CS252736B1 - Vital attenuated vaccine against parvoviral enteritis of minks and method of its preparation - Google Patents

Description

The invention relates to a live attenuated parvovirus enteritis vaccine for mink and a process for its preparation, and to a vaccine component of a bivalent lyophilized vaccine also against distemper prepared on cell cultures.

Mink parvovirus enteritis is a massive digestive tract disease that is clinically manifested by anorexia, malaise, vomiting and diarrhea, which may be bloody. The animals have a fever and, as a rule, a reduced leukocyte count is detected in hematology. The disease causes significant losses in the large-size mink, with mortality accounting for 20 to 50% of the total number of animals. When introduced into the breeding it affects all ages of animals, but especially the young, the onset is steep and progress very fast ·

The disease is caused by a Parvoviridae virus whose particles are isomeric, uncoated and contain deoxyribonucleic acid. The virus replicates in the cell nucleus, and only at certain stages of the cell cycle, making it in vitro difficult to grow.

Specific prophylaxis is based on passive immunization by administration of hyperimmune serum or immune serum globulins, but active immunization is most commonly used. Initially, vaccines (inactivated and live) prepared from cat panleucopenia virus were used for mink, as it was assumed that the two viruses are antigenically related but not identical (FLAGSTAD, A., Paneline Feline Virus and Mink Enteritis Virus. Acta vet. Scand., 18, 1977,: 1-9; CARMAN, PS, POVEY, RC: Comparison of the viral proteins of Canine parvovirus-2, Mink enteritis virus and Feline panleukopenia virus, Veterinary Microbiology, 8, 1983,: GORHAM, JR - HARTSOUGH, GR - BURGER, D. - et al .: The preliminary use of attenuated feline panleukopenia virus to flow cats against panleucopenia and mink against enteritis virus Cornell Vet., 1965,: 559 to 566 POLLOCK, RVH: The Parvoviruses, Piart I. Feline Panleukopenia virus and Mink enteritis virus Compendium on continuing education for practicing veterinarian, Vol. 6, 1984, 3: 227-236.

Later, the principle was adopted that the optimal solution would be if a homologous vaccine was prepared for each disease. Inactivated vaccines prepared from virulent stem cells are used (CARMAN, S. - POVEY, C .: The Inactivated Mink Enteritis Virus Vaccine In Four Preparations To Provide Canine Parvovirus II. Can. J. comp. Med.,

1982, 46: 47-50; SHEN, D.T. - GORHAM, J.R. - RYLAND, L. M. - et al .: Using Jet Injection to Vaccinate Mink and Ferrets Against Canine Distemper, Mink Virus Enteritis, and Botulism, Type C. Vet. Med. (Laughing Animmal Clinician, 1981,: 856-859), but have recently been dominated by live vaccines containing virus attenuated by serial passage on cell cultures. A disadvantage of inactivated vaccines is that their efficacy is significantly influenced in particular by the presence of maternal antibodies and the content of the effective antigen. Immunity lasts only a short time - a few months while protecting only against subclinical infections, but it is important for the control of the infection that such vaccination does not prevent intense virulent virus release into the environment (CARMICHAEL, LE-OLIN, JM: Immunization strategies in puppies - why Compend. Contin. Educ. Work Vet., 5, 1983, 12: 1,043-1,051; POLLOCK,

R.V.H. - CARMICHAEL, L, E,: Canine viral enteritis. Vet. Clin. North. Amer., 13,

1983, 3: 551-556).

In active immunization, live vaccines prepared from attenuated homologous strains gain gradual predominance. After their application, the levels of specific antibodies reach a level as after natural infection with a virulent strain. Antibodies are detected already on day 3 after inoculation and persist for at least 2 years (CARMICHAEL, LE-JOUBERT, JC-POLLOCK, RVH: A modified live canine parvovirus vaccine. II. Irnmune response. Cornell Vet., 73, 1983 ,: 13) KALIN, DE-EMERGY, JB-SMITH, MJ-SPOTTS, AM: Safety and efficacy of modified-live canine parvovirus vaccine, Vet Med Smáli Anim. Clin., 78, 1983 ,: 1,739-1 746).

