CS252747B1 - Vital attenuated vaccine against parvovirosis of dogs and method of its preparation - Google Patents

Description

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

Dog parvovirosis is a mass disease of the digestive tract 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. Heart disease affects exclusively puppies and is characterized by a rapid and very short course with symptoms of dyspnea, nausea, sighing, cardiac arrhythmia and subsequent death. The disease causes significant losses in dog breeding, with mortality of 20 to 60% of the total number of animals. The disease affects all ages of animals, but especially puppies, with onset and rapid onset.

The disease is caused by a virus of the Parvoviridae family 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, so it is relatively difficult to grow in vitro.

Specific prophylaxis is based on pa252747 gray immunization by administration of hyperimmune serum or immune serum globulins, but active immunization is most commonly used. Initially, vaccines (inactivated and alive prepared from cat panleucopenia virus) were used for dogs, as it was assumed that the two viruses were antigenically related but not identical.

Later it was accepted that the optimal solution would be if a homologous vaccine was prepared for each disease. Inactivated vaccines prepared from virulent strains have been used, 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 active antigen. Immunity lasts only a short time - a few months while protecting only against subclinical infection, but it is important for the control of infection that such vaccination does not prevent intense virulent virus secretion into the environment (CARMICHAEL, LE-OLIN, JM: Immunization strategies in puppies - why Compend. Contin. Educ. Pract. Vet., 5, 1983, 12: 1,043-1,051; POLLOCK, RVH-CARMICHAEL, LE: 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 have been present for at least 2 years (CARMICHAEL, LE-JOURBERT, JC-POLLOCK, RVH .: A modified live canine parvovirus vaccine. II. Immune response. Gornell Vet., 73, 1983,: 13-29, KALIN, DE EMERGY, JB-SMITH, MJ-SPOTTS, AM: Safety and efficacy of modified-live canine parvovirus vaccine. Vet. Med. Smal. Anim. Clin. . 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. II. Immune response. Cornell Vet., 73, 1983,: 13 - BAKER, JA - ROBSON, DS - GILLESPIE, JH - ET AL: Cornell Vet., 49, 1959, 158: 167 - CARMICHAEL, LE - ROBSON,

D. S. - BARNES, F. D .: Transfor and Inheritance of 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 culturing parvovirus in cell cultures to propagate viral material for the preparation of experimental vaccines has been described by several antors (CARMICHAEL, LE-JOUBERT, JC-POLLOCK, RVH: A modified live canine parvovirus strain with novel plaque characteristics. I. Viral attenuation and dog response. Cornell Vet., 71, 1981, 4: 408-427; CHURCHILL, AE: A Initiative Test for Inactivated Laughs Animated Parvovirus Vaccines Using Chicks, J. Biol. Standard., 10, 1982, 1-8; PL - WINTERS, K. - RUCE, JB et al .: Systemic and Local Intestinal Antibody Response in Dogs Given Both Infective and Inactivated Canine Parvoviruses, Amer. J. Vet. Res., 44, 1983, 11: 1989-1995; BERGMAN, R. - SUNDQUIST, B. - STRÚMAN, L .: Production of Feline Parvoviruses Vaccine Using Monolayer Cell Systems in Roller Flasks and Microcarriers, Develop. Biol., 55,

1984, 77-78). In particular, they used primary cultures of feline and canine embryoinal cells, or stably cell lines prepared therefrom. The cells were usually infected 4 to 6 hours after seeding into the 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, the immunofluorescent method, or the hemagglutination titer height. Cell infections were often used with saline rinse and culture medium replacement. However, large-scale vaccine production technology requires at least a number of operations and, as far as possible, a primitive, persistent and compelling microscopic control of virus proliferation based on specific cytopathic changes. This task was solved as follows:

A live attenuated canine parvovirus disease vaccine, a monovalent, lyophilized, or vaccine component of a bivalent lyophilized vaccine, also containing canine distemper, containing in one ml an attenuated canine parvovirus strain CPVA-BN 80/82 CAMP V-290 in at least 10 ³ TKID 50 virus particles and in the case of a blvalent vaccine, the distemper virus strain CDV-F-BN 10/83 CAPM V-289 contains at least 10 ³ TKID 50 of distemper virus particles.

Said virus strain was adapted to cultures of stable feline FE cell lines. As a growth medium, bovine serum-enriched at least essential medium was used in which the virus was propagated at 36-38 ° C for 4-8 days. To produce distinct cytopathic changes, the viral material was mixed with the lyophilization medium to contain at least 10 ⁴ TKID 50 of canine enteritis virus particles. in 1 ml.

Virus strain of parvovirus of dogs with the designation CPVA-BN 80/82 and collection number CAPM V-290, deposited in the Czechoslovak Collection of Microorganisms at the Research Institute of Veterinary Medicine in Brno, Hudcova 60, was obtained from flood virulent virus isolated from infectious materials via serial passages on the 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 above strain CPVA-BN 80/82 CAPM V-290 is characterized by the following immunobiological properties:

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 ⁶ TKID 50. ml- ¹ .

2. The vaccine virus is harmless to all ages of animals, 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 confer 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 acquisition, 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. Lack of development of cytopathic changes after infection with the vaccine strain was also a manifestation of insufficient growth properties of the cell line, which did not allow to monitor and evaluate the degree of virus propagation. By serial passage of the FE cell line in selected media (MEM, BEM, M199) containing neonatal serum, after 25 passages in MEM media, we have derived a cell population that exhibits the following properties:

1. The line may be passaged in a ratio of 1:

: 3 to 1: 4 at 72-96 hours intervals using version-trypsin solution.

