CS267936B1 - live attenuated vaccine against parvovirosis, distemper, infectious laryngotracheitis and infectious hepatitis suitable especially for prophylaxis of fur animals and its preparation - Google Patents

Abstract

The solution relates to a live attenuated vaccine against parvovirus, distemper, infectious laryngotracheitis and infectious hepatitis, suitable primarily for prophylaxis of fur animals, and a method for its preparation in cell cultures. The vaccine is intended for protective and emergency vaccination of susceptible animals against the above diseases. The vaccine is lyophilized and is characterized by containing in 1 ml at least 10* TKIď5Q particles of canine parvovirus, strain CPVA-BN 80/82 CAPM V-290, 10·5 TKI0-θ particles of infectious laryngotracheitis virus, strain CAOV-2 H-5/85 MO CK40 CAPM V - - 320, adapted to cell cultures and lyophilization medium. Infectious laryngotracheitis virus is propagated on a stable MDCK line in Eagle's minimal essential medium. After completion of cultivation, the viral materials are mixed with the lyophilized medium and lyophilized.

Description

CS 267936 B1 1

The invention also relates to an attenuated vaccine against parvovirosis, distemper, infectious laryngotracheitis and infectious hepatitis, and to its preparation in cell cultures.

In addition to the well-known viral disease guides, such as fever, canine and infectious hepatitis, reliably controlled by effective attenuated vaccines, there are a number of viruses that are the culprits of frequent alimentary and respiratory diseases. These include, in the first instance, the paravirus of dogs, the causative agent of acute intestinal disease affecting predominantly juveniles, the infectious laryngotracheitis virus and the para-influenza virus 2 dogs, which induce, alone or in association with the bacterial germ of 9orde-tella bronchiseptica! mass respiratory diseases / Kennel cough syn-drome /. In 1978, Appel defined the pathogenicity of parvovirus dog (CPV 2) and described hoako as the epizootic episode of the intestinal disease that first appeared in 1978 in the United States.

The disease caused by this virus is clinically manifested by anorexia, malaise, vomiting and diarrhea, which may be bloody. The animals have fever and, as a rule, a reduced leukocyte count is found in the haematological examination. The cardiac form of the disease affects exclusively puppies and is characterized by a rapid and veto short course and subsequent death. The disease causes significant losses in dog breeds, and death accounts for 20 to 40% of the total number of animals. The disease affects all age groups of animals, but puppies, and the onset is rapid and very fast.

The adenoviruses of serotype 2 (CADV-2), isolated and identified by DITSCHFIELDOM, 1962, are serologically, antigenically, by their pathogens and clinical manifestations of disease different from infectious hepatitis virus (ICH).

The virus-induced disease has the character of infectious laryngotracheitis, tracheobronchitis, inflammation of the nosopharynx and pluc, with clinical signs of rhinitis, spontaneous leakage caused by narrowing of bronchial patency, and swelling hyperventilation accompanied by bronchial lymph node boiling. The pathologically anatomical image is characterized by submucosa infiltration by lymphocytes, plasma cells and neutrophils, proliferative Interstitial pneumonia, bronchial necrosis and bronchioid. CAOV-2 infections provide intradermal inclusions in the epithelium and nasal and pharyngeal mucosa

The specific prophylaxis of said viral diseases, which are a serious health problem in the breed, is based on passive immunization with the administration of hyperimmune sera or immune serum globulins, but the most frequently used active immunization. Inactivated and live monovalent and combined vaccines / CANDURR-P, MULTIMUNR 7, N0BIR VAC-SH, ENDURACELLR 7 - NSR, GALAXYR-VI - USA / First Live cani-parvovirus vaccine, Vet.Rec. 17, 11, 114, No.11 p.255; Stettner, W. - Schutzimpfun-gen beim Hund, Wiener. TierSrztliche Honatsschrift, 72, 1985, Heft 5 S.178-182., Lang-zeituntersuchung zur ImmunogenitSt eines Kombinationipfstoffes ftir Hunde, Zygraich, N., - Charlier, P. Florent, G., Gill, MM.A., et al., Kleintierpraxis 30, 1985, 45-49.

Even if the number of individual virus strains in the vaccines is considerable, it is generally required that one vaccine dose should contain at least 102-55 from each virus.

Due to the general prevalence of parvovirosis in dogs and the increasing number of respiratory lesions, while avoiding the effects of distemper complex viruses in carnivorous fur animals, we have sought to obtain or isolate the corresponding virus strain, using it prospectively to prepare vaccines. The individual virus isolates of the genotypic and parvovirus were identified, cloned, confirmed purity and electron-optically shown by chemical, logical and virological methods. Upon completion of the attenuation of the individual viruses on various cell cultures, and on the basis of a non-uniformity in the susceptible animals, there has been a development of viral vaccinations against the aforementioned species.

