DE2702634A1 - Feline panleukopenia virus attenuated vaccine prodn. - by passaging through cat kidney cells and felid or mustelid permanent cell line - Google Patents

Abstract

In the prodn. of vaccines against feline panleukopenia virus pathogenic panleukopenia virus is passaged at least 80 times through cat kidney cells and then at least 18 times through permanent tissue cells of felids or mustelids, then the attenuated virus thus obtained increased on cat foetal cells and converted into a vaccine. Attenuated panleukopenia virus strain BW 103 is claimed. Used for immunisation for cats against panleukopenia (infectious gastoenteritis). The antigen is highly pure. Since the attenuated virus is raised on homologous tissues, allergic reactions to the vaccine are avoided. The method of raising the virus permits a product of uniform quality to be produced. The attenuated vaccine obtained gives more rapid protection than existing vaccines.

Description

Process for the preparation of a vaccine against the

Cat panleukopenia The invention relates to a vaccine against cat disease, also infectious gastroenteritis or - according to its main symptom - panleukopenia as well as a process for the preparation of such a vaccine.

Katzenseude is a felid-specific viral disease.

The pathogen, a parvovirus, has an electron microscope Size from 20 - 25 nm. The virus is transmitted through the respiratory and digestive market, It is excreted via nasal secretions and feces. After an incubation period * on 2 The disease usually begins up to 6 days with refusal to feed, fever, and vomiting Diarrhea and inclination to jinx with desiccosis due to loss of fluids. The characteristic is severe panleukopenia. The lethality is up to 90 cp.

Cats that survive the disease become immune.

Antibodies can be passed on to kittens through colostrum. But they too are soon without protection, as the maternal antibodies gradually break down will.

The cat disease is spread worldwide, the infectiousness and pathogenicity their pathogen are very large and the cats are therefore very endangered.

Attempts have been made to use a vaccine for the manufacture of a feline disease vaccine to obtain suitable attenuated virus by passages in tissue cultures. At least however, six passages, especially ten to thirty passages, are not sufficient stable attenuated virus, which can serve as an active principle in vaccines obtain.

Namely, it has been observed that the Mmpfoirus excreted and is ingested by other cats. Such animal passages can lead to reverse mutations come.

To multiply the attenuated cat disease virus is also already Embryonic tissue has been used by cats. The cat embryos, however, are very delivering little tissue, so this method is forbidden for economic reasons.

There has now been a method of making a vaccine against found the panleukopenia, which is characterized by the fact that one pathogenes Panleukopenia virus at least 80 times on cat kidney cells, then at least Passed 18 times on permanent tissue cells of felids or mustelids and that attenuated virus obtained in this way is propagated on feline fetal cells and becomes a vaccine worked up. The invention also relates to an attenuated panleukopenia virus, a process for its manufacture and a vaccine against panleukopenia, the contains the attenuated virus.

The first step of attenuation, which is expediently 80 to 100 passages includes, takes place in v-cell cultures of cat kidneys that are in one of the cells responsible for cell reproduction Usual culture media are cultivated. The culture of the cells can be used as a cell lawn as well as a cell suspension. It is a temperature of 35 to 390C, preferably 36.5 to 37.50C * to be observed. Such a culture is early However, at the latest when the cells are about 75 fully grown, to infect. as The supernatant from the previously infected cell culture is used for the infection medium. It is added to the new cell culture in a ratio of 1: 5 to 1:20. 2 to 6 days after After infection, the supernatant can be obtained and transferred to the next cell culture will.

After 80 to 100 passages in the manner described, the virus-containing Transfer the supernatant to a permanent tissue cell culture of felids or mustelids. Corresponding permanent cultures are known from the literature. An example for such permanent cell culture is the Crandell cell, a permanent feline kidney cell. The infection of this cell culture with the supernatant of an infected cell culture and further processing takes place as described above.

After at least 18 passages in permanent tissue cells, fias is virus sufficiently attenuated. It is completely apathogenic, @ harmful and well tolerated. Due to the attenuation, it does not lose immunogenicity and the immunogenicity produced from it Vaccine has excellent immunizing properties.

The attenuated virus, which bears the scientific name "Panleukopenia virus, strain BW 103", was registered at the Institute for Hygiene and Epidemiology, Prague, under the registration number deposited.

