FI61626B - FRAMEWORK FOR THE PREPARATION OF NYA RABIESVIRUSVACCIN - Google Patents

Description

Γβ1 advertisement publication ™ (1) UTLÄGGNINGSSKRIFT 6ΐθΖΟ ^ (^ 5) Patent «granted 10 09 1932 Patent aeddelat ^ J (51) Kv.ik.Vci.3 A 61 K 39/205 * C 12 N 7/02 (21) P »T * nttlh» k * mu «- P» ttnttn »öknlng 800539 (22) HtktmlipUvi - An * 6knln | tdag 22.02.80 (23) Alkuptlvi — Glttlih« ud «| 20.01.T8 (41) Tullut lulklMk ·! - Blhflt offwttllf 22.02.80

Pentent · and the Registry Board Nihtivll «ip« is j. ku «l. | Uik.iM.n pvm.-

Patent and registration authorities Antökin Utli | d och utUkriften publkartd 31.05.82 (32) (33) (31) Pyrri · »/« uolk «jt — B« Hrd prloritM 26.01.77

United Kingdom-Great Britain (GB) 3258/77 (71) Gist-Brocades NV, 1, Wateringseweg, Delft, Netherlands-Nederländerna (NL) (72) Gosse Bijlenga, St. Didier au Mont d'Or, France-France ( FR) (7 * 0 Berggren Oy Ab (5 * 0 Method for the preparation of new rabies virus vaccines -Förfarande för framställning av nya rabiesvirusvaccin (62) Divided by application 7Ö0181 + (patent 59812) -Avdelad frän trap 7888U (patent 59812) for the preparation of active or inactivated rabies vaccines.

Vaccines prepared by the method of the invention may be administered parenterally and non-parenterally. The active type of vaccine is very suitable for oral administration, for example in immunizing foxes. Vaccines may also be administered by other non-parenteral routes, such as intravenous or nasal injection, to other epidemiologically important wildlife, domestic animals, and humans.

It is known to try to control rabies infections in warm-blooded animals by means of parenteral vaccination to prevent / e.g. LEP (Low Egg Passage) Flury or HEP (High Egg Passage) Flury vaccines, obtained as described below.

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On January 29, 1939, a girl named Flury died in the U.S. state of Georgia, the cause of death being diagnosed as rabies, which the girl had contracted from a rabies dog. The rabies virus was isolated from the girl's brain, lacrimal gland and salivary gland and inoculated intracerebrally into white mice as described in:

Leach C.N. & Johnson H.N., Amer. J. Trop. Med. 1940, £ 0: 335.

Mouse brain tissue was inoculated intracerebrally into one-day-old chickens, followed by 136 intracerebral inoculations into chickens as described by Koprowski H. & Cox H.R., J. Immunol. 1948, 60: 533. After two more intracerebral inoculations in chickens, the rabies virus was transferred to chicken-chick embryos by inoculation in the yolk sac.

After 60 yolk sac inoculations, the virus proved to be non-pathogenic to several mammals, and the virus is available at this level of transplantation as a rabies vaccine for dogs under the name LEP Flury vaccine. When further transplants were performed on chick embryos (up to 170-174 inoculations), the virus was shown to have lost much more pathogenicity, so that two-week-old laboratory mice did not die after intracerebral inoculation of the virus, even though infant mice died. This vaccine is available as Hep Flury.

Although these LEP and HEP vaccines have been effective in preventing rabies infection when used as pre-exposure vaccines, there is a need for other types of rabies vaccines, as demonstrated by recent new control measures at the national and international levels. For example, the management of sylvatic rabies in developed countries, where the disease has been present for a long time or to which it has recently entered, is one of the difficult and ecologically complex problems for which no effective and economical method is available.

Post-infection human vaccination has also caused several problems in rabies-infected countries around the world.

For these reasons, rabies research has focused on the problems of 61 626 3 for post-infection and pre-infection vaccination as well as immunization by the non-parenteral route, preferably orally.

As a result of research and experiments, a new vaccine has been developed that can be administered in a variety of ways, i.e., can be administered parenterally or non-parenterally, and has been found to provide full protection when administered either before or after rabies infection. In the method of the invention, vaccines for parenteral and non-parenteral administration are prepared by growing an in vitro rabies virus strain in a manner known per se in cell systems of avian or mammalian origin and freezing the resulting cell suspension at -70 ° C or below and sonicating and / or sonicating the cells. and then removing the cells and cell debris by filtration or centrifugation under sterile conditions, and finally possibly inactivating the virus in the culture broths using known methods and preparing a vaccine using known methods, and the method of the invention is characterized by the use of a novel rabies virus strain No. 675 , deposited in the collection "Czechoslovak National Collection of Type Cultures" of the Department of Hygiene and Epidemiology of Prague under number CNC TC AO 4/77.

