SK129694A3 - A vaccine against hydrophobia and preparation method thereof - Google Patents

Abstract

The hydrophobia vaccine is applied for prophylactics related to virus diseases of dogs, cats and other agricultural and household animals. This vaccine is applied for vaccination of sensitive animals against the above mentioned diseases. This vaccine is liquid and 1,0 cm3 contains at least 1 international unit of virus VNUKOVO 32 1994 S 1, at least 10exp(4) inactivated particles of virus VEKO TL9/89 1994 S 2 and aluminium hydroxide gel. The hydrophobia virus is reproduced in a stabile line BHK-21, virus VEKO TL9 /89 is reproduced based on the cultures related to the calf's kidney cells. Infection virus liquids are semi purified with the use of ultra-filtration, they are inactivated with the use of beta-propiolactone, they are homogenised in the defined ratio, on aluminium hydroxide gel absorbed and this vaccine is purified so that, instead of the cultivation medium a physiological solution is applied.

Description

Technical field

The invention relates to a rabies vaccine and to a method for its preparation. The vaccine is used in immunoprophylaxis of dogs and carnivorous fur animals.

BACKGROUND OF THE INVENTION

The rabies of animals and humans is one of the oldest known infectious diseases, which still present an extremely serious epidemiological, epidemiological and economic problem. As the disease is fatal in humans, it is also a serious ethical problem.

The disease caused by a virus of the family Rhabdoviridae, genus Lyssavirus, has an acute course, mainly affecting the central nervous system.

The history of specific prophylaxis of rabies is associated with the famous personalities of Pasteur, Galdier and Roux of the Pasteur Institute in Paris. Since the preparation of the first vaccine in 1885 PASTEUR, L. Methode pour prévenir la rage apres morsure, C.R.Acad.Sci. (Paris) 101, 1885, p. 765-772, preparations intended for the specific prophylaxis of rabies have undergone substantial changes. To date, they can basically be divided into inactivated and live vaccines prepared from attenuated rabies virus strains. Older inactivated vaccines prepared from organs, respectively. brain of FERMI, or HEMPT, FERMI, C type animals. Ober die Immunisierung Gegen Wutkrankenheit, Ztschr. F. Hyg. und Infektionskr., 58, 1908, pp.233-276, HEMPT, A., Sur une methode rapide de traitment of the anti-revolution, Ann.Inst. Pasteur 39, 1925, pp.632-640, replaced vaccines prepared from viral strains propagated on cell cultures (Delcavac - R. of Gist Brocades n in Oelft, Holland) and vaccines prepared by genetic engineering methods. Developments in biology and technology have allowed widespread use of inactivated vaccines in the human population of World Health O'rganisation (WHO), Symposium, Lyon, March 10-12, 1980. In 1984, WHO recommended replacing live anti -rabial vaccines for animals with inactivated vaccines. These, despite the variety of strains of rabies virus used, different preparation technology and composition, must be:

- immune and provide maximum protection for as long as possible

- harmless to all species of vaccinated animals, without causing local or systemic reactions.

Under the WHO Health for All by Year 1000 program, the development of vaccines for both human and veterinary use is aimed at maximizing quality and efficacy while minimizing cost.

Possibilities of utilization of different types of cell cultures are being searched for, WHO Acceptability of cell substrates for production of biologicals. WHO Tech. Rep. Ser. 1987, 747.5; GRACHEV, V.- MAGRATH, D., Status of WHO concerning continuous cell lines and substrates for the duction of biologicals. Dev. Biol. Stand. 1989, 70, 215; MONTAGNON, B., Polio and rabies vaccines produced in continuous cell lines: a reality for VERO cell line. Dev. Biol. Stand. 1989, 70, 27; optimal methods of inactivation and purification of rabies virus, respectively. his antigens BUDOWSKY, E.- ZALESSKAYA, M .: Principles of selective inactivation of viral genome. V. Rational selection of conditions for inactivation of viral suspension of infectivity to a given extent by action of beta-propiolactone. Vaccine 1991, 9, 319; MORGEAUXjS.- T0RD0, N.- GONTIER.C. and PERRIN, P .: / β-propiolactone treatment of the biological activity of residual ONA from BHK-21 cells infected with rabies virus, Vaccine, 11, 1, 1993, p. B2-9O. The possibilities of maximal stimulation of humoral and cell-mediated immunity are studied. WUNDERLI, P.- SHADD0K, 3.- SCHMID, D.- MILLER, T., - BAER, G .: The protective role of humoral neutralizing antibody in the NIH potency test for rabies vaccines, Vaccin, 1991, 9, 638; JOFFRET.M.L., - ZANETTI.C.- MORGEAUX, S. and col. Appraisal of rabies vaccine potency by in vitro, specific interleukin-2 production. Biologicals, 1991, 19,113; and minimizing residues such as cellular ONA in a van WEZEL vaccine dose, A. van der MAREL, P.- van BEVEREN, C. and col. Detection and elimination of cellular nucleic acids in biologicals produced on continuous cell lines. Dev. Biol. Stand. 1982, 50, 59. The inactivated vaccine technology is based on the preparation of viral suspensions that are suitably inactivated. The immunostimulatory effect of inactivated viral suspensions is usually potentiated by the addition of suitable mineral carriers or adjuvants.