The virus attenuated in this way can reproduce in tissue cultures of cat fetuses will. Particularly suitable for this are fetuses which: '. In the stage of organogenesis, approximately in the last third of pregnancy. For the creation of tissue cultures all or part of the fetuses can be processed. It is advisable, not to use extremities, head and skin for cell culture. the Cells are processed in the usual way, e.g. by trinification and work-up as before be-J as a stationary culture, as a roller culture or as a suspension culture wrote infected with the attenuated virus for the purpose of reproduction. The harvest of the supernatant takes place after 2 to 6 days. The infected cell cultures can with the addition of new Nutrient solution can be further cultivated. This operation can be repeated several times, E.g. with stationary culture at least 4-6 times and with roller culture at least 20-25 times. After that, the cells are generally exhausted. At the last work-up the cells can be used, for example, by deep freezing to obtain a greater yield or ultrasound treatment can be destroyed.

The material obtained by working up the propagation culture can be processed into a vaccine in the usual way. It is advisable, a stabilizer, for example gelatin, meat extract, peptone or sugar solutions to be added, especially if the vaccine is to be lyophilized. This is recommended because of the better half-ability. The vaccine can, however can also be used as a liquid vaccine. The addition of one of the usual adjuvants is possible if desired.

The vaccine can - if it is in lyophilized form, after Dissolve in one of the solvents customary for this purpose - subcutaneously or intramuscularly administered. It has the following advantages over known vaccines on: 1.) Great purity of the antigens.

2.) No foreign cracking reactions due to reproduction on homologous tissue. No shock reactions or allergies with repeated administration.

3.) Consistent quality and purity of the vaccine viruses as they multiply takes place on a uniform, tested cell substrate.

4.) Fast protection.

The panleukopenia vaccine according to the invention was in a Series of examinations and tests in the laboratory and in animal experiments on his Properties, tolerance, harmlessness and effectiveness tested.

Experiments and results: Harmlessness The test of harmlessness was carried out on white mice and guinea pigs. 5 mice are each with o, ml of the redissolved Vaccinc vaccinated subcutaneously. 2 guinea pigs each receive 2 ml intraperitoneally. The test animals must remain healthy during an observation period of 10 days. Particular importance was attached to the harmlessness and compatibility test Cats laid. In controlled laboratory and field tests, 962, predominantly isolated cats and 174 large cats - such as lions, tigers, cheetahs, etc. vaccinated with the vaccine according to the invention subcutaneously or intramus culture and usually Observed for 14 days, sometimes for several weeks. In addition to the control of local and general responses were also leukocyte counts in the laboratory tests performed. It was found that the vaccine was well tolerated. Also the Vaccination of SPF. (SPF = specified pathogen-free) - cats, some of which over 2 Were kept in isolation for years, gave no cause for complaint.

Stability For a consistently good effectiveness of the invention In addition to the virus titer, the vaccine must have a good shelf life. The exam the same was done after determining the content of vaccine virus after storage of the vaccine at different temperatures up to 24 months after manufacture. The received Antigen titers can be read from Table 1 (Table 1).

T able 1 Shelf life test of the vaccine after storage at different temperatures. Initial titre after production: 104.833ID50 / m1 Storage time Virus content in ID50 at storage temperatures from + 37 ° C + 20 ° C + 10 ° C + 4 ° C -35 ° C 1. Wick 103,633 3 pods 103,375 1 month 103,375 104,166 2 months 101.50 104.375 3 months 101.35 104.375 104.633 104.833 4 months no virus 104,166 6 months 103.50 104.833 104.625 104.833 9 months 103.50 10 12 months 103.5 104.625 104.833 104.833 18 months 102.625 104.625 104.833 104.833 24 months 102.625 104.5 104.833 104.833 Effectiveness and tolerance In addition to testing the content of live vaccine virus by virus titration in the cell culture, the product according to the invention was tested in a series of immunization tests on cats. The widespread distribution of the panleukopenia virus and other felid pathogens must be taken into account when carrying out experiments with purchased cats by keeping them in quarantine. In order to avoid foreign infections and to obtain exact test results, it is advisable to use specified pathogen-free cats, so-called SPF cats.

Animal experiment no. 1 In a first animal experiment, the effectiveness and compatibility of the vaccine tested on 6 SPF cats. From those in an isolation facility 4 cats housed together were vaccinated subcutaneously with the vaccine. 2 cats remained as unvaccinated controls. In addition to an examination of the general In terms of health, particular attention was paid to whether or not after administration of the Vaccine showed a significant decrease in white blood cell counts compared to those who were not vaccinated Cats occurs. This was not the case in the present and in other experiments.

The effectiveness was determined serologically by antibody measurement and following Stress infection checked. The result of this test is summarized in Table 2 reproduced. It can be seen from it that the cats were susceptible to panleukopenia prior to vaccination 2 weeks later the vaccinated cats were already serologically immune and resilient, the controls, however, remained defenseless and succumbed to the infection.

