CS252746B1 - Live attenuated vaccine against, panleukopenia cats and its preparation - Google Patents

Abstract

The solution relates to a live attenuated vaccine against feline panleukopenia and a method for its preparation on cell cultures. The vaccine is intended for protective and emergency vaccination of cats against the above disease. The live attenuated vaccine against feline panleukopenia is lyophilized and is characterized in that it contains in 1 ml at least 103 TKIDso particles of feline parvovirus, strain FPVA-BN 110/83 CAPM V-291 adapted to cell cultures. The attenuated viral strain of feline panleukopenia is propagated on a stable feline FE cell line in Eagle's minimal essential medium enriched with bovine serum at a temperature of 37 °C, for 4 to 8 days. After the formation of significant cytopathic changes, the cultivation is terminated and the viral material is mixed with the lyophilization medium so that it contains at least 104 TKIDso. ml"1 virus particles. After lyophilization, the vaccine virus content in 1 ml is at least 103 TKID50.

Description

The invention relates to a live attenuated vaccine against cat panleucopenia and to a process for its preparation.

Cat panleucopenia 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 a reduced leukocyte count is detected in the hematalog.

Ochoreinia causes significant losses in cat conditions, with mortality accounting for 20 to 50% of the total number of animals. The disease affects all ages of animals, but especially the young, with the onset being abrupt and progressing rapidly (JOHNSON, R. H .: Feline panleucopenia. Vet. Rec., March 29th, 1969 ,: 338-340).

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 in certain stages of the cell cycle, making its in vitro cultivation quite difficult (OREREYY, K.J. - WHITAKER,

A. M .: Development of feline cell lines for the growth of infectious enteritis / panleucopaenia / virus feline. J. Hyg. Camb., 67, 1969, 115; FLAGSTAD, A .: Studies on the growth of panleucopaenia virus in cell cultures. Acta. Path. Microbiol. Scand. Sect.

B, 1975, 83: 71-78; O‘SHEA, J.D. - STUDDERT, M. J .: Growth of autonomously replicating parvovirus / Feline Panleukopenia /: Kinetics aind morphogenesis. Arch. Virol. 57 (1978): 107-122; ENDO, M. SHINAGAWA, M. - GOTO, H. - ET AL .: Growth Characteristics of Feline Panleucopenia Virus in Synchronized Kitten Kidney Cells. Jpn. J. Vet. Sci., 43, 1981, 65-70).

Specific prophylaxis is based on passive immunization by administration of hyperimmune serum or immunoglobulins of serum globulins, but active immunization is most commonly used. Initially, vaccines were used, inactivated and live skins prepared from the cat panleucopenia virus (ERHARDT, W .: Ober die Anwendung von Passiv- und Aktiviflstoffen gegen die Panleucopenia der Katzen. Berl. Munch. Tierarztl. Wschr., 90, 1977, 337) - 340; CHAPPUIS, G. - PETERMANN, HG: Zur Prophylaxis von Viruskrankheiten der Katze, Prakt Tierarzt 2, 1979: 112-115, POVEY, R. CH - KOONSE, H. - HAYS, B. B. : Immunogenicity and Safety of an Inactivated Vaccine for the Prevention of Rhinotracheitis, Caliciviral Disease, and Panleucopenia in Cats (JAVMA, 177, 1981 4: 347-350).

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 - several months, while protecting lein against subclinical infection, but in terms of attenuating the disease, it is important that such vaccination does not prevent the intensive secretion of the virulent virus into the environment. Recently, live vaccines that contain virus attenuated by serial passages on cell cultures have prevailed. After their application, the levels of specific antibodies reach a level as after natural infection with a virulent strain. Antibodies are detected as early as 3 days after inoculation and persist for at least 2 years. The motherhood of immune mothers is 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 (SICOTT, F.W. - CSIZA, C.K. - GILLESPIE, J.H. 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 parvovirus cultivation in cell cultures for the propagation of viral material for the preparation of experimental vaccines has been described by several authors (GORMAN, JR - HARTSOUGH, GR - BURGER, D. - ET AL. mink against enteritis virus Cornell Vet., 55, 1965, 559-556, MOCHIZUKI, M. - KONISHI, S. - OGATA, M.: Studies on feline panleucopenia, II Antigenicities of the virus Jpn. Sci., 40, 1978, 4: 375-383, BERGMAN, R. - SUNDQUIST, B. - STROMAN, L. Production of a feline parvovirus vaccine using a monalayer cell system in roller flasks and microcarriers. 55, 1984, 77-78). In particular, they used primary kits of feline and canine embryonic cells or stably cell lines prepared therefrom. The cells were usually infected 4 to 6 hours after seeding in the culture flasks. Since virus multiplication was not accompanied by regular development of cytopathic changes, they checked the virus content in tissue fluids based on the presence of intranuclear bodies, by immunofluorescent method or hemagglutination titer height. Saline rinsing and culture medium replacement were often used for cell infection. 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:

She was ready to live freeze-dried vaccine against feline cats, lyophilized, containing 1 ml of at least 10 ³

252748

Cat Panleucopenia Virus TKID 50 strain FPVA-BN 110/83 CAPM V-291.

Said virus strain was adapted to cultures of stable feline FE cell lines. The growth medium used was at least essentially the bovine serum-enriched podium Eagle medium in which the virus was propagated at 36-38 ° C for 4-8 days. After making clear eytopathic changes, the viral material was mixed with the lyophilization medium to contain at least 10 ⁴ TKID 50 of the cat enteritis virus particles per ml.