SUMMARY OF THE INVENTION

The present invention consists in a process for the preparation of an inactivated rabies adsorbate vaccine based on the production of rabies virus, VNUKOVO 32 1994 S 1 strain propagated on stable line cultures of BHK-21 cells cultured statically and on microcarriers, and production of sheep parapoxvirus / SKO strain TL9 1994 2 are propagated on kidney cell culture cultures, clarification of rabies virus infectious fluids by ultrafiltration, gentle virus inactivation, adsorption of inactivated viral antigens on aluminum hydroxide gel, and further purification by replacing the culture medium with saline. Adherence to defined technological procedures will ensure the preparation of an inactivated vaccine with high quality parameters that effectively acts on the humoral and cell-mediated component of the immune system.

An inactivated rabies adsorbate vaccine containing 1.0 cm @ 2 of rabies virus, VNUKOVO 32 1994 S strain 1. was propagated on a stable BHK-21 line with an infectious titer prior to inactivation of at least 10 ^ TK10 _sn / ml, sheep parapoxvirus immunostimulator , strain VEKO TL9 / 89 · 1994 S 2 propagated on kidney cell carcasses with infectious titer before inactivation of at least 10 * TKID / g, traces of antibiotics, thiomersal preservative and mineral carrier, aluminum hydroxide gel. The vaccine is prepared from bacteriologically sterile infectious fluids of VNUKOVO 32 virus; which, after clarification by filtration through bacteriological filters with a porosity of 0.80 and 0.45 µM, are inactivated by the addition of 1: 4000 beta-propiolactone and the same inactivated VEKO TL9 / 89 fluids which are homogenized in a 4: 1/80 volume ratio parts by volume of inactivated virus VNUKOVO 32 + 20 parts by volume of inactivated virus VEKO TL9 / 89 and adsorbed on the mineral carrier Al (OH) j so that the final concentration of Al ²⁺ is 1.5 mg to 2.0 mg per milliliter of vaccine. After complete adsorption of the inactivated viral antigens to the mineral support and its sedimentation, the supernatant is gently removed and replaced with sterile buffered saline (PBS) pH 6.8-7.0. To the purified pH-stable purified vaccine, a fungisone solution is added and the formulation is preserved by the addition of thiomersal to a final concentration of 1:10,000.

The inactivated rabies adsorbate vaccine thus prepared exhibits the following immunobiological parameters:

(a) Since it contains only inactivated viral antigens bound to a mineral carrier and a Thiomersal preservative, it is harmless to different types of domestic animals, regardless of age.

(b) It is effective, each vaccine dose has at least one International Unit (1 TU).

c) Vaccination of susceptible animals induces specific virus-neutralizing antibodies to titers of 1:32 to 1:64 within 14 days.

d) A biological immunostimulator actively affects the cell-mediated immunity component and induces early and long-term protection of vaccinated animals against disease.

e) The offspring of immunized mothers are passively protected by maternal antibodies until they can be actively immunized.

f) The method of preparation of the vaccine allows it to be combined with other inactivated and live vaccines, while maintaining the immunogenic efficacy of all preparations administered.

A simple method of preparing inactivated rabies adsorbate vaccine from the VNUKOVO 32 strain ensures a high purity of the formulation, free of residual substances that may have a negative effect on the formation of immunity in vaccinated animals. The vaccine induces humoral antibody production to high titers in vaccinated animals and, unlike other similar preparations, actively acts through a biostimulator to the cellular component of the immune system. It is effective and protects against disease.