The possibility of administering the substance orally, "oral vaccination", to wild animals, which are the main vectors of rabies infection and / or sources of this disease, is to be considered as a characteristic feature of the vaccine prepared by the method of this invention.

The active vaccine virus strain 675 has the least pathogenic properties of all hitherto known rabies vaccine viruses. It allows protection against viral infection after infection with even just one sufficiently large dose of vaccine. An inactivated vaccine made from virus strain 675 also protects after infection, but in a smaller amount of 4,61626. The active vaccine has a very high degree of antigenic properties and the inactivated vaccine type almost as much.

Both active and inactivated vaccines are cell culture vaccines that are almost completely free of foreign proteins, which is capable of at least substantially reducing the number of unwanted side effects, if not eliminating them completely. The rabies virus strain 675 used in the method of the invention is prepared by growing HEP Flury virus and isolating a plaque having the characteristics set forth below. The production method is described in FI patent application 780184.

The originally isolated plaque was given the number 675 and the strain obtained after purification was marked: rabies vaccine strain no. 675. A sample of this strain was deposited on 13.01.1977 in the collection of the Czechoslovak National Collection of Type Cultures, where it was registered under CNC TC AO 4/77.

The strain used in the method according to the invention differs from the original HEP Flury virus in the following respects: a) Very clear cytopathic effect on cell cultures in a test tube, which differs in intensity from the cytopathic effect caused by HEP Flury virus.

b) A shortened primary reproductive cycle ranging from 9 to 11 hours (12 to 16 hours for the HEP strain), causing a faster onset of plaques, the so-called absence of interference, due to the purity of the virus strain (cloning), thus the virus strain 675 does not contain the so-called Dl particles (defective interfering particles).

c) There are no living cells in the area of the plaques, whereas plaques caused by the HEP virus always contain some living 5,61626 cells. Therefore, the plaques of virus strain 675 are very bright (transparent) and the plaques of HEP virus are cloudy. Morphologically, the plaques of virus strain 675 are larger and generally more uniformly shaped than the plaques of HEP virus.

d) Better adhesion to the cell surface in the test tube, a property essential for adequate oral vaccination. In vitro studies have shown that no differences in cell adhesion were observed for virus strain 675 using DEAE-dextran. In the case of HEP virus, on the other hand, it was clearly observed that several cells became infected with the virus using DEAE-dextran. This phenomenon can be explained by the fact that the virus strain 675 is cleaner, but there may also be small differences in its surface compared to the HEP virus.

e) Faster formation of interferon (approximately 60 minutes) in the body after vaccination due to the above properties. This feature is important for post-infection vaccination.

f) Higher concentrations are obtained in cell cultures.

(g) Cloning results in homogeneous virus strains, with the lower risk of the virus species returning to its original pathogenicity, which is virtually non-existent. Examination of the virus strain in 675 young (4 weeks) mice showed no increase in viral pathogenicity after ten consecutive intracerebral inoculations, whereas HEP virus showed a marked increase in pathogenicity after only one transfection (see HF Clark, Science, 199 (1978)). ), p. 1072 and H. Koprowski, Bull. WHO, 10 (1954), p. 709). This "stability" of virus strain 675 is a very important property when used as bait for immunization of wild animals for sex. In this case, there should be no increase in the pathogenicity of the vaccine · h) This rabies virus strain has the least pathogenic properties of the strains available so far. In investigations (Staatliches Veterinär Untersuchungsamt, 6 61 626

Frankfurt am Main) has been shown to be pathogenic to mice. Replication of the HEP virus could be detected in the brains of flies and brown rats. The new virus strain 675 did not show pathogenicity in the above-mentioned animal species, nor in the numerous other animal species studied.

It should be noted that the characteristics presented in (a) to (h) above show a clear difference from the original HEP Flury virus.

The above-mentioned properties, in particular feature (g), facilitate the preparation of vaccine batches of constant quality, i.e. vaccines with an improved composition can be prepared according to the invention from virus strain No. 675.

Like the original HEP Flury vaccine, virus 675 of the active viral vaccine of the invention does not multiply after vaccination, nor can viruses be detected after vaccination.