Table 2 Testing the vaccine for efficacy in SPF cats Katzo No. PL Antibody - Clinical PL Antibody Result titer before d. vaccinated with: course up to titer MO50 / ml lactation infection of the Vaccination 14 dpv 14 dpv load 59 0 oB 681 o. B. Vaccine according to @ 1 ml per 62 0 Invention oB 1.466 o. B. 65 0 1 Ds. = 1 ml oB 1.466 kg Kgw. if. subcutaneous pathogenes 67 0 oB 3,162 o. B. PL virus 61 0 oB 0 kr. F. Controls paroral 64 0 oB 0 kr.

Sign clearing: d.p.V. = this post vaccinationem o.B. = without particularity kr = sick with panleukopenia kr # = sick with panleukopenia and perish PL = Panelukopenia KGW = body weight ND50 = 50% neutralizing final value Animal experiment No. 2: Vaccines containing live non-pathogenic panleukopenia viruses result in usually to a rapid vaccination protection, especially the phenomenon of interference is attributable. Thereby the multiplication of pathogenic virus in the host cell prevented by the vaccine virus. In a test on 12 young cats it was tested whether this already 72 hours after administration of the vaccine according to the invention are adequately protected against exposure to contamination. The result of these investigations is shown in Table 3. The experiment shows that the vaccine is in the Is able to combat panleukopenia in the short term - namely within 3 days - to protect. All cats were unprotected from vaccination. 3 days later w1, stood there the vaccinated animals of a stress infection with pathogenic panleukopenia virus. Through a daily teucocyte count carried out after the stress infection, which stretched over two weeks, the cats were found to be outwardly remained healthy and showed no significant decrease in leukocytes. The during The initial and minimum values determined during the examination period were entered in the table 3. / The non-vaccinated control cats became more or less ill parleucopenia, with 4 out of 6 animals succumbing to the infection.

e A decrease in leukocytes below 2,000 / mm3 is generally a characteristic Panleukopenia symptoms.

Table 3 Effectiveness test of the vaccine 72 hours after vaccination Katzo No. PL anti-exposure 72h pv result of the load infection body inoculated with: leukocyte values up to 2 weeks p. before d. Vaccination Generally- the comparison values are the minimum values 268 0 0.8. 13,400 10,200 mm³ vaccine 598 0 according to 0.8. 20,400 10,600 mm³ Invention 1 ml each 599 0 0.8. 12,100 5,600 mm³ 1 oo each. = = 100 ID 602 0 50 0.8. 14,600 10,300 mm³ 1 ml 604 0 subcutaneously per kg 0.8. 11,300 10,100 mm³ 606 0 body 0.8. 18,800 10,200 mm³ 269 0 weight kr. 11,400 600 mm³ 600 0 pathogenic kr. 10,100 5,700 mm³ Controls 601 0 PL virus kr. 13,200 1,200 mm³ 605 0 No. 1 kr. 12,300 1,200 mm³ 603 0 porarel kr. 10,600 4,200 mm³ 607 0 kr. 15,800 3,800 mm³ oB = without peculiarity Kr = suffered from panleukopenia # = died of panleukopenia ID50 = 50% infectious dose Animal experiment no. 3 According to animal experiment 2, the vaccine produced according to the invention protects cats from developing panleukopenia as early as 3 days after vaccination (3 dpv) ( PL). To determine whether the same vaccine gives protection against PL even before the 3rd dpv; The experiment described below was carried out.

16 cats susceptible to panleukopenia were given 1 dose each of the Vaccine vaccinated and in 4 groups of 4 animals each.

Splits. A group of them was already immediately after the vaccination, the other groups one after the other with an interval of one day with pathogenic Panleukopenia virus strained, six untreated control cats were- together with the last group on the 3rd d.p.v. infected. As shown in Table 4, this shows Try to have the vaccine part of the day after the vaccination can protect vaccinated cats from panleukopenia. From four Cats of the group concerned fell ill and only one animal died of panleukopenia, the other three animals remained healthy.

Of the animals that were challenged two days after vaccination, only one of animal cats showed one on the 4th and 5th day after infection slight decrease in leukocytes (to .4. 200 or 5,100 / mm3). Those cats those infected three days after vaccination withstood the challenge unresponsive.

Of the control cats, all died of panleukopenia.