Thus, the present invention is a live parvovlrosis vaccinated vaccine and a CPVA-BM 80782 CAPN V-290 vaccine at 103 TKIDg, distemper, both CDV-F-BN10 / 83 CAPN V-289 at 103 103 θ, InfakCnaj laryngotraeheltld · a Infectious hepatitis D, which is characterized in that it binds to 2 alveolar parvovirus dogs, CPVA-BN 80 / 82CAPN V-290, in the ingested 103 TK1D $ O, canine distemper virus CDV-F-BN 10/83 CAPN V -289 in an inhaled 103 TKI · S0 and a virus infectious laryngotraehid virus CAOV-2H-5/85 MDCK40 CAPN V-320 in an 102 * 5 TKIOjg virus particle. The vaccine contains lyophilizate aldose and 25 to 50 percent by weight of sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or other source of bleach and gelatin, or daxtrene. The production of the vaccine is characterized by the cultivation of the parvovlrua dogs and the willow molluscs prepared by the present invention of the infectious laryngotrachdis CADV-2 H-5/85 HDCK40 obtained by propagation on the atelial Hn11 ROCK. Infectious laryngotraehel virus and on said stable Hnil anozil at 37 ° C for 3-6 days, when aa formed a significant cytoplasmic virus and aaerophilic and lyophilized aloid and lyophilized.

Infectious laryngotracheid virus is a virulent strain of foreign tissue obtained in the form of lyophilisate of infectious tissue fluids in the 5th passage to cell cultures. Virus attenuation, homogeneity of the viral population, and stability of the immuno-biological properties were achieved in passage 35 at b.l. MDCK. A virus was used in the 40th passage on the said tissue type attenuated and defined on the comp.

Parvovirus virus strains of dogs CPVA-BN 80/82 CAPM V-290, COV-F-BN 10 / 83CAPM V-289 infectious laryngotrache1 CADV-2 h-5/85 MDCK 40 CARM V-320, are stored in the Czechoslovak Collection of microorganisms at the Research Institute of Veterinary Medicine Brno, Hudcova 60.

Vaccine Production Procedure:

The method of producing vaccine is based on the fact that said vaccine strains, for all age categories of dogs and carnivorous fur animals, are (at random) propagated on defined cellular substrates grown on glass, plastic, cultured by stationary or rotated Rollerball / Rollerballs on Micro Carriers.

Infection of FE Cell Infections with Vaccine Parvovirus Virus: 6 · 1

A cell suspension of 5 to 10 10 cells density. ml in 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 min. Thereafter, the mixture of virus and cells is diluted in growth medium, which is at least essential medium of Eagle's pH 6.8-7.3 with the addition of well-knit and susceptible paraffin-free pareant of high quality.

It is also possible to add a vaccine virus inoculum with a multiplicity of infection of 0.0001 to 0.001 into the cell suspension in the growth medium and pour the virus / cell mixture into the culture vessel.

The cell suspension prepared in this way is prepared on glass, plastic, stationary or rotary flasks on roller carriers at 36 to 38 ° C for 4 to 8 days.

It is also possible to attach the attached cells to the surface cultivator - incubating the FE line cells for four to six hours at 36-38 ° C. In the growth medium, the vaccine virus is added to a multiplicity of infection of 0.0001 to 0.001. Incubation at 36-38 ° C for 4-8 days. The propagation of the virus, as in point A and B, is accompanied by significant cytopathic changes.

The method of infection of KEB chicken embryo fibroblast cells and distemper cell lines.

Cell cultures of KEB or VĚRO are infected with canine distemper virus strains primultiplicity of infection of at least 0.005. Cells grown on the culture surface are infected 24 to 48 hours by changing the growth medium to the maintenance to which the infectious inoculum is added. The maintenance medium is for a single cell type of the same composition as the growth medium, but does not contain serum and has a adjusted sodium bicarbonate content of 0.1 to 0.2 weight percent.

Alternatively, the mixture of virus and cells is allowed to incubate for 60 to 120 min. at 37 ° C, the cell suspension is diluted in growth medium to contain 4-8 x 10 6 cells. 1 ml. cells.