The quality parameters of the vaccine were defended on August 31, 1994 in the Commission for Veterinary Biologicals and Medicines of SVS SR and the registered product will be the subject of commercial sale. The purchase of a license of an inactivated rabies adsorbate vaccine and the method of its preparation has shown serious interest to a foreign partner who has already received the preliminary terms of the license agreement and the vaccine sample. DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by the following examples which are not intended to be limiting or exhaustive:

Example 1

I

Method of cell culture

BHK-21 cell suspension prepared by proteolytic ferments and bovine kidney cell suspension prepared from kidneys of freshly drawn calves prepared by proteolytic ferments in a growth medium containing defined amounts of amino acids, vitamins and addition of 5 volume percent serum for cell cultures are cultured for glass, plastic, in a stationary manner, in rotated bottles or in suspension on microcarriers.

.5

2000 ml of growth medium, which is a minimum essential medium according to Eagle, pH 7.2, enriched with the addition of five percent by volume of precolateral calf serum (PKS) and antibiotic solution, is poured into a sterile vessel of suspension cell culture apparatus with 4000 ml capacity. Subsequently, 54 ml of a microcarrier stock type solution is added

D

CYTODEX 3 (Pharmacia Bio Process, Uppsala, Sweden) treated according to the manufacturer ' s instructions to make a stock suspension in 30 ml of buffered saline 1.0 grams of originally dry collagen-dextrate microcarriers, representing a culture surface 9 of approximately 4800 cm. Cells of the BHK-21 line, obtained from Roux and Lys, respectively. roller bottles using proteolytic ferments resuspended in a small volume of the minimum essential medium according to Eagle with the addition of 5 volume percent precolostral calf serum represent a biological substrate concentrate with a defined number of cells. From the concentrate thus prepared, a number of cells are added to the culture vessel of the apparatus to a final concentration of 4.7 x 10 ⁵ . ml ^-1 growth medium. Cells are cultured at 37 ° C with stirring at 0.4 stirrer per second for 48 hours.

Cells prepared from bovine kidneys at concentration

6-1 x 10 ml Hanks culture medium supplemented with five volume percent of precolostral calf serum were cultured in a stationary manner on Roux flasks at 37 ° C for 5 days.

Example 2

A method of infecting cell cultures and multiplying viruses

BHK-21 cell cultures prepared and propagated according to section 1 are infected with VNUKOVO 32 1994 S 1 vaccine virus as follows:

For example, hourly cultures of cells that formed a complete monolayer on the microcarrier culture surface, after fusion of the growth culture medium with the serum-free medium prepared according to Hanks, are infected directly in the culture vessel by VNUKOVO 32 virus adsorption at a multiplicity of infection of 0.01. After 60 minutes of adsorption at 22 ° C with occasional mixing of the infected material, the culture vessel is made up to a volume of 4000 ml with Hanks' maintenance medium enriched by the addition of 1 volume per 6 cents of precostral calf serum. The virus multiplies for 5 days at 32 ° C and 0.4 stirrers per second. After five days of culture, the propagated virus culture medium is gently harvested after microcarrier sedimentation and the infected cells are repeatedly watered with fresh maintenance medium. A second virus harvest is performed in the same manner 48 hours after the medium change.

Bovine kidney cultures prepared according to point 1 are infected with VEKO TL9 / 89 virus as follows:

Growth medium is removed from the monolayer of cells grown on Roux flasks after five days, the cultures are fed with a smaller volume of Hanks prepared medium and infected with VEKO TL9 / / 89 by adsorption for 30 minutes at 22 ° C. The multiplicity of infection is 0.04. After adsorption is complete, maintenance medium is added with the addition of 1 percent bovine fetal serum (BOFES). The virus is propagated at 37 ° C until extensive cytopathic changes are determined microscopically.

