RU2242246C2 - Tableted live recombinant bivaccine "revaks vkt" against natural smallpox and hepatitis b and method for its preparing - Google Patents

Abstract

FIELD: biotechnology, medicine, virology, pharmacy.

SUBSTANCE: invention proposes bivaccine comprising in a single tablet lyophilized viral material produced on the basis of the recombinant smallpox vaccine virus strain GKV № 2131 with typical properties of smallpox virus expressing preS2-Ag and HbsAg of hepatitis B virus with activity 6.5-8.0 lg CPD₅₀ per a tablet, lactose, sucrose, calcium stearate, vanillin, peptone, gelatose and buffer. Smallpox virus is produced in transplanted mammalian cell cultures in the following ratio of components per one tablet, wt.-%: smallpox lyophilized viral material with activity 6.5-8.0 lg CPD₅₀ per a tablet, 1.04-31.3; peptone, 0.12-3.8; gelatose, 0.06-1.9; sucrose, 10.1-11.55; buffer, 0.001-0.027; calcium stearate, 1.94-2.0; vanillin, 0.17-0.2; lactose, the balance, up to 100%. Method for preparing bivaccine involves passaging the strain GKV № 2131 of recombinant smallpox virus in biological system used for culturing viruses, preparing virus-containing material with titer 8.0 lg CPD₅₀/ml, not less, its lyophilization with stabilizing additives, milling, mixing with filling agent and pressing. The transplanted mammalian cell culture 4647 or VERO are used as a biological system with the infection dose with the recombinant strain of smallpox virus 1.0 CPD₅₀ per a cell, not above. Virus is produced in monolayer of indicated mammalian cells in rollers on nutrient medium up to 50% value of cytopathic effect (CPE) followed by exchange of this medium for buffer solution with less volume as compared with volume of the nutrient medium. Preparing virus-containing material is carried out after exchange of the nutrient medium for buffer solution by extraction of viruses in three-fold freezing and thawing the cellular monolayer. As stabilizing additives in lyophilic drying peptone, sucrose and gelatose are used in their final concentration in mixture, wt.-%: 2.0-4.0; 2.0-3.0, and 1.0-3.0, respectively. Vaccine retains activity during processes of drying and tableting and comprises lesser amounts of foreign impurities. Method provides the improved technology in preparing vaccine.

EFFECT: improved preparing method.

5 cl, 4 tbl, 7 ex

Description

The invention relates to medicine, in particular to immunology, and can be used for specific prophylaxis of infectious viral hepatitis B and smallpox.

Known live recombinant vaccine for the prevention of hepatitis B and smallpox for cutaneous use (RF patent No. 2073524, IPC A 61 K 39/29; C 12 N 15/86, publ. 02.20.97). The vaccine contains a strain of recombinant smallpox vaccine virus (BWA) with bills No. 2131 with a titer of (3-5) × 10 ⁸ OOE / ml, capable of synthesizing preS2 and HbsAg of hepatitis B virus and peptone as a stabilizer in the following ratio of components, wt.%:

recombinant smallpox virus strain

STB vaccine No. 2131 with a titer of 90-95

peptone 5-10

A disadvantage of the known vaccine is that it uses only peptone as a stabilizing additive, which does not sufficiently ensure the preservation of the activity of the viral material during its lyophilization. In addition, the productivity of this drug in case of skin vaccination is low, the need for qualified medical personnel for vaccination, the threat of infection of the vaccinated patient with immunodeficiency viruses, hepatitis, etc. When vaccinated with scarification, vaccinia virus can be released into the environment, which threatens its uncontrolled transmission to unvaccinated people with the risk of developing serious complications.

A known method of producing a live recombinant vaccine for the prevention of hepatitis B and smallpox for cutaneous application, including the passage of the recombinant strain of Great Patriotic War Bills No. 2131 twice on chicken embryos and once on the skin of the calf, then the virus-containing material is accumulated on the skin of the calf and collected by scraping the homogenized smallpox with freon, the virus is isolated by centrifugation in a supernatant containing a virus with a titer of (3-5) × 10 ⁸ OOE / ml, 5-10 wt.% peptone is added, after which the resulting vaccine is decomposed they are poured into ampoules and freeze-dried (RF patent No. 2073524, IPC A 61 K 39/29; C 12 N 15/86, publ. 02.20.97).