2. The most suitable growth medium is at least essentially the medium according to Eagle 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 achieved virus titers allow the effective production of a vaccine against parvovirosis in dogs.

Selection of culture medium:

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.

The suspension cell lines of the FE of the density of 5 to 10.10 ⁶ cells .ml ^-1 in the growth medium was treated 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 mixture of virus and cells is diluted in a growth medium which is at least essentially essential medium according to Eagle having a pH of 6.8 to 7.3 and the addition of five to ten masses. % bovine serum of indicated quality.

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 and immediately poured into the culture vessel. The cell suspension thus prepared for vaccine preparation is cultured on glass, plastic, in a stationary manner, or in rotated flasks, optionally on microcarriers, at 36-38 ° C for 4-8 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 paste 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 to 90 percent 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 can 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, airtight sealed and stored at refrigerator temperature. The vaccine prepared as described above contains at least 10 ³ TKID 50. ml ^-1 vaccine virus, which represents a single immunization dose.

Lyophilic 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 risketsiae. J. Bacteriol 59, 1950, 509-622) 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 any 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 vitamins with an addition of 10 wt. % of the tissue culture serum is cultured on glass, plastic, in a stationary manner or in rotated flasks, optionally in suspension on microcarriers.

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

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

a) the cell suspension density the FE line ⁶ 10:10 .ml ¹ cells in 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 the Eagle medium at pH 7.1 with the addition of 10 wt. % bovine serum.

(b) a vaccine virus inoculum with a multiplicity of infection of 0.001 is added to a suspension of FE cells of a density of 5.10 ⁵ ml ^-1 in the growth medium, and the infected suspension is immediately poured into a culture vessel. The cell suspension thus prepared for vaccine preparation is cultured in Roux flasks at 37 ° C for 6 days. Virus multiplication is accompanied by marked cytopathic changes.

(c) attachment of adherent cells 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, determined microscopically, the culture is terminated and a suitable lyophilization medium in a ratio of 67 wt. . % viral suspension and 33 wt. % lyophilization medium. The mixture is then stored frozen in culture containers until lyophilized.

Homogenised virus material with the required content of infectious units (at least 10 ⁴ TKID 50 ml ^-1 ), free from contaminating microorganisms, is filled into ampoules, lyophilized, sealed and refrigerated. The vaccine prepared as described above contains at least 10 ³ · ⁰ TKID 50.

. ml ^-1 vaccine virus.

4. Preparation of the combination vaccine:

The 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 canine parvovirosis vaccine is mixed with live attenuated canine vaccine at 1 'v / v ratio, filled into ampoules, lyophilized, sealed and refrigerated. The bivalent vaccine prepared as described above comprises at least 10 ³ TKID 50. ml ^-1 vaccine viruses · canine distemper and canine parvovirus.

Claims (6)

1. A live attenuated vaccine against parvovirosis in dogs of a monovalent, lyophilized or vaccine component of a bivalent lyophilized vaccine also against distemper, characterized in that it contains in 1 ml an attenuated canine parvovirus strain CPVA-BN 80/82 CAPN V-290 in an amount of at least 10 ³ The TKID 50 of the virus particles and, in the case of a bivalent vaccine, also comprises distemper virus strain CDV-F-BN 10/83 CAPM V-289 in an amount of at least 10 ³ TKID 50 of the distemper virus particles. 2. A live attenuated vaccine according to item 1, or a vaccine component of a bivalent lyophilized vaccine also against distemper, comprising a lyophilization medium in an amount of 25 to 50% by weight. %, based on the weight of the whole vaccine. 3. A method for producing a vaccine according to claim 1 or claim 2, wherein the attenuated canine parvovirus strain is propagated on a stable feline cell line of FE cells in minimal essential medium of Eaglea pod at pH 6.8-7.3 enriched with bovine serum at 36-38 ^Q C for 4-8 days, until a significant cytopathic changes and the virus material mixed with the freeze-drying medium to contain at least 10 ⁴ TKIDso .ml ^'1. 4. A method according to claim 3, characterized in that bovine serum with a defined content of non-specific inhibitors against the corresponding virus is used as an ingredient in the culture medium. INVENTION 5. The vaccine production method according to items 3 and 4, characterized in that the infection of the cells for the production of the virus is carried out either by a cell suspension containing from 5 to 10.10 ^s . ml per ¹ cells in growth medium are infected with virus at an MOI of at least 0 0001 per cell, incubated at 36-38 ° C for 30-60 minutes, then diluted 10-20 times in growth medium, poured into culture flasks and cultured at at 36 to 38 ° C and collecting the virus and the cell suspension containing 4 to 8.10 ^5th ml of ¹ cell in growth medium is infected with a virus at an MOI of at least 0.0001 per cell and incubated at 36-38 ° C until harvesting the virus or the cell suspension is seeded in a growth medium containing 4 to 8 × ^{10 5} . . ml of ¹ cell, and the cultures are infected directly into the growth medium with a virus at an MOI of at least 0.0001 per cell after incubation for 4-6 hours at 36-38 ° C and incubated until the virus is harvested. 6. A method according to any one of Claims 3 to 5, characterized in that after the incubation the virus suspensions are added with a lyophilization medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other protein source and gelatin or dextran and the mixture is stored frozen at temperatures above -20 degrees Celsius until lyophilized or lyophilized immediately.