Further details can be found in Table 4. Table 4 Efficacy test 0.1, 2 and 3 days after vaccination Cat No. PL antibody vaccinated on strain infection in ND50 / ml 11/26. av with: D atum M aterial Result on 11/26/74 1017 0 + 7th dpi 1022 32 on November 26th + 12. dpi 1028 0 1 ml each 0 dpv 1 ml each + 8th dpi 1166 0 + 10. Vaccine PL-Infek- oB 1018 0 on 11/27 functional oB 1023 0 1 dpv material, + 9th dpi 1029 0 oB 1167 0 subcutaneous No. 1, 1019 0 on 11/28 if 1024 0 2 dpv 1: 5 diluted, oB 1160 0 per kg Kgw. L (4,200) 4.dpi 1168 0 oB 1020 0 on 11/29 by os oB 1025 0 3 dpvoB 1165 0 above 1169 0 oB 1021 0 on 11/29 + 6th dpi 1031 0 + 6th dpi 1158 0 controls + 6th dpi 1159 0 + 6th dpi 1163 7 + 6th dpi 1164 0 + 8. Dpi

a.v = ante vaccinationen d.p.v. = this post vaccination + = died an PL L = leukocyte drop (...) = lower leukocyte value d.p.i. = this post infections Animal experiment no. 4: To check the duration of protection Another attempt was made on SPF cats kept under strict isolation have been performed. Out of 10 SPF cats, -8 each received one dose of the vaccine subcutaneous. The two non-vaccinated control cats were used to check shedding and a possible further spread of the vaccine virus or PL virus introduction housed in the same room. Blood samples were used in all cats to measure antibodies taken. The results of this investigation are shown in Table 5.

The experiment shows that the vaccine has an active immunity a high-quality antibody titer that the vaccinated cats over the entire Period of observation gives reliable protection against panleukopenia. He also shows that the control cats remained antibody free.

This means that during the trial period neither a retransmission of the vaccine virus, there was also an introduction of PL viruses that occurred in the vaccinated individuals to a natural booster or, in the controls, to panleukopenia with subsequent antibody development. Since the test cats daily were waited and observed, it could be stated that during the entire During the test period, no reactions that indicate an intolerance or Let harmfulness close.

Table 5 Testing the duration of protection on SPF cats after vaccination Set no. Vaccinated with: before vaccination, penleukopenic body titre in NO56 / ml 2 weeks pv 1 month pv 2 months pv 4 weeks pv 6 months pv 8 weeks pv 151 0 4,217 6,813 4,210 6,813 3,162 6,813 152 1 Os each. 0 3,162 3,813 6,813 3,162 3,162 3,162 154 (= 1 ml) 0 3,162 6,813 1,466 4,217 1,466 * of the inven- 155 used 0 4,217 14,660 6,813 3,162 2,371 4,217 have to 156 vaccine 0 4,217 4,217 4,217 3,162 2,371 3,162 157 subcutaneous 0 3,162 6,813 4,217 3,162 3,162 4,217 158 0 3,162 6,813 14,660 4,217 6,813 4,217 159 0 14,660 23,710 23,710 4,217 2,371 6,162 153 0 0 0 0 0 0 0 Control 160 len 0 0 0 0 0 0 0 * = Cat is condensed during cardiac puncture where. pv = weeks post vaccination Mon pv = months post vaccination cont. Table 5 11 Mon. pv 14 Mon. pv 17 Mon. pv 23 Mon. pv 26 Mon. pv 3,162 422 2,371 1,466 6,813 3,162 1,466 3,162 3,162 3,162 4,217 316 422 1,466 4,217 3,162 1,466 1,466 3,162 3,162 3,162 2,371 3,162 6,813 6,813 14,660 3,162 3,162 6,813 6,813 6,813 2,371 4,217 4,217 6,813 0 0 0 0 0 0 0 0 # 0 # perishes intercurrently Example 1) Production of a culture of feline fetal cells A pregnant cat is killed painlessly and the ketones are removed under sterile conditions. The head, extremity ends and skin are removed from a fetus and the remaining body is cut into pieces about 2-4 mm in size with scissors.

The pieces are placed in a 0125C, o1ige trypsin solution and heated to 370C with stirring. The trypsin solution is added at intervals of about 10-20 minutes with the suspended cells contained therein poured off and replaced by new typesin solution replaced.

6 ml of the poured off cell sediment are diluted with 1: 300 suspended in a medium of the following composition: 10% Eagle's Medium (10-fold) 10 C, o mutton or calf serum 2 v6 lactalbumin hydrolyzate (5%) 1% NSP solution (neomycin-streptomycin-penicillin 20,000 µg / ml or 20,000 IU / ml) 2% sodium hydrogen carbonate (5%) Aqua double dist. ad 100% The mixture is in placed in a culture flask and maintained at 37 ° C.