Offspring from immune mothers are protected against infection by maternal antibodies, of which 10% are transplacental and 90% colostromal. On the other hand, these antibodies adversely affect the immune response to the administration of live or inactivated vaccines (CARMICHAEL, LE-JOUBERT, JC-POLLOCK, RVH: A modified live canine parvovirus vaccine, fl. Immune response. Cornell Vet., 73, 1983): BAKER, JA-ROBSON, DS-GILLESPIE, JH-ET AL: Cornell Vet., 49 (1959), 185-167, CARMICHAEL, LE-ROBSON , D.

S. - BARNES, F. D .: Transfer and decline of maternal infectious canine hepatitis antibody in puppies. Why. Soc. Exp. Biol. Med., 109, 1962, 677-681).

Experimental and practical experience suggests that vaccination of pups at the age of about 16 to 18 weeks is a precondition for achieving long-term protection.

The method of cultivating parvovirus in cell cultures for the propagation of viral material for the preparation of experimental vaccines has been described by veterinarians (GORHAM, JR - HARTSOUGH, GR - BURGER, D. - ET AL. Cornell Vet., 55, 1965, 559-556, MOCHIZUKI, M. - KONISHI, S. - OGATA, M.: Studies on Feline Panleucopenia, II Antigenicities of the Virus, Jpn, J. vet Sci., 40, 1978, 4: 375-383; CARMICHAEL, LE-JOUBERT, JC-POLLOCK, RVH: A modified live canine parvovirus strain with novel plague characteristics I. Viral Attenuation and Dog Response Cornell Vet., 71 , 1981, 4: 408-427; CHURCHILL, AE: A potency test for inactivated laughing animal parvovirus vaccines using chicks, J. Biol. Standard., 10, 1982,: 1-8; NARA, PL-WINTERS, K.- RICE, JB et al .: Systemic and Local Intestinal Antibody r esponse in dogs given both infective and inactivated canine parvovirus. Amer. J. vet. Res., 44, 1983, 11: 1,989-1,995; BERGMAN, R. - SUNDQUIST

B. - STROMAN, L .: Production of Feline Parvovirus Vaccine Using Monolayer Cell Systems in Roller Flasks and Microcarriers. Develop. Biol. Standard., 55, 1984, 77-78.

In particular, they used primary cultures of feline and canine embryonic cells or stably cell lines prepared therefrom. The cells were usually infected 4 to 6 hours after they were plated in culture flasks. Since virus multiplication was not accompanied by regular development of cytopathic changes, they checked the virus content in tissue fluids by the presence of intranuclear bodies, by immunofluorescent method, or by hemagglutination titer height. Cell infections were often used with saline rinse and replacement of the culture medium. However, large-scale vaccine production technology requires at least a number of operations and, as far as possible, direct, prompt and convincing microscopic control of virus propagation based on specific cytopathic changes.

This task was solved as follows:

A live attenuated parvovirus enteric mink vaccine, a monovalent, phyophilized, or vaccine component of a bivalent lyophilized vaccine, also containing distemper, containing at least 10 ³ TKID 50 of the attenuated mink enteric virus strain MEVA-BN 63/82 CAMP V-292 was prepared per ml. virus strain and, in the case of a bivalent vaccine, the distemper virus strain CDV-F-BN 10/83 CAPM V-289 also contains at least 10 ³ TKID 50 of distemper virus particles.

Said virus strain was adapted to cultures of stabile feline cells

FE. As the growth medium, mini252738, essentially essential medium according to Eagle, enriched in bovine serum, was used. the virus was propagated at 36-38 ^° C for 4-8 days. After making clear cytopathic changes, the viral material was mixed with the lyophilization medium to contain at least 10 ⁴ TKID 50 mink enteritis virus particles per ml.