Also, infectious inoculum can be added to the cell suspension in the growth medium and poured into the culture vessel without prior incubation. After 24 to 48 hours, the growth medium is changed, without rinsing the monolayer, with maintenance and incubation under the same conditions. After the detection of the first cytopathic changes (usually 72-96 hours), the medium is exchanged at 24 hour intervals, the lyophilization medium is added to it and stored frozen until the combined vaccine is prepared before lyophilization, or the culture is allowed to proceed for as long as possible. more than 50 percent of the monolayer is affected by virus-specific changes and the virus suspension is collected only once.

Method of Infecting MDCK Cells with Infectious Laryngotracheitis Virus.

Cell cultures are infected with the infectious laryngotraeheitis virus strain at a multiplicity of infection of 0.1 to 0.001. 24-48 hour cultures grown on the culture surface are infected by removing the growth medium and adding a maintenance medium after 30-60 minutes of virus uptake to cells at 36-38 ° C. The maintenance medium is the same composition as the growth medium but does not contain serum. Incubation is continued at 36-38 ° C for 3-5 days.

It is also possible to add the infectious inoculum to the cell suspension in the growth medium and pour it into the culture vessel without prior incubation. After 24 to 48 hours, the growth medium is replaced, without rinsing the monolayer, with maintenance and incubation continued for 3 to 5 days. Cultivation in both cases is continued until more than 80 percent of the monolayer is affected by virus specific changes. . Upon completion of the culture, the infectious fluids of the individual viruses are 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; maintaining the same weight ratios until just before correlation and lyophilization of the vaccine. Infectious fluids containing at least 10 ΤΚΙΟ θ are used for the preparation of the lyophilized vaccine. ml of CPVA-BN 80 / 82CAMP V-290, 104 TKID ^ q particles of CDV-F-BN 10/83 CAPM V-289 and 103 ΤΚΙ05θ particles vi-rus CADV-2 HS / 85 MDCK 40 CAMP V-320 , bacteriologically sterile. The thus prepared viral suspensions are mixed in a proportion of 0.5 parts of parvovirus and 1 part of distemper virus and a diet of infectious laryngotracheitis virus with Lyofiti-start medium containing sucrose, phosphate buffer, sodium glutamate or potassium, bovine albumin or protein source, dextran or gelatin / BOVARNICK, HR - MILLER, J.C. - SNYDER, J.C., The influence of certain salts, aminoacids, sugars, and proteins on the stability ofrickettsiae. J. Baeteriol., 59, 1950: 509-522] are packed into a vial or vial in the form of a homogenate, lyophilized, sealed and stored in a refrigerator. The vaccine prepared according to the above procedure contains at least 10 3 ΤΚΙΟ θ n -1 1 · n · -1 · parvovirus, 10 ΤΚΙύ ΤΚΙύ, -θ. canine distemper virus and 10 ^ '® γκιο ^ θ. infectious laryngotracheitis virus, which represents One Immune Girl. The live parvovirus, infectious laryngotracheitis and infectious hepatitis vaccine may be advantageously used for the prophylaxis of fever and fowl disease, with clinical signs of rhinitis, discharge from the eye, laryngotracheitis, tracheobronchltis, and paralysis of any limbs accompanied by 30-50 percent death from total count. The wild-type vaccine prepared according to the invention provides the desired protection against all current viral infections in said fur animals.

In the current state of the art, the broadening of the spectrum of vaccine efficacy has no merit, while the addition of additional antigenic components would significantly increase the presumed cost of the product (vaccine), and negatively affect the economy in large-scale cochlea breeds.

AND

The following examples are not intended to limit or exemplify the invention: Example 1

A suspension of FE, MDCK, VERO cells prepared by proteolytic fermentation of ambryonic cell smokers (KEB) prepared by proteolytic ferments from 11-day germs in growth media containing a defined number of amino acids and vitamins with the addition of 5 weight percent serum in KEB and VĚRO and 10% by weight of sera intended for tissue culture cultures ofFE and MDCK are cultured on glass, in a conventional manner and in suspension. Example 2

Method of Cell Culture Infection with Parvovirus Vaccine Virus:

Cell cultures prepared and cultured according to step 1 are infected with the vaccine virus CPVA-BN 80/82 at a multiplicity of infection of at least 0.001 as follows: 6 a. 10.10 cell Union FE cell suspension. ml in growth medium is mixed with 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 growth medium, which is at least the esophageal medium of Eagle's pH 7.2 and the addition of 10 weight percent bovine serum. 5-1 b. In the growth cell suspension of the FE line cells at a density of 5x10ml, a growth virus virus of multiplicity of infection of 0.001 is added and the infected suspension is immediately poured into a culture vessel. The thus prepared cell suspension for virus vaccination is cultured in Roux flasks at 37 ° C for 6 days. c. Infection of adherent cells on the culture surface after a six hour culture of FE cells at 37 ° C is added to the growth medium by the inoculum of the vaccine virus at a multiplicity of infection of 0.001. Incubation is continued at 37 ° C for 6 days. Propagation of the virus as in a / a and b) is accompanied by significant cytopathic changes.