Vaccines prepared from this new virus by the method of the invention cause very rapid interferon formation as well as other unknown inhibitory properties, e.g. cell-mediated immunity. Thus, a single vaccination with the above-mentioned vaccine after infection with a pathogenic rabies virus can provide complete protection. Due to the very rapid formation of interferon and cell-mediated immunity, the use of the vaccines of the invention for the protection of newly infected animals during a rabies epidemic is now possible. In particular, the possibility of vaccinating humans after infection has been greatly improved because vaccines have proven to be very effective in animals after infection, and because they can even cure animals when they are in a very advanced infected condition. Non-parenteral, e.g. oral, administration of an active viral vaccine facilitates its use in an organized vaccination campaign.

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An active viral vaccine prepared from virus strain 675 has the following potential advantages in pre-infection vaccination.

A) In animals: 1. Immunization of foxes and other epidemiologically important animals is possible with baits containing the rabies vaccine.

2. Oral vaccination of other animals, such as stray dogs, in countries where their killing is prohibited for religious and / or legal reasons.

3. Large-scale active administration of the viral vaccine by bait so that the vaccination campaign can be greatly simplified. For example, the distribution of baits containing a viral vaccine from an aircraft.

4. Effective protection of domestic animals after infection for use in countries where such vaccination methods are legalized or in countries where animals can be kept alive after infection, provided that they have already been immunized.

B. In humans: 1. "Oral vaccination" is painless for the patient. In many countries, vaccination is feared, causing many patients not to return to take the required number of vaccinations.

2. Oral vaccination shall not cause local reactions or irritations which constitute a real disadvantage with the use of previous commercially available vaccines.

3. Easy large-scale vaccination by non-medically trained staff has become possible (compare oral poliomyelitis vaccination).

In that context, the fact that a single vaccination has proved sufficient must be regarded as an important factor.

‘’ Rt 61626

New vaccines are prepared by standard methods, i.e., in vitro cell cultures, using conventional and controlled control procedures to eliminate bacterial and / or viral contamination in accordance with known principles and international standard requirements.

The novel vaccines of this invention are preferably prepared by growing rabies virus 675 in primary or secondary chick embryo fibroblasts in unicellular layers derived from 10-day-old chicken-chick embryos (SPF). However, other cell systems that are sufficiently susceptible to rabies infection can also be used, such as other avian or mammalian cells, e.g., Baby Hamster Kidney 21 (21 transplants) for single culture or BHK-21 13S (13 suspension culture transplants) suspended. for cultivation, but in this case only for animal vaccines. The suspended cell culture system has the great advantage of enabling the production of viral vaccines in high-volume fermentors, which facilitates production.

Vaccines can also be prepared in cells grown on microcarriers (pearl-cell culture system). For example, polydextran beads treated with high molecular weight anions before and / or during cell growth and having a sufficient charge density on the surface for cell growth can be used. In this way, the desired virus can be implanted in a stirred liquid medium containing cells and beads, and after virus propagation, the beads are allowed to settle to the bottom and separate from the virus suspension.

When culturing rabies virus 675 in a chicken embryo fibroblast, these are infected with rabies virus strain No. 675, which m.o.i. is suitably (a figure indicating how many viruses infect each cell, i.e. the ability to infect) is 0.02-1 pfu / cell (plaque forming unit, indicating the ability of the viruses to form plaques), the infection time preferably being at least 1 hour. After removal of the unattached virus, a medium is added, preferably consisting of Basal Medium Eagle (BME) in Earle's saline supplemented with appropriate amounts of antibiotics 9,61626 (e.g., 100 IU penicillin and 100 micrograms streptomycin) and 0.2-0.5 % albumin. Care must be taken to maintain the pH of the medium at 8.0-8.2. Virus-infected cells are incubated at 32-39 ° C for 3-10 days, after which the medium is frozen at -70 ° C or lower to disrupt the cells and release the virus (increasing the concentration). Cells and cellular debris are removed by filtration or centrifugation under sterile conditions. The filtrate or centrifugation fluid is stored at -70 ° C, and samples are taken for titration using the above-mentioned in vitro plaque formation method.

Other methods can also be used to disrupt cells after incubation, e.g., subjecting the cell suspension to ultrasonic vibration.