Example 3

Preparation of inactivated adsorbate vaccine

Infectious fluids containing at least 10 µg / ml VNUKOVO 32 virus particles and 10 µg / ml VEKO TL9 / 89 particles, bacteriologically sterile, are used to prepare the inactivated adsorbate vaccine. VNUKOVO 32 virus infectious fluids of the desired quality are purified by filtration through filters with a porosity of 0.80 and 0.45 µM (Millipore, Millipore Corporation, Bedford, MA 01730) and then inactivated by the addition of 1: 4000 beta-propiolactone. The same beta-propiolactone concentration inactivates VEKO TL9 / 89. The inactivated viral fluids are mixed at a ratio of 1: 4 (80 parts by volume of rabies virus + 20 parts by volume of parapoxvirus) and homogenized perfectly. The inactivated viral antigens are adsorbed onto a mineral carrier - high strength aluminum hydroxide gel so that the final concentration of A1 is 1.5, maximum 2.0 mg per 1.0 milliliter of vaccine. Upon complete adsorption of the inactivated antigens to the aluminum hydroxide gel, the vaccine is further purified by gently removing from the sedimented gel the supernatant, which is a culture medium with traces of serum and other impurities, and this is replaced by sterile physiological saline (PBS) pH 6, 8 to 7.0. To the vaccine thus prepared is added a solution of fungisone and thiomersal to a final concentration of 1:10,000.

Test results

Result of the vaccine toxicity study The toxicity study of the inactivated rabies adsorbate vaccine (IAV) focused on:

- Verifying the innocuity of the inactivating agent on the cells

- Examining the possibility of mixing the vaccine with other bioproducts

- Clinical testing of vaccine harmlessness for laboratory and domestic animals.

a) Filtered rabies virus infectious fluids were inactivated by beta-propiolactone at a ratio of 1: 4000 (recommended concentration), 1: 1000 and 1: 500 at +4 ° C. after termination of the inactivation, cell suspensions of BHK-21 (Stable Syrian Hamster Kidney Line), MDCK (Stable Canine Kidney Cell Line), and PE (Stable Cat Kidney Cell Line) were infected with 0.1 ml of inactivated virus. Dynamics of growth and appearance of cells, resp. monolayer, compared to unvaccinated control cultures, was followed for 5 days. The BHK-21 cell line was also evaluated for the effect of virus inactivation relative to individual inactivating agent concentrations by direct immunofluorescence. The results are shown in Table 1.

TAB.č.l

concentration BPL Dilution of inactivated virus inoculum 10 ° 10 ¹ 10 ' ² 1: 4000 x - - 1: 1000 - - - 1: 500 average changes to BHK-21

x cultures showed normal growth

The stable feline kidney cell line FE was used for its high sensitivity to any change, respectively. negative impact on the quality of culture conditions.

The recommended concentration of 1: 4000, or concentrations of BPL 4 and 7 times higher, did not affect the cell cultures used, with the recommended concentration of 0.00025 ml / ml of the viral suspension reliably inactivating rabies virus.

b) Possibility of mixing, resp. Combined rabies IAV with live vaccine documented results with attenuated distemper vaccine - DISTEVAC. Ten minutes after mixing the vaccines, 5 puppies aged 7 to 8 weeks were vaccinated containing anti-distemper antibodies and three seronegative pups at the same time with the same vaccine mixture. Sera, obtained one month after vaccination, were screened for VN antibodies against rabies virus and distemper. The results are shown in Table 2. Thus, it is clear that the inactivated vaccine can be combined with a live canine vaccine or other live vaccines and administered by a single injection without fear of the immunogenic efficacy of the two vaccines administered.

table 2

Applied vaccine Seropositivity against distemper Wall number VN antibody titers against virus rabies distemper 1 7.33 5.0 2 8.0 5.5 positive 3 7.5 4.33 4 7.33 6.5 IAV + 5 7.0 5.33 DISTEVAC 6 7.5 6.5 negative 7 7.5 7.33 8 7.33 7.0

c) Toxicity of a complete adsorbate vaccine characterized by physicochemical parameters: actual acidity of 7, 15, Thiomersal 0.096 g / l, Al ⁺ 4.080 g / l was verified in mice and rabbits and in veterinary hospital facilities in Trnava on target animals - dogs and cats.

The vaccine was administered to mice weighing 12 to 18 g intraperitoneally at a dose of 0.5 ml, subcutaneously 0.2 ml, intradermally not 0.05 ml, and intracerebrally at a dose of 0.03 ml. In the intracerebral route of administration, two of the 10 animals died within 24 hours of dosing. This mortality was apparently caused by mechanical disruption of nerve tissue and is also permitted by the OIE methodology, which defines the conditions for controlling the efficacy of an inactivated vaccine. For the other routes of administration, the dose administered did not elicit any overall response in the monitored animals for 14 days and had no effect on their growth (body weight).