The viral strain of cat panleucopenia designated FPVA-BN 110/83 and the collection number CAPM V-291 deposited at the Czechoslovak Microorganism Collection at the Veterinary Research Institute in Brno, Hudcova 60, was obtained from a flood virulent virus isolated from infectious materials via serial passages on a feline cell line designated FE. The homogeneity of the viral population and the stability of the immunohiological properties of the attenuated strain were achieved by isolating the line by a six-fold borderline dilution method.

The strain FPVA-BN 110/83 CAPM V-291 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 ^e TKID 50. ml- ¹ .

2. The vaccine virus is harmless to all ages of the 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 lasts for at least one year and is accompanied by the presence of Specific Antibodies.

The stable FE cell line showed 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. Lack of development of cytopathic changes after infection with the vaccine strain was also a manifestation of insufficient growth characteristics of the cell line, which did not allow to monitor and evaluate the degree of virus propagation. Serial passage of the FE cell line in selected media (MEM, BEM,

M199) with neonatal serum content, after 25 passages in MEN medium, 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 to 96 hour intervals using a version-trypsin solution.

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

3. The cell line can be stored frozen at temperatures below -100 ° C, using a protection medium containing 10 to 20 weight percent 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 cat panleucopenia.

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.

The suspension lines of the FE bunieik a density ^of 5 cells Iaz 10:10 .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 virus-cell mixture is diluted in a growth medium which is at least essentially essential according to Eagle's pH of 6.8 to 7.3 and the addition of five to ten percent by weight of 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 preparation is cultured on glass, plastic, in a stationary manner, or in roller bottles, optionally on microcarriers, at 36 to 38% 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 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. Upon completion of the culture, the viral material is mixed with the lyophilization medium at a ratio of 50 to 75 weight percent of the virus to 50 to 25 weight percent of the 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 dispensed into ampoules or vials, lyophilized, sealed and stored at refrigerated temperature. The vaccine prepared as described above contains at least 10 ³ TKID 50. ml ^-1 vaccine virus, which represents a single immunization dose.

Used lyophilic medium 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, 509-552) is also suitable for the freeze-drying of other vaccine virus species. 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 at any rate and lyophilized.

The following examples are not intended to limit or limit the invention in any way:

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 the addition of 10 wt. % of the tissue culture serum is cultured on glass, plastic, in a stationary manner or in rotated bottles, optionally in suspension on 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:

a)

Suspension of FE 10 cells.

. 10 .ml ^{-1 those} cells in 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 the Eagle medium at pH 7.1 with the addition of 10 wt. % bovine serum.

bj

De cell suspension FE-0 density 5.10 ⁵ . ml ^-1 in the growth medium, a vaccine virus inoculum with a multiplicity of infection of 0.0001 is added, 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]

Infection of the adherent cells on the culture surface after six hours of culturing the FE-line cells at 37 ° C was added to the growth medium with a vaccine virus inoculum at a multiplicity of infection of 0.001. Incubation is continued at 37 ^and C for 6 days. As in a) and b), the multiplication of the virus is 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 appropriate lyophilization medium is added directly to the culture vessels in a 67 wt. % viral suspension and 33 wt. percent lyophilization medium. The mixture is then stored frozen in culture flasks until lyophilized.

Homogenized viral material with the required content of infectious units (at least 10 ⁴ TKID 50 ml ^-1 ), which does not contain contaminating microorganisms, is dispensed into ampoules, lyophilized, sealed and refrigerated. The vaccine prepared according to the above procedure comprises at least 10 ^-3 ° TKID 50. . ml ^-1 vaccine virus.

Claims (6)

A live attenuated vaccine against feline panleucopenia comprising at least 10 ³ TKID 50 particles of feline panleucopenia virus in 1 ml, strain FPVA-BN 110/83 CAPM V-291 adapted to cell cultures. 2. A live attenuated vaccine against cat panleucopenia according to claim 1, characterized in that it contains a lyophilization medium in an amount of 25 to 50% by weight. percent, based on the weight of the whole vaccine. 3. The vaccine production method according to claims 1 and 2, characterized in that the attenuated cat panleucopenia strain is propagated on a stable feline FE cell line in Eagle's minimum essential medium at pH 6.8-7.3 supplemented with bovine serum at 36 to 38 ° C for 4 to 8 days, until significant cytopathic changes are made, and then the viral material is mixed with the lyophilization medium to contain at least 10 ⁴ TKID 50. ml “ ¹ . 4. The vaccine production method of claim 3, wherein bovine serum with a defined content of non-specific inhibitors against the corresponding virus is used as an additive in the culture medium. 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 of 5-10.10 ^B. Ml ^-1 cells in the growth medium being infected with the virus at an MOI of at least 0.0001 per cell, incubated at 36-38 ° C for 30-60 minutes, then diluted 10- to 20-fold in growth medium, poured into a culture vessel and cultured at 36-38 ° C until virus harvest, or cellular suspension containing 4 to 8.10 ⁵ .ml ^_1 cells in growth medium were infected with virus at an MOI of less than 0.0001 per cell and incubated at 36 to 38 ° C and the virus harvest, and the culture vessels are deployed in a cell suspension in growth medium obsahorn 4 to 8.10 ⁵ .ml ^_1 cells and culture at the 4 to 6 hour incubation at 36 to 38 DEG C. are infected directly into the growth medium virus at MOI less than 0.0001 per cell and incubated until the virus harvest. 6. The vaccine production method according to claims 3 to 5, characterized in that a lyophilization medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other protein source and gelatin or dextran is added to the virus suspensions after the incubation. the mixture is kept frozen at temperatures above -20 degrees Celsius until freeze-dried or freeze-dried immediately.