The disadvantage of this method is the use of an expensive and scarce biological system (producer) for the production of the virus (calves and developing chicken embryos - RCE), the parameters of which are difficult to standardize and maintain. In Russia, there are practically no farms for the production of RCEs in accordance with international standards that guarantee the absence of extraneous microflora in the RCEs and standard indicators of embryo development. This, in turn, leads to marriage in the production of vaccines, and can also adversely affect its properties. To an even greater extent, these shortcomings are manifested when the virus is produced on calves.

The closest analogue prototype of the vaccine is a tablet live recombinant bivaccine against smallpox and hepatitis B based on the smallpox vaccine virus for oral use, containing in one tablet coated with an acid-resistant coating, lyophilized viral material, developed on the basis of the recombinant BOB strain GKV No. 2 vaccinia virus expressing preS 2-Ag and HbsAg of hepatitis B virus with activity of 6.5-8.0 lg OOE per tablet, lactose, sucrose, calcium stearate and vanillin (RF patent 2076735, IPC A 61 K 39/00 C 12 N 15/00, published 10.04.97 g)..

A disadvantage of the known vaccine is that it uses lactose alone as a stabilizing additive, which insufficiently ensures the preservation of the activity of the viral material during its lyophilization and especially tabletting. In addition, active viral material is produced at the RCE, which does not exclude the content of additional impurities in the preparation and its contamination with extraneous microflora, and requires additional biological control.

The closest prototype method for producing a tablet hepatitis B and smallpox bivaccine is a method for producing a live, recombinant hepatitis B smallpox and hepatitis B tablet, including passaging the recombinant strain of Great Patriotic Bacteria bills №2131 on the RCA, obtaining a virus-containing material with a titer of at least 8.0 lg OOE / ml, its lyophilization with a stabilizing additive, grinding, mixing with a filler, pressing and coating with an acid-resistant coating (RF patent No. 2076735, IPC A 61 K 39/00; C 12 N 15/00, publ. 1 04/04/97).

The disadvantage of this prototype method is the use of an expensive and scarce biological system (producer) for the production of the virus (RCE), the parameters of which are difficult to standardize and maintain. In Russia, there are practically no farms for the production of RCEs in accordance with international standards that guarantee the absence of extraneous microflora in the RCEs and standard indicators of embryo development. This, in turn, leads to marriage in the production of vaccines, and can also adversely affect its properties.

The technical result of the proposed inventions is the creation of such a vaccine against smallpox and hepatitis B and a method for its preparation, which would make it possible to obtain the virus-containing material in a more concentrated and purified form, to maintain the activity of this virus-containing material during lyophilization and tabletting, and to reduce the likelihood of contamination of the finished product with an extraneous microflora, as well as simplify production technology and provide the ability to standardize the main technological stages of obtaining the vaccine.

The specified technical result is achieved in that in a tablet live recombinant bivaccine against smallpox and hepatitis B based on the vaccinia virus for oral use, containing in one tablet lyophilized viral material, produced on the basis of the recombinant BOB strain BKB No. 2131 with typical properties of BOB expressing preS 2-Ag and HbsAg of hepatitis B virus with an activity of 6.5-8.0 lg CPD ₅₀ per tablet, lactose, sucrose, calcium stearate and vanillin, according to the invention, the vaccine further comprises peptone , gelatose and buffer, and WWII was developed on transplantable cultures of animal cells in the following ratio of components per tablet, wt.%:

lyophilized viral

vaccinia material with activity

6.5-8.0 lg CPD ₅₀ per tablet 1.04-31.3

peptone 0.12-3.8

gelatin 0.06-1.9

sucrose 10.1-11.55

buffer 0.001-0.027

calcium stearate 1.94-2.0

vanillin 0.17-0.2

lactose rest up to 100%

Bivaccine may additionally contain urea in an amount of 0.01-0.2 wt.%.

