Method of obtaining foot-and-mouth disease vaccine and foot-and-mouth disease vaccine

FIELD: medicine; veterinary.

SUBSTANCE: method of vaccine obtaining involves virus antigen cultivation in BHK-21 suspension cell culture at the temperature of 36-37°C, purification of virus suspension from ballast admixtures, inactivation, concentration of foot-and-mouth disease virus antigen obtained and combination of antigen concentrate with adjuvant. After 6-8 hours of virus antigen cultivation, percentage of dead cells is registered every 2 hours. When the level of dead cells reaches 95% or more, further cultivation is performed for 2-8 hours depending on the virus type. Foot-and-mouth disease vaccine obtained by this method contains antigen material of a-type and/or O-type and/or Asia-1 type foot-and-mouth disease viruses in effective quantity of 146S-component, aluminium hydroxide gel, saponin and maintenance medium.

EFFECT: increased volume of antigen material during foot-and-mouth disease virus cultivation and obtaining harmless immunogenic vaccine.

13 cl, 1 tbl, 14 ex

 

The group of inventions relates to the field of veterinary Virology and biotechnology. The invention can be used in the manufacture of vaccines against FMD.

There is a method of making a vaccine against FMD type a and monovalent vaccine against FMD type a (see EN 2143921 C1, 10.01.2000).

The method of manufacture of this vaccine includes a reproduction of viral antigen in suspension culture cells KSS-21, cleaning vaccinated suspension from the ballast impurities, the inactivation of the virus, the concentration of the obtained antigen and subsequent connection of the concentrate antigen with adjuvant. Received the vaccine contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type a, gel, aluminum hydroxide, saponin and supportive environment.

A known method of manufacturing a monovalent emulsion vaccine against FMD type a, type O, type C, type Asia-1 and monovalent vaccines against FMD type a, type O, type C, type Asia-1 (see Instructions on ishullanu and control vaccines monovalent emulsion against FMD type a, type O, type C, type Asia-1 (from virus grown in cell KSS-21), appr. The BS of the state Commission of the USSR food and procurement 24.01.1991). The method of manufacture of these vaccines also includes a reproduction of viral antigen in suspension culture cells KSS-21, the cleaning vaccinated suspension from the ballast impurities, inactivation of the virus, the concentration of the obtained antigen and subsequent connection of the concentrate antigen with adjuvant. Each of the obtained monovaccines contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type a or type O or type C, or of type Asia-1, gel aluminium hydroxide, saponin and supportive environment.

A known method of manufacturing a multivalent vaccine and the polyvalent vaccine against foot and mouth disease (see Vaccine trivalent FMD of the virus "O"-"And"-"With"cultivated epithelium of the tongue of cattle. Technical conditions TU 10-09-25-89, appr. Zam. The head of the Main Department of veterinary medicine 11.12.1989 entered from 01.01.1990). This vaccine is a mixture of monovalent vaccines three types, making use of a monovalent vaccine against FMD type O (THE 10-19-403-87), a monovalent vaccine against FMD type a (TU 10-19-404-87), a monovalent vaccine against FMD type C (TU 45-21-1528-84).

The closest analogue is a method of making a vaccine against FMD type O, including the cultivation of viral antigen in suspension culture cells KSS-21 for 10-15 hours at 36-37°and culturing stop when reaching the number of dead cleeton 90-95%, after which the virus is subjected to inactivation, clear of ballast note is this, concentrate the resulting antigen and connect the concentrate antigen with adjuvant. The vaccine obtained by this method contains avirulent and purified antigenic material in the form of immunogenic components (146S+75S) of FMD virus type O gel aluminium hydroxide, saponin and a supportive environment (see EN 2212895 C1, 27.09.2003).

A disadvantage of the known methods of manufacture of the vaccine is not guaranteed level derived antigenic material in connection with the evaluation of the number of method definitions (146S+75S)components, since the capsid of the virus 75S as the partial structure of the antigen, prone to destruction to capsomeres 12S by temperature fluctuations and other physico-chemical factors.

The aim of the present group of inventions is to develop methods of producing vaccines against FMD, allowing to increase the level of antigenic material in the form of 146S-immunogenic component in the cultivation of the virus of foot and mouth disease, and the development of highly immunogenic vaccines against FMD obtained by this method.

The task in part of the way is solved in that in the method of manufacturing a vaccine against FMD, including the cultivation of viral antigen in suspension culture cells KSS-21 at a temperature of 36-37°, purification of virus suspension from the ballast impurities, inactivation, concentration recip is spent antigen of FMDV and the connection of the concentrate antigen with adjuvant, according to the invention, starting with 6-8 hours of cultivation viral antigen every 2 hours to determine the percentage of dead cells, and upon reaching his level of 95% and above take further cultivation within 2-8 hours depending on the virus type.