According to an alternative preparation method, BHK-21 13S cells are grown in suspension, e.g. in a spinner flask with a hanging rod, using BHK-21 medium supplemented with small amounts of tryptose phosphate nutrient solution (7-15%), inactivated calf serum (7-15%) for culture. ) and antibiotics (eg 100 IU penicillin and 100 micrograms streptomycin). The cells are grown until a level of approximately 2 x 10 6 cells / ml is reached, while the cell suspension is mixed sufficiently to keep the cells in suspension and to avoid agglomeration.

After centrifugation of the cells, infection is performed for 30-60 minutes with rabies virus strain 675, which m.o.i. is between 0.02-1 pfu / cell, with constant agitation.

After infection, the cells are resuspended in spinner flask medium containing BHK-21 culture medium supplemented with small amounts of bovine serum albumin fraction V, e.g. 0.1-0.4% by weight, and antibiotics (e.g. kanamycin or neomycin, e.g. 50-300 micrograms) while adjusting the pH to 7.5-8.0. During infection, the temperature is maintained at 32-35 ° C, more preferably at 33 ° C, and a small sample of the spinner is taken daily for analysis by the 4 4 Å, 6162 6 immunofluorescence method. 2-3 days after infection, 80-100% of the cells in the sample show rabies virus-specific immune fluorescence 89 sensitivity in the cytoplasm, corresponding to a titer of 10 -10 pfu / ml in recovery 4-6 days after infection. When recovering the virus, the liquid is removed from the flask and stored frozen at -70 ° C.

After removal of cells and cellular debris, a small sample is titrated by the plaque titration method. If an acceptable concentration (between 10 -10 'pfu / ml or higher) is reached, cells and cell debris are removed from the product by either filtration or centrifugation under sterile conditions.

After the addition of a suitable stabilizer, the liquid is ready, for example, to be dispensed into small vials for freeze-drying the final product.

Inactivated rabies vaccines, which are best suited for use in humans, are also within the scope of this invention.

These inactivated rabies vaccines can be prepared by inactivating the virus in the prepared culture media by known methods or variants thereof, e.g. by adding beta-propiolactone, formalin or acetylethyleneimine, or by ultraviolet radiation.

The rabies vaccines prepared by the method of the invention may contain one or more suitable stabilizers, preservatives, buffer salts and excipients.

The term vaccination via the gut, means the "vaccine" administration form which will release the intestine.

The invention is illustrated by the following examples.

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Example I

Single cell layers of primary or secondary chick embryo fibroblasts (SPF) grown in stationary or rotating flasks are infected with rabies virus strain 675, which m.o.i. is between 0.02-1 pfu / cell.

Smaller amounts of virus can also be used, but the incubation time is longer. An infection time of at least 1 hour is required to attach the envelope virus to the cells. The unbound virus is then removed and the medium formed by Basal Medium Eagle (BME) in Earle's saline supplemented with appropriate amounts of antibiotics and 0.2% albumin of the species to which the vaccine will be added is added, ensuring that: the pH of the final medium mixture is 8.2.

Incubation of virus-infected cells at 33-35 ° C

4-8 days produces a clear cytopathic (CPE) effect, and the medium is then frozen at -70 ° C.

After thawing the product, cells and cell debris are removed by centrifugation at 1000 g or filtration through a membrane filter (pore size 5 micrometers) under sterile conditions. The liquid obtained by either centrifugation or filtration is stored at -70 ° C, and samples are taken by titration using the in vitro plaque formation method Bijlenga, G., Joubert, L., Bull. Soc. Sci. V§t. et Möd. ComparSe, 1974, 76: 429.

For post-infection vaccination, a titer of 10 pfu / ml is required for vaccine lots to achieve effective vaccination. For pre-infection vaccination 7, a titer of 10 pfu / ml can still be accepted.

Vaccine batch production as described above is monitored by standard efficacy trials (NIH and Habel trial), and a new efficacy trial has also been introduced for post-infection batches as described in Bijlenga, G., Symposium on Advances in Rabies Research, Atlanta, Georgia, USA, September 7, 1976, page 14.

In this new efficacy trial, local wild-type virus is injected intramuscularly (i.m.) into 12-week-old 12 61 626 mice, followed by one intraperitoneal vaccination with 5 ml of vaccine within 24 hours. All vaccinated mice should survive, while 50% of control mice should die before the end of the three-week test period.

In addition, routine surveillance tests for possible bacterial and viral contamination of vaccine batches and inspections of primary and secondary cells and cell generations used in the manufacture of vaccines shall be performed in accordance with internationally agreed minimum standards.