Another laboratory animal tested was the rabbit. The test rabbits were vaccinated with the same vaccine as mice intramuscularly and subcutaneously with a 0.2 ml dose. Vaccination did not cause any local or general reactions in the animals studied.

The harmlessness of the vaccine to the target animals was confirmed by the results of closer clinical testing in dogs, cats without breeding, performed at the veterinary hospital in Trnava. Eight 7-8 weeks old pups, five 6-7 month-old dogs and nine cats aged 3 to about 8 months were vaccinated intramuscularly and subcutaneously (approximately half of the animals) with a 1.0 ml dose. All vaccinated animals, irrespective of the route of administration, were free from local and any general reactions. In a wider clinical trial of vaccine harmlessness, 216 dogs of different ages and breeds and 31 cats were vaccinated. Of the 84 dogs vaccinated at VELAZ Prague were predominant dogs of breed German Shepherd and Beagle. 132 dogs of private breeders, vaccinated by veterinary surgeons of Trnava Hospital, formed a heterogeneous group of animals of different ages and breeds. In the wider clinical trial of the vaccine, no negative reactions were found after vaccination.

(

Also for cats, the vaccine at the prescribed vaccine dose, irrespective of the route of administration (i.m., s.c.), was harmless.

CLINICAL VERIFICATION OF THE MEDICINAL PRODUCT

The quality of the vaccine was determined in accordance with internationally applicable guidelines: OIE Manual, Rabies (B8) p. 205-215, European Pharmacopoeia, Second Edition (1985), Part II (9), 451, Vaccinum rabiei inactivatum ad usum veterinarium, and WHO Expert Committee on Rabies, 1992.

The vaccine with the required IU was tested in mice, dogs and cats, and its immunogenic and protective effect was compared with that of commercial vaccines of foreign origin.

a) Three groups of 12-18 g mice (10 mice per group) were injected in parallel intraperitoneally with two lots of rabies IAV and a commercial vaccine RAB1SIN. Half of the mice from each treatment group were bled 14 days after immunization and the remaining animals were revaccinated with the same dose (0.05 ml). Blood from revaccinated mice was collected 28 days after the first immunization. The sera obtained were, as pooled samples, screened for antibody content by the VN BHK-21 cell culture assay. Test Method - According to b) Virus Neutralization Test in Cells (RFFIT), CIE Manual 1992, p. 209-210. The results of examinations are expressed in Table 3.

TAB.č.3

Group Applied Titres against: .átok vaccine after vaccination after revaccination First IAV 06 5.33 8.50 Second IAV 07 5.00 8.33 Third RABISIN 5.50 8.33

b) The results of testing the protective effect of IAV against rabies and a comparison with the protective effect of DEFENSOR are shown in Table 4.

For challenge infection of all immunized mice, virulent CVS virus was used as a brain suspension, administered at a dose of 0.03 ml containing 50 TKID / g.

TAB.č.4

vaccine Dilution of the vaccine died / survived Control titration prac.vírusu 3VS 10 ° 10 ¹ 10 ' ² w ⁰ 10 ^-1 10 ^-2 IAV 0/10 3/5 5/5 5/5 3/5 0/5 DEFENSOR 0/10 3/5 5/5

Quote! - number of dead mice Denominator - number of infected mice

The test results confirmed the good protective efficacy of the prepared vaccine.

The immunogenic efficacy of the vaccine was examined for

- puppies aged 7 + 28 weeks

- puppies aged 13 to 16 weeks

- dogs and cats over 4 months of age

(c) Control of the immunogenic efficacy of the vaccine on 7-8 weeks of age:

seronegative and 9 seropositive puppies with a colostral antibody titer of 1: 8 to 1:64, were immunized intramuscularly against rabies. All pups immunized with 1.0 ml were revaccinated with the same dose at 13 to 14 weeks of age. Blood samples were taken at 2, 5, 7 weeks, 3 and months after vaccination. The immune response of seronegative and seropositive puppies is shown in Fig. 1.

Puppies with no starting antibody titres responded to priming with VN antibody formation up to 1:64 to

1: 512 and for subsequent revaccination with a further rise in antibodies to titers of 1: 256 to 1: 1024.