The nutrient solution is renewed on the 2nd, 4th and 7th day. On the 10th day will the nutrient solution is poured off the cells and replaced with a solution that is 95% of a trypsinization medium, 1% a 1% solution of disodium dihydrogen ethylenediamine tetra acetate, 1% of a 1% trypsin solution and 3% of a 5% sodium carbonate solution contains.

hydrogen The trypsinization medium contains 1 liter of double-distilled water.

7.27 g sodium chloride 0.36 g potassium chloride 0.91 g dextrose In this Solution, the cell clusters dissolve into single cells or smaller groups. the cells thus obtained are cultivated several times in the manner described above.

2) Production of Panleukopenia Seed Virus A cat is infected with pathogenic Panleukopenia virus, obtained from a cat that has suffered from panleukopenia and has died was isolated, infected and 7 days later in the seriously ill state with a white blood cell count of 200 / mm3 killed. The kidneys are removed and placed in a Treated 25 igen trypsin solution to obtain a cell suspension. The received Virus suspension is sedimented and the sediment in a ratio of 1: 300 in one Suspended culture medium of the following composition: 52.2% Hanks' medium 5.8% lactalbumin hydrolyzate 20.0% tissue culture medium 199 20.0 calf serum 1.0% sodium hydrogen carbonate (5%) 1.0% NSP solution The suspension is kept at 37.degree. This leads to a multiplication of the panleukopenia virus in the cells. The virus can be detected by transferring the culture supernatant to healthy cats.

The virus continues to be cultivated as follows: From healthy Kidneys are removed from cats and trypsinized in the manner described above suspended in nutrient solution.

When the lawn of cells is about 75% full, it will become with the virus-containing supernatant of the panleukopenia virus culture inoculated.

For this purpose, the supernatant is mixed with fresh nutrient medium in a ratio of 1:10 mixed and layered over the cells. After 4 days, the supernatant that contains the contains increased panleukopenia virus, poured off. After about 80 repetitions the cat kidney cells used until then are replaced by a permanent cat kidney cell line after Crandell replaced and the passages grounded again 17 times.

The following is used for the cultivation of stable renal kidney cells Medium used: 10% Eagle's Medium 10% calf serum 1.5% sodium hydrogen carbonate (5%) 1.0% NSP solution to 100% double-distilled water.

With the virus inoculation, the serum proportion is reduced to 3%.

After the 103rd passage, the virus has lost its pathogenicity, but retain its good immunizing properties. It can be used for making of a vaccine.

3) Manufacture of the vaccine A feline fetal cell culture made after (1) was propagated, is suspended in a medium of the following composition: 10 9 Eagle's Medium (10-fold) 10% calf serum 2% lactalbumin hydrolyzate (5 epig) hydrogen 2 /% sodium / carbonate (5% i6) 1- * NSP solution 75% aqua bidist.

The nutrient solution is renewed after 1 - 2 days. When the cell lawn 50-75% has grown out, a 10% virus suspension is applied to the cell culture - obtained according to (2) - pipetted.

After 4 days, the virus-containing supernatant is poured off and the cell culture cultivated further with the addition of new nutrient solution. The supernatant has a virus titer of about This virus supernatant is in a known manner by adding a Stabilizer of the following composition processed into a vaccine.

4 parts gelatin broth 2% 1 part glucose 50%.

The vaccine is supplied in 1 ml portions in 3 ml crimp neck vials bottled, freeze-dried and sealed under vacuum.

Virus replication can also be carried out in the manner described above be carried out in roller or suspension cultures.

Claims (6)

Patent Claims 1. Process for the preparation of a vaccine against the panleukopenia of the felids, characterized in that one pathogenic panleukopenia virus at least 80 times on cat kidney cells, then at least 18 times on permanent tissue cells passaged by felids or mustelids and the attenuated virus obtained in this way Cat fetal cells increased and processed into a vaccine. 2. Process for the production of an attenuated pankeukopenia virus, characterized in that one pathogenic panleukopenia virus at least 80 times from cat kidney cells and then at least 18 times to permanent tissue cells Passed by felids or mustelids. 3. The method according to claim 1 and 2, characterized in that one the cultures of feline fetal cells used to propagate the attenuated virus harvested several times. 4. The method according to claim 3, characterized in that one for Propagation of the attenuated virus used stationary cultures from feline fetal cells and harvest them at least 4 - 6 times. 5. The method according to claim 3, characterized in that one for Propagation of the attenuated virus Roller cultures from feline fetal cells were used and harvest them at least 20-25 times. 6. Vaccine against panloucopenia of the Fslids, characterized in that that it contains a panleukopenia virus attenuated according to claim 2. ii Attenuated panleukopenia virus, strain BW 103.