Mink enteritis virus strain designated MEVA-BN 63/82 and collection number CAMP V-292, deposited at the Czechoslovak Collection of Microorganisms at the Veterinary Research Institute in Brno, Hudcova 60, was obtained from a flood virulent virus isolated from infectious materials via serial passages to a feline cell line designated " FE. The homogeneity of the viral population and the stability of the immunobiological properties of the attenuated strain were achieved by isolating the line by a six-fold repetition dilution method.

The strain MEVA-BN 63/82 CAPM V-292 is characterized by the following immunobiological characteristics:

1. Following the defined culture conditions, virus multiplication on a stable FE cell line is accompanied by the development of significant cytopathic changes containing 10 '· ⁵ to 10 ⁵ · ⁶ TKID 50. ml- ¹ .

2. The vaccine virus is harmless to all age categories of mink, and pups without maternal antibodies can be successfully immunized from the age of 6 weeks.

3. The vaccine virus shall not be excluded from the organism of vaccinated animals and shall not be transmitted to co-susceptible susceptible animals.

4. Vaccination of susceptible animals will induce protection starting on day 4 after vaccine administration.

5. The immunity induced by a single vaccination of animals over 12 weeks of age shall last for at least one year and be accompanied by the presence of specific antibodies.

The stable FE cell line exhibited insufficient growth characteristics in its recovery characterized by slow cell proliferation and inability to form a continuous monolayer. Said cell line properties did not correspond to the claims for intensely multiplying said virus to obtain virus suspensions! with sufficient titers to produce the vaccine. The lack of paste properties of the cell line was also manifested by the lack of development of cytopathic changes after infection with the vaccine strain, which did not allow monitoring and evaluation of the degree of virus propagation. By serial passage of the FE cell line in selected media (MEM, BEM, M199) containing neonatal serum we are po. The 25 passages in MEM medium derived a population of cells that exhibited the following properties: 1. The line can be passaged at a ratio of 1: 3 to 1: 4 at 72-96 hour intervals using a version of trypsin solution.

2. The most suitable growth medium is at least essentially an Eagle medium with an addition of 5 to 15 wt. % bovine. serum.

3. The cell line may be stored frozen at temperatures below -100 ° C, using a protection medium containing 10 to 20 wt. % Dimethylsulfoxide.

4. Vaccine virus multiplication is periodically accompanied by marked cytopathic changes at days 4-8 post infection depending on virus dose.

The virus titres achieved make it possible to efficiently produce a vaccine against parvovirus enteritis of mink.

Selection of culture serum:

Culture serum with good growth properties for said cell line may contain non-specific inhibitors at a maximum of 1: 32 as determined by the haemagglutination-inhibition assay.

Vaccine production procedure:

Method of infection of FE cells with vaccine virus:

A.

FE cell suspension at a density of 5 to 10.

. 10 ^B cells. ml ^-1 in the growth medium is mixed with the vaccine virus at a multiplicity of infection of 0.0001 to 0.001. The virus-cell mixture is incubated at 36-38 ° C for 30-60 minutes. Thereafter, the virus-cell mixture is diluted in a growth medium which is at least essentially essential according to Eagle at pH 6.8-7.3 i and the addition of five to ten masses. ° / o bovine series with the quality indicated in the section "Selection of culture serum".

B.

A vaccine virus inoculum with a multiplicity of infection of 0.0001 to 0.001 is added to the cell suspension in the growth medium, which is immediately poured into a culture vessel. The cell suspension thus prepared to prepare the vaccine is cultured on glass, plastic, in a stationary manner, or in culture. roller bottles, optionally on microcarriers, at 36 to 38 ° C for 4 to 8 days. Virus multiplication is accompanied by marked cytopathic changes.

C.

Infection of adherent cells on the culture surface - after a 4 to 6 hour incubation of FE-line cells at 36-38 ° C. Vaccine virus inoculum is added to the growth medium at a multiplicity of infection of 0.0001 to 0.001. Incubation is continued at 36-38 ° C for 4-8 days. As in A and B, virus multiplication is accompanied by significant cytopathic changes.

