit (Example 1) by the above method.

 Example 3. Tests of a vaccine in a production environment.

 To test the new vaccine under production conditions, 580 dogs, 18640 minks, 14032 Arctic foxes and 628 raccoons were immunized. The vaccination dose of the VNIIVViM-88 vaccine was: for dogs - from 1000 to 3000 TCD 50 / cm3, minks - from 200 to 470 TCD 50 / cm3, Arctic foxes and raccoons - from 400 to 940 TCD 50/2 cm cube vaccine virus. Post-vaccination reactions and complications were not observed among immunized animals.

 9-12 months after vaccination of fur-bearing animals, BH antibodies to the PE virus were detected in titers sufficient to protect against field virus at the level of 1: 8 - 1: 32 in 82% of samples and 1: 4 in 13.5% of serum samples blood. During vaccine tests in vaccinated dogs and fur animals, there were no cases of breakthrough immunity during the year of observation.

 An interdepartmental check of the immunobiological properties of the vaccine was carried out with positive results, there is a positive decision of the State Veterinary Commission, technical specifications TU 9384-005-0000864-96 for the pilot industrial series of the drug and temporary instruction for its use are approved. Pilot production of the vaccine has been established according to the new method.

 The vaccine from the strain "VNIIVViM-88" can be used for the preventive immunization of domestic and service dogs, fur animals in large animal complexes and farms. The use of the vaccine is safe in veterinary and sanitary terms. The introduction of the proposed method into production will expand the range of preventive drugs and increase the effectiveness of anti-epizootic measures against carnivorous plague.

Claims (1)

A method of obtaining a vaccine against carnivore plague, including growing the virus in a cell culture, collecting virus-containing material, mixing it with a protective medium and lyophilization, characterized in that the virus is an attenuated strain of VNIIVViM-88 virus of the carnivorous plague virus, from cell cultures - transplantable CV-1 subline cell culture from an African green monkey kidney, from protective media — a medium containing an enzymatic peptone or whey protein hydrolyzate or an enzymatic muscle protein hydrolyzate, sor um or sucrose and hydrolyzed gelatin, with collection of virus-containing material is performed when the accumulation level virus 3.5 - 4.5 lg TCD 50 / cm ³ and mixed with the protective medium in a volume ratio of 3: 2.

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