The vaccine virus is produced in the growth medium in cell cultures FE grown according to point 1 and infected according to point 2. After large-scale cytopathic CS 267936 81 5 alterations have been made microscopically, the culture is terminated and added directly to the growth virus culture medium. culture flasks, suitable lyophilization medium at a ratio of 50 weight percent virus suspension and 50 weight percent lyophilization medium. The mixture is kept frozen in culture vessels. Example 3

Method of cell culture infection by distemper vaccine virus:

The vaccine virus inoculum in which the virus is stabilized by the addition of 10 weight percent dimethylsulfoxide is stored frozen at -70 ° C. Cell cultures prepared and cultured at step 1 are infected with CDV-F-BN10 / 83 vaccine virus at a multiplicity of infection of at least 0.005 by the following methods: a. Growth of single cell KEB monolayer: growth medium and cell growth are removed from the cell monolayer after 24 hours growth the culture is infected with an inoculum added to the holding medium to replace the growth medium. b. VERA cell suspension in growth medium with a density of about 10? . ml of cells are mixed with the vaccine virus inoculum, and the mixture is incubated at 37 ° C for 30 min. After the infected cell suspension is terminated, it is diluted in growth medium to contain 4.15 µl cells per ml and cultured at 37 ° C for 48 hours, i.e. until complete monolayer of cells on the culture surface is formed. Thereafter, the growth medium is replaced with a maintenance medium. 5 -1 c. To KEB cell suspension in growth medium with density of 4.10. ml of the vaccine virus is added. The suspension is poured into a culture vessel and incubated at 37 ° C for 24 hours, i.e. until a monolayer is formed. This is followed by a replacement of the growth medium with the maintenance medium.

The vaccine virus is produced in a maintenance medium which, by its basic composition, is the same as the growth medium, except that it does not contain serum and the sodium carbonate concentration is up to 0.30 weight percent. virus from cell cultures grown according to step 1 is infected as described in point 3 by the following methods: a. Maintenance medium is left unchanged in cultures until extensive cytopathic changes are established microscopically. Appropriate lyophilization medium is added directly to the culture flasks into the virus maintenance medium in a ratio of 2 di-virulent suspension and 1 µl lyophilization medium. The mixture is further stored frozen in the flasks. b. Upon appearance of the first signs of specific cytopathic changes, maintenance media is discharged and equal amounts of maintenance medium are poured into the culture vessels. The sequence is repeated at 24 hour intervals until a large-scale cytopathic effect is produced, wherein the final treatment is carried out according to a. A lyophilization medium is added to the virus suspension obtained from the repeated maintenance medium changes and stored in the vial. frozen state. Example 4

Method of Cell Culture Infection with Infectious Laryngotracheitis Virus:

Cell cultures prepared and cultured according to section 1 are infected with the vaccine virus CADV-2 H-5/85 MD CK40 at a multiplicity of infection of at least 0.01 by the following methods: a. The 48 hour growth at 37 ° C removes the growth medium that represents at least Eagle's essential medium and the cell culture is infected with the virus adsorption to the cells for 60 minutes at 37 ° C. Thereafter, a maintenance medium is added, which is of the same composition as the growth medium but does not contain serum. Oo of cell suspension in growth medium with density of 4.10. ml is added to one virus of the virus. The suspension is immediately poured into a culture vessel and incubated at 37 ° C for 48 hours, i.e. until a single-layer cell is formed. Thereafter, the replacement of the growth medium with the maintenance is followed, and the incubation is continued at 37 ° C for 2 days. Virus destruction in most cases is accompanied by marked cytopathic changes in cell monolayer.

The vaccinating virus is produced in growth medium in cell cultures of MOCs grown under the point 1 and infected with step 4. After large cytopathic blisters have been formed, the culture is terminated, and appropriate lyophilization media is added to the culture medium with the propagated virus in a 2-liter ratio of the viral suspension. the lysis medium. The mixture is further stored in culture flasks in a frozen state. Example 5 Preparation of Combined Vaccine:

Infectious fluids containing at least 104 TCID50 were used to prepare lyophilized vaccines. ml "1 particle CPVA-BN 80/82, 104 TKIDSO. ml" 1 COV-F-BN 10/83 and 103 TKIKjq virus particles CAOV-2 H-5/85 M0CK40 bacteriologically sterile. are mixed in a ratio of 0.5 parts of parvovirus and 1 dlelvirus of distemper and 1 dl of virus of infectious laryngotrachia Homogenized virus material with the desired content Infectious units are adjusted, lyophilized, air-sealed and stored refrigerated.