The problem is solved also by the fact that:

as viral antigen using the FMD virus type a and/or the FMD virus type O, and/or the FMD virus type Asia-1;

as of FMDV type And use the strain And22No. 550, and as of FMDV type Of use strain O1Manisa or strain O1No. 1618, and as FMD virus type Asia-1 use strain Shamir;

after completion of the cultivation process of viral antigen is added chloroform to a final concentration of 0.7-0.9%;

purification of viral suspension is carried out by separation.

The task in the part of the vaccine is solved by the fact that the vaccine obtained the claimed method contains avirulent and purified antigenic material of FMD virus type a and/or FMD virus type O, and/or FMD virus type Asia-1 in an effective amount 146S-component gel aluminium hydroxide, saponin and supportive environment.

While the task is in terms of vaccine is solved also by the fact that:

as avirulent and purified antigenic material virus is FMD type a vaccine contains avirulent and purified antigenic material strain of FMD virus And 22No. 550 in the amount of not less than 0.8 µg 146S component in vaccination dose;

as avirulent and purified antigenic material of FMD virus type O vaccine contains avirulent and purified antigenic material of FMDV O1Manisa in the amount of not less than 1.6 µg 146S component in vaccination dose;

as avirulent and purified antigenic material of FMD virus type O vaccine contains avirulent and purified antigenic material of FMDV O1No. 1618, in the amount not less than 1.5 µg 146S component in vaccination dose;

as avirulent and purified antigenic material of FMD virus type Asia-1 vaccine contains avirulent and purified antigenic material of FMDV Asia 1 Shamir) in an amount of not less than 1.5 µg 146S component in vaccination dose;

the vaccine contains a gel of aluminum hydroxide, saponin and supportive environment at the next number in the vaccination dose:

gel aluminium hydroxide0,5-1,4 ml
saponin1.5-2.5 mg
support the environmentto 2 ml

In addition, the task in the part of the vaccine is solved in that the polyvalent vaccine received the claimed method contains avirulent and cleaned and Tihany material, at least two types of FMD virus is selected from a virus of type a of FMDV type O and FMD virus type Asia-1, gel aluminium hydroxide, saponin and supportive environment.

The technical result of the claimed group of inventions is to increase the level of accumulation of antigenic material, as measured by the content of 146S - component of the FMD virus, and that the vaccine obtained by the proposed method has high immunogenicity and provides reliable protection of animals against FMD.

The invention is illustrated by the following examples.

Example 1.

For the manufacture of monovalent vaccines use the FMD virus And22No. 550, which has been cultivated in suspension culture cells KSS-21 at a temperature of 36°C. After 6 hours of cultivation takes samples every 2 hours and counting live and dead cells. When you reach the number of dead cells, more than 95%, the sampling stops and the cultivation carried out within 2 hours.

It is established that within 2-8 hours the virus continues to evolve in dead cells, moreover, at this time finally formed its protein shell, responsible for antigenic properties, and several times increases the "harvest" of the virus.

Then in the bioreactor add chloroform to a final concentration of 0.7% and before the Ute viral suspension in the collection with a temperature of 4° C. After accumulating the required number of viral suspension is subjected to separation.

Purification of viral suspension by adding chloroform and subsequent separation allow you to get rid of cell fragments and ballast proteins that are reactive factors.

The precipitate, containing fragments of cells and chloroform, cast away, and the clarified viral suspension sent to inactivation.

The inactivation is carried out by adding under stirring dimer etilenimina to a concentration of 0.04% and temperatures up to 30°C.

The required contents of the 146S component in vaccination the vaccine dose is obtained by concentration of the antigen on the gel of aluminium hydroxide. The final concentration 146S-component of the FMD virus must be at least 0.8 µg/ml Add saponin adjuvant.

Received the vaccine Packed and checked for sterility, avirulence, harmlessness, immunogenic activity.

Example 2.

For the manufacture of monovalent vaccine using virus O1Manisa, which are cultivated in suspension culture cells KSS-21 at a temperature of 37°C. After 6 hours of cultivation takes samples every 2 hours and counting live and dead cells. When you reach the number of dead cells, more than 95% of the sampling of termination is t, and cultivation carried out for 6 hours. Then in the bioreactor add chloroform to a final concentration of 0.7% and passed the viral suspension in the collection with a temperature of 8°C. After accumulating the required number of viral suspension is subjected to separation. The precipitate, containing fragments of cells and chloroform, cast away, and the clarified viral suspension sent to inactivation. The inactivation is carried out by adding under stirring dimer etilenimina to a concentration of 0.04% and a temperature of up to 30°C.

The required contents of the 146S component in vaccination the vaccine dose is obtained by concentration of the antigen on the gel of aluminium hydroxide. The final concentration 146S-component of the FMD virus must be at least 1.6 ĩg/ml Add saponin adjuvant. Received the vaccine Packed and checked for sterility, avirulence, harmlessness, immunogenic activity.

Comparison of indicators of FMD virus at different periods of cultivation are shown in table 1.

Example 3.