Example II

BHK-21 13S cells are grown in suspension in a swirling flask equipped with a stir bar, using BHK-21 medium supplemented with 10% tryptose phosphate nutrient solution and 10% inactivated calf serum for culture in the presence of antibiotics (100 IU penicillin and 100 micrograms streptomycin). The cells are grown until a cell density of 2 x 10 6 cells / ml is reached, while stirring the cells sufficiently to keep them in suspension and to avoid agglomeration.

After centrifugation of the cells at 800 rpm, infection is performed for 45 minutes with rabies virus strain 675 having an m.o.i. is between 0.01 and 1 pfu / cell, with constant agitation. After infection, the cells are resuspended in spinner flask medium consisting of BHK-21 culture medium supplemented with 0.2% bovine serum albumin fraction V and appropriate amounts of acceptable antibiotics (e.g., kanamycin or neomycin) while adjusting the pH to 7.8. During infection, the temperature is maintained at 33 ° C and a small sample is taken from the spinner flask for analysis by immunofluorescence. 2-3 days after infection, 80-100% of the cells in these samples should show rabies virus-specific immunofluorescence in the cytoplasm, ensuring sufficient tiit-89 ter 10-10 pfu / ml for virus recovery 4-6 days after infection.

i 13 61 6 2 6

When the virus is recovered, the liquid is removed from the flask and stored frozen at -70 ° C. After removal of cells and cell debris, a small sample of the product is kept separate for titration by the plaque titration method. If the titer is acceptable (between 10 '' -10 'pfu / ml or higher), the cells and cell debris are removed from the whole product as described above, and the liquid is ready to be dispensed into small vials for lyophilization of the final product after addition of a suitable stabilizer.

An alternative method, to achieve even higher titer values, can be used by infecting either stationary monolayers or such layers in rotating culture flasks. 2-8 hours after infection, the cells are trypsinized and transferred to suspension culture as described above. Care must be taken that no accumulation of cells occurs. In this way, a concentration higher than 2 x 10 cells / ml can be used, which provides higher titer values for the product depending on the number of infected cells introduced into the suspension culture.

Pharmacological Study To investigate the efficacy of the active and inactive rabies vaccines of this invention, the following are some of the results obtained in pre-infection and post-infection vaccination trials: A: Oral vaccination of foxes with active rabies vaccine

Q

strain 675 (2 ml / fox) at a vaccine titer of 2 x 10 pfu / ml.

λ 14 61 626

Age of the fox ro- Serum titer (IU *) Infection- identification- home- Q 1Λ ....

Oral 84 5 months. 4.0 7.0 8.2 secured through 86 6 months. 1.3 1.6 1.4 protected vaccine- 88 6 months. 2.0 2.4 3.2 protected animals Köntrol- 83 6 months. died 15 days.

li- ie after vaccination- 85 6 17 days · after mats __ 92 6 months. died 18 days.

animals after * IU (International Unit) = 1/300 dilution of serum, which dilution results in a 50% reduction in plaque formation.

A very high dose of infectious virus (obtained from the salivary gland of a naturally infected fox), namely 1,391,610 MLD ^ units, was used to determine the protective capacity of the vaccine.

This experiment proves the effectiveness of oral vaccination of young foxes. Immune reactions in foxes show adequate seroconversion and good titer values. More than five months after vaccination, all three foxes were fully protected, and the three unvaccinated three foxes died within a very short incubation period due to a very high dose of infectious virus implanted intramuscularly in the right hind leg. The usual dose of infection for foxes is 3000 MLD ^ Q units.

B: Serological results after vaccination with inactivated strain 675 either intramuscularly or subcutaneously: is 616 2 6

Animal Animal Vaccine Serum Titers 4 Weeks Species Weight (kg) Dose After Vaccination

Dog 1 6.5 2 ml i.m. 6.8 28 2 ml i.m. 6.3 3 12 2 ml i.m. 5.5 4 10 2 ml i.m. 12.3 5 15 2 ml i.m. 5.9 6 14 2 ml i.m. 4.5 7 17 2 ml i.m. 3.8 8 17 2 ml i.m. 20.5

Cow 1,200 5 ml s.c. 4.5 2,400 5 ml s.c. 8.5 3 300 5 ml s.c. 15.5 4,500 5 ml s.c. 4.8 5 400 5 ml s.c. 7.6 6,600 5 ml s.c. 8.9 7,500 5 ml s.c. 3.8 8 400 5 ml s.c. 4.7 i.m. = intramuscularly, s.c. = subcutaneously

Only one vaccine injection was given, and titer values are reported in IU as in the previous table.