The vaccine-prepared vaccine vaccine contains at least 10 µ TKID / qq of parvovirus dogs, 103 TKIO / q distemper virus and 10 µ / 5 µg / ml infectious laryngotracheitis virus at a single dose. comparing the efficacy of live attenuated vaccinia against parvovlrosis, distemper, infectious laryngotracheitis and infectious hepatitis suitable in particular for the prophylaxis of monofunctional laryngotrachelecta virus-infected laryngotrachelidea furry vesicles, showing the picture 1. The values shown graphically expressed Mean Tltre of Hemagglutinating-Antibodies to Parvovirus and Virus Infectious Laryngotracheitis and Neutralizing Antibody Virus (VN) Against Canine Distemper. It is evident from the presented results that the vaccine vaccinated with polyvafcent vaccine hurts against the expected value of the antibody at the same level as after the application of mono-resp. blvalentnej vakdny. The induction of such an immune response has not been anticipated and has not yet been found in carnivorous fur animals. The results shown in Table 1 indicate that vaccinated and control animals did not respond to infection with a canine parvovirus strain with a noticeable overall health disorder. These findings are also supported by the data found by McAdaragh et al. Am, J.Vet.R., Vol. 43, Ho.4, 1982, p. 693-696.

After the virulent canine distemper infection, all vaccinated animals remained healthy, while the control foxes died of characteristic psoriasis symptoms.

With complete protection of vaccinated animals for Infectious Laryngotra-Cheitis Infection, two of the plateau controls responded to transient but obviously diseases of the respiratory tract. Similarly, all vaccinated animals remained infected with the infected worm-infectious hepatitis, which is the cause of fox encephalitis, a manifestation of the krill

Claims (4)

An infectious laryngotracheitis virus vaccinated against virus-infectious hepatitis (in encephalitis foxes). Both unvaccinated controls reactive to infection by developing clinical signs characteristic of viral encephalitis. ANIMAL RINFECTION VIRUS NUMBER OF ANIMALS RESULTS Grain. Healthy Wedge. ill Death CPV 5 5 0 0 Vaccine - CDV 5 5 0 0 n CADV-2 5 5 0 0 CADV-1 5 5 0 0 CPV 5 5 0 0 Checks CDV 2 0 0 2 CADV-2 5 3 2 0 CADV-1 2 0 2 0 Tab. no. 1 ? BACKGROUND OF THE INVENTION 1. Live attenuated parvovirosis vaccine containing CPVA-BN 80/82 strain CAPH V-290 at 103 ΤΚΙΟ ^ θ, CDV-F-BN strain 10/83 10/83 CAPH V-289 pituitary 103 TKID ^ θ , infectious laryngotracheitis and infectious hepatitis, characterized in that it contains CPVA-BN 80/82 CAPH V-290, at least 103 TKID, n, distemper strain CDV-F-BN 10/83 in a 2 ml strain of CPV-BN dog parvovirus CAPM V-289 in the amount of at least 10 ΤΚΙΟ, -θ and infectious laryngotracheitis virus strain CADV-2 H-5 / 85MDCK40 CAPH V-320 in the amount of 10 µl of virus particles, and lyophilization medium. 2. A live attenuated vaccine according to claim 1, characterized in that it comprises a lyophilizing medium in a maturity of 25 to 50 weight percent based on the weight of the whole vaccine. 3. A live attenuated vaccine according to any one of claims 1 to 3, wherein the lyophilizing medium comprises sachrosis, phosphate buffer, sodium or potassium glutamate, bovine albumin or other source of protein and gelatin or dextran. 4. A method of producing a stage 1 vaccine by culturing parvovirus CPVA-BN 80/82 CAPM V-290 and distemper virus No. CDV-F-BN 10/83 CAPH V-289 by a known method, characterized in that the parvovirus and virus cultures are distemper mixes with infectious laryngotracheitis virus cultivator CADV-2 H-5/85 MDCK40 CAPH V-320 obtained by propagation of unstable HDCK booth line after 30-60 minute infection of 24-48 hour mono-layer in minimal Eagle medium essential medium at 36 to 38 ° C for 3 to 6 days, until cytopathic changes are formed, and then the viral material is mixed with lyophilized medium to a minimum content of 10 µl / ml, and the mixture is lyophilized. The process of claim 4, wherein the medium is added to the virulent suspension immediately after recovery, and the mixture is stored frozen or freeze-dried. . 1 drawing