Puppies containing antibodies at the time of the start of the experiment showed a further decrease in maternal antibody titers after priming and were almost seronegative at the time of revaccination (1: 2 - 1: 4). The revaccination carried out in these animals at weeks 13 to 14 initiated active antibody production, with the dynamics of their production and the titers being basically the same as in the primed negative pups.

1.2

The VN antibody titers detected in the sera of both initially seronegative and seropositive puppies were approximately the same at 6 months after vaccination and ranged from 1: 128 to 1: 512.

(d) Control of the immunogenic efficacy of the vaccine on puppies aged 13 to 16 weeks:

Eleven seronegative pups aged 13 to 16 weeks were immunized subcutaneously with the inactivated vaccine.

All pups immunized with 1.0 ml were revaccinated with the same dose and in the same manner at 6 months of age, with 7 of the puppies being simultaneously immunized with live attenuated parvovirosis, distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2 commercially labeled TETRA inj. a.u.v. from MEVAK a.s.

TETRACAN was administered simultaneously according to the instructions for use. Blood samples were taken at the time of vaccination at the age of 6 months Puppies and one month after revaccination (7 months of age).

The vaccination results of this group of animals are shown in Fig. 2. Seronegative pups responded to primary vaccination with the production of VN antibodies, which were at titers of 1: 128 to 1: 256 2 to 3 months after vaccination (6 months of age). Subsequent revaccination stimulated a further rise in antibodies to titers of 1: 128 to 1: 1024, while simultaneous administration of TETRACAN did not affect the intensity of the antibody response against rabies virus.

TAB.č.5

Wall applied vaccine Titer antirabic antibodies after revaccination 1 8.5 2 8.0 3 10.0 4 IAV + 8.33 5 TETRACAN 8.0 6 9.0 7 average ^7.33 8.44 8 9.0 9 10 IAV 9.5 8.0 11 8.0 average B, 62

VNT expressed in

(e) Control of immunogenic efficacy of the vaccine in adult dogs and cats:

Immunogenic efficacy of the vaccine for adult dogs (5 stateless animals housed in private breeders); VELAZ Lysa nad Labem, race 04 (6 pcs Labrador, 7 pcs German Shepherd, 10 dogs Beagle) and cats placed by private breeders, is shown in Fig. 3.

Clinical verification of vaccine efficacy continued and sufficient protection titers of VN antibodies were demonstrated in vaccinated animals after 12 months.

Claims (4)

Patent claims An anti-rabies vaccine, characterized in that it contains 1-5 international units (IU) of VNUKOVO 32 1994 S 1 virus at 1.0 cm ^, 10 ^4-10 ^ chemically inactivated virus particles LID TL9 / 89 1994 S 2 and the aluminum hydroxide gel. 2. The vaccine of claim 1, wherein 1.0 cm @ 2 contains 1.0 mg to 2.0 mg ^Al5 +. 3. The vaccine production method according to claim 1 or 2, characterized in that the VNUKOVO 32 virus and the VEKO TL9 / 89 virus are propagated on a Syrian hamster kidney cell culture and cultures prepared from glass kidney grown on plastic, cultured in a stationary manner. , in rotated flasks, or on microcarriers. 4. The method of claim 1, wherein the rabies virus VNUKOVO 32 1994 S1 culture is obtained by propagation on a stable BHK-21 cell line after a 30-60 minute infection of a 24-72 hour monolayer in a culture medium at 31 ° C. to 33 ° C for 3 to 6 days and subsequent culture medium change and further multiplication at the same temperature for 1 to 3 days, with a minimum content of 10 µg / ml, semi-purified by filtration and inactivated with o-propiolactone and cultivate parapoxvirus VEKO TL9 / 89 1994 S 2 propagated on calf kidney cell cultures after 30 to 60 minute infection of 4 to 6 day monolayers in culture medium at 36 to 38 ° C for 3 to 6 days, until clear cytopathic changes are observed with minimal containing 10 ⁴ TKID? g / ml, inactivated by? -propiolactone is mixed in a ratio of 70 to 90 parts by volume of VNUKOVO virus 32:30 to 10 parts by volume of VEKO TL9 / 89, h The omogenizate adsorbs onto the aluminum hydroxide gel and is subsequently purified by removing the supernatant and diluting the sediment with saline to 0.5 to 1.5 times the original volume. it Ή C it > C about about 2C • H XJ 2C E O tn C Jč 03 r4 tn tn U c to (U rM> o + -> h it 03> E -H to Ή N E E 03 JC it