A mixture of infectious fluids is obtained when 80-90% of the monolayer exhibits specific changes. The virus suspension thus prepared is checked for the presence of undesirable contaminating microorganisms and the virus content is determined by titration on FE-cell cells. After the cultivation is complete, the viral material is mixed with the lyophilization medium in a ratio of 50 to 75% by weight. % virus to 50 to 25 wt. % lyophilization medium and stored at -15 to -20 ° C until lyophilization. The lyophilization medium may be added just prior to lyophilization, while maintaining the same weight ratios. Infectious fluids containing at least 10 ⁴ TKID 50 are used to prepare the lyophilized vaccine. ml ^-1 virus, bacteriologically sterile. Homogenised virus suspension with lyophilization medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other protein source, dextran or gelatin (BOVARNICK, MR - MILLER, JC - SYNDER, JC: J. Bacteriol., 59, 1950, 509-522] is filled into ampoules or vials, lyophilized, air tightly sealed, and stored at refrigerated temperature. 10 ^{3 -1} TKIDso.ml vaccine virus, which is a single immunizing dose.

Lyophilization medium used according to BOVARNICK, M.R. - MILLER, J.C. - SNYDER, J.C .: The influence of certain salts, aminoacids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol 59, 1950,: 509-522] is also suitable for the lyophilization of other types of vaccine viruses. Therefore, when added to an infectious suspension containing another type of vaccine virus, such as distemper virus, these suspensions may be mixed with each other in an arbitrary ratio and lyophilized.

The following examples are not intended to limit or exhaust:

1. Method of cultivation:

A cell suspension prepared by proteolytic ferments in a growth medium containing a defined amount of amino acids and of vitamins with an addition of 10 wt. % of the serum intended for tissue culture is processed on glass, plastic, in a stationary manner or in roller bottles, or suspended in microcarriers.

2. Method of infection of cell cultures with vaccine virus:

Cell cultures prepared and cultured according to point 1 are infected with the vaccine virus at a multiplicity of infection of at least 0.001 in the following ways:

as well as a cell suspension of FE cells with a density of 10.

. 10 ⁶ cells ^-1 .ml the growth medium was treated with the vaccine virus at a multiplicity of infection of 0.001. The virus-cell mixture is incubated at 37 ^° C for 30 minutes. Thereafter, the mixture of virus and cells is diluted in a growth medium which is at least essentially essential according to Eaglea pH 7.1 with addition of 10 wt. % Mandatory serum.

(b) into a suspension of FE cells of density 5.

. 10 ⁵ . ¹ ml of growth medium was added to the vaccine virus inoculum on the multiplicity of infection of 0.001, and the suspension was infected immediately poured into the culture vessel. The cell suspension thus prepared for vaccine preparation is cultured in Roux bottles at 37 ° C for 6 days. Virus multiplication is accompanied by marked cytopathic changes.

c) Attached cell infection on the culture surface - after culturing FE cells at 37 ° C for six hours, the vaccine virus inoculum is added to the growth medium at a multiplicity of infection of 0.001. Incubation is continued at 37 ° C for 6 days. The multiplication of the virus is, as in i and b, accompanied by significant cytopathic changes.

3. Collection of vaccine virus:

The vaccine virus is produced in growth medium, on cell cultures grown under point 1 and infected under point 2. After extensive cytopathic changes have been established microscopically, the culture is terminated and a suitable lyophilization medium in a ratio of 67 is added directly to the culture vessels. wt. % viral suspension and 33 wt. % lyophilization medium. The mixture is then stored frozen in culture flasks until lyophilized.

252738

Homogenised virus material with the required content of infectious units [at least 10 ⁴ TKID 50. ml ' ¹ ), which does not contain contaminating micro-organisms, is filled into ampoules, lyophilized, sealed and refrigerated. The vaccine prepared according to the above procedure comprises at least 10 ^-3 ° TKID 50. . mto ¹ vaccine virus.