The specified technical result is also achieved by the fact that in the method for producing a tablet live recombinant bivaccine against smallpox and hepatitis B, which includes the passage of the recombinant strain of Great Patriotic War Bills No. 2131 in a biological system for the cultivation of viruses, obtaining a virus-containing material with a titer of at least 8.0 lg CPD ₅₀ / ml, its lyophilization with a stabilizing additive, grinding, mixing with a filler, compression, according to the invention as a biological system for culturing the virus using m continuous culture of animal cells with a dose of vaccinia infection of not more than 1.0 CPD ₅₀ per cell, the virus is carried to an operating time of said monolayer of animal cells in roller on the medium before the 50% cytopathic effect (CPE) followed by replacement of the medium in buffer a solution with a volume less than the volume of the nutrient medium; the preparation of the virus-containing material is carried out after replacing the nutrient medium with a buffer solution by extracting the virus by freezing and thawing the cell monolayer three times, and peptone, sucrose and gelatose in the final concentration in the mixture are used as stabilizing additives for freeze drying, wt.%: 2.0 -4.0; 2.0-3.0; 1.0-3.0 respectively.

As a transplantable culture of animal cells, green monkey kidney cells No. 4647 or VERO are used, as a nutrient medium for virus production, DMEM medium or MEM Needle with 2% cattle serum or 2% fetal calf serum as a buffer the solution is used 0.01 M Tris, pH 8, and when changing the nutrient medium to a buffer solution, its volume is reduced by at least 10 times.

Urea in a final concentration in a mixture of 1.0-2.0 wt.% Is used as an additional component of the stabilizing additive during freeze drying of the bivaccine.

Below are examples of 1-4 formulations of a tablet live recombinant bivaccine against smallpox and hepatitis B.

Example 1

lyophilized viral material

smallpox vaccines with an activity of 6.5 lg

₅₀ CPP per tablet 1.04

peptone 0.12

gelatin 0.06

sucrose 10.10

urea 0.20

buffer 0.001

calcium stearate 2.0

vanillin 0.20

lactose rest up to 100%

Example 2

lyophilized viral material

smallpox vaccines with an activity of 7.0 lg

₅₀ CPP per tablet 3.20

peptone 0.38

gelatin 0.19

sucrose 10.29

urea 0.01

buffer 0.003

calcium stearate 1.99

vanillin 0.2

lactose rest up to 100%

Example 3

lyophilized viral material

7.5 smallpox vaccines

lg CPA ₅₀ per tablet 10.40

peptone 1.20

gelatin 0.60

sucrose 10.85

buffer 0.009

calcium stearate 1.97

vanillin 0.19

lactose rest up to 100%

Example 4

lyophilized viral material

smallpox vaccines with an activity of 8.0 lg

₅₀ CPP per tablet 31.30

peptone 3.80

gelatin 1.90

sucrose 11.55

buffer 0.027

calcium stearate 1.94

vanillin 0.17

lactose rest up to 100%

The recombinant strain of the Great Patriotic War of Bills No. 2131 (b7.5S2-S BOB) was deposited in the State Virus Collection of the Institute of Virology named after D. I. Ivanovsky (RF patent No. 1575576, C 12 N 15/00, publ. 1994).

Gelatose - partially hydrolyzed gelatin - a component of the stabilizer; urea is a chelating agent, a component of the stabilizer. Lactose (TU 6-09-2293-77), stabilizer and filler; sucrose (GOST 5833-75), stabilizer and organoleptic; calcium stearate (TU 6-09-4233-76) - a sliding additive; vanillin (GOST 16599-71), organoleptic.

Example 5. A method of obtaining a tablet live recombinant bivaccine "Revax VKT"

First, the homogeneity of the viral materials obtained by 10-fold passage of the museum strain of STB No. 2131 (b7,5S2-S BOB) on transplantable cultures of Vero and 4647 cells was tested. For this purpose, studies of the passive material (5th and 10th passages) by analysis expression of HBs antigen by ELISA and the presence of an insert of the pre-S2-S gene in viral DNA by PCR. The need for these studies is dictated by the requirements for the production of the drug, when the number of passages of the production strain in producer cell cultures can reach 10. As a result of these studies, it was noted that the passage material (5th and 10th passages in Vero and 4647 cell cultures) do not contain BOB impurities wild-type and expresses the HBs antigen at a level of 100-200 ng / 10 ⁶ cells. This testifies to the high stability of the created genetic construct and opens up prospects for using any of the tested cell cultures for the production of bivaccine against smallpox and hepatitis B.