Four weeks after vaccination, eight dogs and eight cattle showed very high titer seroconversion. The minimum titer required for seroconversion has been set by the European Pharmacopoeia at 0.2 IU / ml.

C: Protection of laboratory mice after infection with active and inactivated strain 675 by intraperitoneal administration of wild-type active virus (fox-derived): 16 61 626

Time between infection and vaccination

Vaccine- 12345678 9 *** type 1 h day. d. d. d. d. d. d. d. d.

Active vaccine 675 *

Unlawful 0/10 ** 1/10 1/10 1/10 2/10 0/10 0/10 4/10 5/10 3/10 10_1 3/10 10 "2 4/10 10 ~ 3 6 / 10 10 "4 8/10

Check 6/10 8/10 7/10 7/10 7/10 7/10 6/10 6/10 6/10 8/10

Inactive vaccine 675 *

Undiluted 0/10 1/10 5/10 10 "1 1/10 10-2 6/10 10" 3 8/10 10 "4 6/10 X o o

Vaccine titer 10 'pfu / ml, (inactive vaccine titer was determined before inactivation)

XX

Number of dead / number of mice vaccinated

XXX

Two vaccinations (on days 9 and 11).

All mice listed in this table were inoculated intramuscularly with rabies salivary gland virus (0.1 ml of solution) that killed 60-80% of control mice with a germination time of 9-11 days.

Active and inactivated vaccine virus 675 was administered in a single dose (0.5 ml) intraperitoneally at various times after initial infection, as shown in the table.

The active undiluted vaccine protected for a further 6 days after infection, which has never been seen before with other commercially available vaccines.

17 61 626 This protection indicates that mice can be cured even after the rabies virus enters the central nervous system.

Using two vaccinations every one day and starting 9 days after infection, a few of the mice became still protected.

This active vaccine could also be diluted tenfold and provided protection, but not for more than 24 hours after infection.

The inactivated vaccine protected only when administered within 24 hours of infection and could also be diluted, but not more than tenfold.

Claims (2)

18 61 626 A method for preparing active and inactivated rabies virus vaccines suitable for administration, both parenterally and non-parenterally, by growing an in vitro rabies virus strain in a manner known per se in cell systems of avian or mammalian origin and freezing the resulting cell suspension at 70 ° C or lowering and / or sonicating the cell suspension to disrupt the cells and release the virus, and then removing the cells and cell debris by filtration or centrifugation under sterile conditions, and finally optionally inactivating the culture broth using known methods and preparing a vaccine using known methods, the method uses a new rabies virus strain No 675 deposited in the collection of the "Czechoslovak National Collection of Type Cultures" of the Prague Institute of Hygiene and Epidemiology under number CNC TC AO 4/77. A method according to claim 1, characterized in that the chick embryo fibroblasts are infected for at least one hour with rabies virus strain No. 675, the infectivity of which, i.e. m.o.i. is 0.02-1 pfu / cell, and after removal of the unbound virus, a medium having a pH of 8.0-8.2 is added, preferably Basal Medium Eagle (bME) in Earle's saline supplemented with appropriate amounts of antibiotics, and 0.2-0.5% albumin, and the virus-infected cells are incubated at 32-39 ° C for 3-10 days, after which the cells and medium are frozen at -70 ° C or lower and the cells and cell debris are removed by filtration or centrifugation after sterilization in sterile conditions, and finally the virus in the culture media may be inactivated, and a vaccine is prepared using known methods. The method according to claim 1, characterized in that the cells of Baby Hamster Kidney 21 13 S (13 suspension cultures) grown in a spinner flask in a suspension of culture medium BHK-21 supplemented with small amounts of tryptose phosphate nutrient solution, inactivated calf serum and antibiotic approximately 8 x 10 6 cells / ml is reached, is infected for about 45 minutes with rabies virus strain 675 with a moi of 0.01-1 pfu / cell while stirring, after which the virus is incubated in 6162 6 BHK-21 culture medium containing 0.2% bovine serum albumin fraction V and appropriate amounts of antibiotics have been added at pH 7.5-8.0 and 32-35 ° C for 4-6 days, and at the end of the incubation the cells and medium are frozen at -70 ° C or at a lower temperature, followed by filtration or centrifugation under sterile conditions to remove cells and cellular debris, after which the virus in the culture media may be inactivated. and a vaccine is prepared using known methods.