4. Preparation of the combination vaccine:

A lyophilization medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other protein source and gelatin or dextran is also suitable for lyophilization of other types of vaccine viruses. The prepared mink virus enteritis vaccine is mixed with live attenuated canine distemper vaccine at any rate, filled into ampoules, lyophilized, airtight sealed and stored at refrigerator temperature. Bivalent vaccine prepared according to the procedure contains at least ³ TKIDso IQ. mto ¹ distemper vaccine viruses and mink virus enteritis.

Claims (6)

252738 10 Homogenized viral material with the desired content of infectious units [at least 104 TCID50. ml "1), which does not contain contaminating microorganisms, is dispensed into ampoules, lyophilized, sealed airtight and stored in a refrigerator. The vaccine-prepared vaccine vaccine vaccine virus vaccine vaccine vaccine virus preparation contains at least 10 3 TKID 50 ml ml vaccine virus. What is claimed is: 1. A live attenuated vaccine against parvus parasite enteritis, a monovalent, lyophilized, or a vaccine component of a bi-valent lyophilized vaccine, both against and against psinitis, comprising v1 mutated enteric strain of normeMEVA-BN 63/82 virus. CAPM-292 in at least 103 TCID 50 virus particles and in the equivalent vaccine still contains the viVirus strain of CDV-F-BN 10/83 CAPM V-289 in a quantity of at least 103 TCID 50 virus-drug particles. 2. A live attenuated vaccine of step 1, or a vaccine component of a bivalent lyophilized vaccine against distemper, comprising a lyophilization medium in an amount of 25 to 50 wt. % of the total vaccine. 3. A method of producing a vaccine according to clauses 1 and 2, wherein the attenuated enteric strain of mink enteritis is propagated in a stable feline FE cell line in a minimal essential medium of Eagle's pH 6.8 to 7.3 enriched with bovine serum priteplote from 36 to 38 ° C for 4-8 days, until significant cytopathic changes are formed and then the viral material is mixed with the lyophilization medium to contain at least 10 4 TCID 50. ml “1. 4. A method of producing a vaccine according to claim 3, wherein bovine serum is defined as an additive to the culture medium as defined by the non-specific inhibitor content of the corresponding virus. Y N A L E Z U 4. Preparation of the Combined Vaccine: A lyophilizing medium that contains sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or any other source of protein and gelatin or dextran is also suitable for lyophilizing other species vaccines. The prepared viral enteritis virus vaccine is mixed with live attenuated canine distemper vaccine at any rate, dispensed into the ampoule, lyophilized, airtight sealed and stored at refrigerator temperature. The bivalent vaccine prepared in the above procedure comprises at least IQ3 TKID50. ml "1 Vaccine distemper virus and viral mink virus. PŘEĎME T 5. A method of producing a vaccine according to clauses 3 and 4 wherein the infection of the cell with virus production is done either by a cell suspension containing from 5 to 10 @ 10 6. ml "1 cell in growth medium is infected with a vortex at an MOI of at least 0.0001 per cell, incubates at 36-38 ° C for 30-60 minutes, then 10-20 times diluted in growth medium, poured into a culture vessel and cultures at 36-38 ° C until viral harvest, or a cell suspension containing 4 to 8 105 ml of 1 cell in growth medium infects the virus at an MOI of at least 0.0001 cells and incubates at 36-38 ° C C to virus harvesting, a cell suspension in growth medium containing 4 to 8.10 5 is loaded into the culture flasks. ml "1 cells, the cultures are infected directly into the growth medium with a virus at MOI of at least 0.0001 per cell after incubation for 4-6 hours at 36-38 ° C and incubated until virus collection. 6. A method for producing a vaccine according to clauses 1 to 5, characterized in that an incubation medium comprising sachrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or any other source of protein and gelatin or dextran mixture is added to the virus suspension. is stored until freeze-dried without loss of infectious virus content at temperatures above -20 ° C, or lyophilized immediately.