After this, monolayers of transplantable cultures of green monkey kidney cells No. 4647 or VERO are grown on DMEM or MEM Needle with 10% cattle or 10% fetal calf serum in roll bottles, for example, 2 liters. The growth medium used is drained and the cell monolayer is poured with DMEM or Eagle MEM maintenance nutrient medium with 2% cattle serum or 2% fetal calf serum in an amount of 200 ml per bottle. Cells are infected with a recombinant strain of the Great Patriotic War of bills bills No. 2131 (b7,5S2-S BOB) with a dose of infection of not more than 1.0 CPD ₅₀ per cell. Cultivation is carried out at a bottle rotation speed of 3-12 revolutions per minute up to 50% of the CPP (under this condition, the highest virus yield is achieved). Table 1 below shows the results of cultivation.

Next, the nutrient medium (200 ml in one bottle) is replaced with a buffer solution with a volume (20 ml), i.e. 10 times less than the volume of the nutrient medium. As a buffer solution, 0.01 M Tris is used to maintain pH 8. The nutrient medium is replaced with a smaller buffer (0.01 M Tris with pH 8.0) before the virus is extracted from animal cells in order to reduce the volume of viral biomass (while maintaining the total number of viable viral particles), which reduces financial and time costs during the process of subsequent lyophilization of viral material. Obtaining a virus-containing material is carried out after replacing the nutrient medium with a buffer solution by extracting the virus by freezing three times at a temperature of minus 20 ° C for at least 5 hours and thawing the cell monolayer at a temperature of plus 20 ° C for at least 1 hour. Next, the resulting virus-containing suspension is defended and the target supernatant fraction is separated with a titer of at least 8.0 lg CPD ₅₀ / ml, into which stabilizing additives are introduced: peptone, sucrose and gelatin in their final concentration in the mixture, wt.%: 2.0-4-4 0; 2.0-3.0; 1.0-3.0, respectively, or urea, peptone, sucrose and gelatose in their final concentration in the mixture, wt.%: 1.0-2.0; 2.0-4.0; 2.0-3.0; 1.0-3.0 respectively. The resulting liquid preparative form is poured into containers, which are placed in a lyophilic type SUPER-4. After carrying out the standard lyophilization procedure, the infectious viral activity practically does not decrease in the preparation, which indicates a high resistance of the b7.5S2-S BOB strain to lyophilization in the presence of the claimed stabilizing additives. Next, the dry material is ground and mixed with a filler. Table 2 below shows the prescription composition of tablets with different activity of the vaccine material (in accordance with examples 1-4).

Compression into tablets of the drug is carried out on a twelve-punch tablet press model K-VIII (Dresden, Germany). The amount of virus-containing material in each tablet is laid so that the concentration of viable virus in them, according to the calculated data based on the activity of lyophilized materials, is in the range from 6.5 to 8.0 lg CPD ₅₀ / tablet.

Example 6. Data on the study of the activity of the tablet form of the bivaccine "Revax VKT"

Experimental studies of the activity of the obtained tablets showed that during the preparation of the Revax BKT vaccine tablets on the basis of lyophilized viral material in the presence of a stabilizing additive, no significant inactivation of the viral component was observed. The calculated data on the concentration of the virus in the tablets coincided with the results of virus measurements in tablets: 7.2; 7.2; 7.2; 7.0 lg CPD ₅₀ / tablet, and the tablet form itself corresponded in its physical and biological properties to all the requirements for such drugs, which follows from table 3 below.

Example 7. Data on the evaluation of the reactogenicity of the substance of the claimed bivaccine "Revax VKT"

To assess the reactogenicity of the substance of the claimed bivaccine, we studied the dynamics of the development of infiltrates in rabbits vaccinated intradermally with substances of the vaccine virus strains obtained in different ways:

- on the basis of the proposed method (when cultivating a strain of STB No. 2131 BOB on cell cultures);

- on the basis of the prototype method (during cultivation of the STB strain No. 2131 BOB at RCE);

- on the basis of the prototype method (during cultivation of the parent strain of L-IVP at RCE);

The data are shown below in table 4.

Animals were infected by the Grott method. The maximum development of infiltrate in rabbits was observed on the 4th day after vaccination at the injection site of 5 lg OOE / 0.1 ml and 4 lg OOE / 0.1 ml of strains of b7.5S2-S embryonic and L-IVP. Moreover, the appearance of infiltrates in both cases was recorded on the second day after infection. In this case, the smallest infiltrates in area were formed upon administration of the embryonic strain b7.5S2-S (P <0.05) as compared with the L-IVP strains. At the same time, intradermal administration to rabbits of 5 lg TCD ₅₀ / 0.1 ml and 4 lg TCD ₅₀ / 0.1 ml of culture strain b7,5S2-S led to the appearance of infiltrates at the injection site only on the 7th day with a maximum area on the 8th day. In addition, the sizes of these infiltrates were significantly smaller (P <0.05) compared to even those for the b7.5S2-S embryonic strain.

The results obtained indicate that the recombinant strain b7,5S2-S of cultural origin has a significantly lower reactogenicity compared to that of embryonic origin.

Thus, the inventive tabletted bivaccine "Revax VKT", developed on transplantable cultures of animal cells, and the method of its preparation in comparison with the same tablet form of the vaccine "Revax BT", developed on RKE, is less reactogenic, as it contains less impurities, is not contaminated by extraneous microflora, more technological in the manufacture on an industrial scale.

Claims (5)

1. Tableted live recombinant smallpox and hepatitis B vaccine based on the smallpox vaccine virus for oral use, containing in one tablet, lyophilized viral material, produced on the basis of the recombinant strain of the smallpox vaccine HBV vaccine No. 2131 with typical properties of the smallpox vaccine, HbsAg hepatitis B virus, with an activity of ₅₀ CPE 6,5-8,0 lg per tablet, lactose, sucrose, calcium stearate, and vanillin, characterized in that the vaccine additionally contains peptone, and salt used zhelatozu Fer and vaccinia virus gained on transplantable animal cell cultures with the following ratio of components per tablet, wt.%: Lyophilized viral material smallpox vaccines with activity 6.5-8.0 lg CPD ₅₀ per tablet 1.04-31.3 Peptone 0.12-3.8 Gelatosis 0.06-1.9 Sucrose 10.1-11.55 Buffer 0.001-0.027 Calcium Stearate 1.94-2.0 Vanillin 0.17-0.2 Lactose The rest is up to 100% 2. Tableted live recombinant bivaccine according to claim 1, characterized in that it further comprises urea in an amount of 0.01-0.2 wt.%. 3. A method for producing a tablet live recombinant bivaccine against smallpox and hepatitis B, comprising passaging the strain of the recombinant vaccinia vaccine bills of vaccine No. 2131 in a biological system for the cultivation of viruses, obtaining a virus-containing material with a titer of at least 8.0 lg CPD ₅₀ / ml, lyophilization with stabilizing additives, grinding, mixing with a filler and pressing, characterized in that as a biological system for the cultivation of vaccinia virus use transplantable cell culture to animals with a dose of vaccinia virus vaccine of not more than 1.0 CPD ₅₀ per cell, the virus is produced on a monolayer of these animal cells in scooters on a nutrient medium with up to 50% CPP, followed by replacing this medium with a buffer solution with a volume less than the volume nutrient medium; the preparation of the virus-containing material is carried out after replacing the nutrient medium with a buffer solution by extracting the virus by freezing and thawing the cell monolayer three times, and peptone, sucrose and gelatose are used as stabilizing additives during freeze drying in the final concentration in a mixture of wt.%: 2.0- 4.0; 2.0-3.0; 1.0-3.0 respectively. 4. The method according to claim 3, characterized in that as a transplantable culture of animal cells, green monkey kidney cells No. 4647 or VERO are used, as a nutrient medium for virus production, DMEM or MEM Needle with 2% cattle serum or 2% fetal calf serum, 0.01 M Tris, pH = 8, is used as the buffer solution, and when changing the nutrient medium to the buffer solution, its volume is reduced by at least 10 times. 5. The method according to claim 3, characterized in that as an additional component of the stabilizing additive during freeze drying of the bivaccine, urea is used in a final concentration in a mixture of 1.0-2.0 wt.%.