For the manufacture of monovalent vaccine using virus O1No. 1618, which are cultivated in suspension culture cells KSS-21 at a temperature of 37°C. After 6 hours of cultivation takes samples every 2 hours and counting live and dead cells. the ri the number of dead cells, more than 95% of the sampling stop, and cultivation carried out for 7 hours. Then in the bioreactor add chloroform to a final concentration of 0.7% and passed the viral suspension in the collection with a temperature of 8°C. After accumulating the required number of viral suspension is subjected to separation. The precipitate, containing fragments of cells and chloroform, cast away, and the clarified viral suspension sent to inactivation. The inactivation is carried out by adding under stirring dimer etilenimina to a concentration of 0.04% and a temperature of up to 30°C. Required contents of the 146S component in vaccination the vaccine dose is obtained by concentration of the antigen on the gel of aluminium hydroxide. The final concentration 146S-component of the FMD virus must be at least 1.5 mcg/ml Add saponin adjuvant.

Received the vaccine Packed and checked for sterility, avirulence, harmlessness, immunogenic activity.

Example 4.

For the manufacture of monovalent vaccine using virus Asia 1 (Shamir), which are cultivated in suspension culture cells VPK-21 at a temperature of 37°C. After 6 hours of cultivation takes samples every 2 hours and counting live and dead cells. When you reach the number of dead cells, more than 95% of the sampling stops and the cultivation of implementing tlaut for 8 hours. Then in the bioreactor add chloroform to a final concentration of 0.7% and passed the viral suspension in the collection with a temperature of 10°C. After accumulating the required number of viral suspension is subjected to separation. The precipitate, containing fragments of cells and chloroform, cast away, and the clarified viral suspension sent to inactivation.

The inactivation is carried out by adding under stirring dimer etilenimina to a concentration of 0.04% and a temperature of up to 30°C.

The required contents of the 146S component in vaccination the vaccine dose is obtained by concentration of the antigen on the gel of aluminium hydroxide. The final concentration 146S-component of the FMD virus must be at least 1.5 mcg/ml Add saponin adjuvant. Received the vaccine Packed and checked for sterility, avirulence, harmlessness, immunogenic activity.

Example 5.

The formation of multivalent vaccines.

For production of multivalent vaccines after accumulation of the raw materials for all valences they are mixed prior to the addition of saponin. Other operations are carried out analogously to examples 1-4.

Example 6.

Monovalent vaccine against FMD type a, obtained by the above method (example 1), contains in vaccination dose:

- the virulent and purified antigenic
the material of the virus And22No. 5500.8 µg 146S-component
- gel aluminum hydroxide0.5 ml
- saponin1.5 mg
- support the environmentto 2 ml

Example 7.

Monovalent vaccine against FMD type O, obtained by the above method (example 2), contains in vaccination dose:

- avirulent and purified antigenic
the material of FMDV O1Manisa1.6 ĩg 146S-component
- gel aluminum hydroxide0.8 ml
- saponin2.0 mg
- support the environmentto 2 ml

Example 8.

Monovalent vaccine against FMD type O, obtained by the above method (example 3), contains in vaccination dose:

- avirulent and purified antigenic
the material of FMDV O1No. 16181.5 mcg 146S-component
- gel aluminum hydroxide1.2 ml
- saponin2,3 mg
- support the environment to 2 ml

Example 9.

Monovalent vaccine against FMD type Asia-1 obtained by the above method (example 4), contains in vaccination dose:

- avirulent and purified antigenic
the material of FMDV Asia 1 Shamir)1.5 mcg 146S-component
- gel aluminum hydroxide1.3 ml
- saponin2.5 mg
- support the environmentto 2 ml

Example 10.

Bivalent vaccine against foot and mouth disease types a and O, obtained by the above method (example 5), contains in vaccination dose:

- avirulent and purified antigenic
the material of the virus And22No. 5500.8 µg 146S-component
- avirulent and purified antigenic
the material of FMDV O1Manisa1.6 ĩg 146S-component
- gel aluminum hydroxide1.2 ml
- saponin2.5 mg
- support the environmentto 2 ml

Example 11.

Bivalent vaccine against foot and mouth disease types a and O poluchennaya the above-described method (example 5), contains vaccination dose:

- avirulent and purified antigenic
the material of the virus And22No. 5500.8 µg 146S-component
- avirulent and purified antigenic
the material of FMDV O1No. 16181.5 mcg 146S-component
- gel aluminum hydroxide1.3 ml
- saponin2.5 mg
- support the environmentto 2 ml

Example 12.

Bivalent vaccine against FMD types O and Asia-1 obtained by the above method (example 5), contains in vaccination dose:

- avirulent and purified antigenic
the material of FMDV O1Manisa1.6 ĩg 146S-component
- avirulent and purified antigenic
the material of FMDV Asia 1 Shamir)1.5 mcg 146S-component
- gel aluminum hydroxide1,4 ml
- saponin2.5 mg
- support the environmentto 2 ml

Example 13.

Bivalent vaccine is Rotel FMD types a and Asia 1, obtained by the above method (example 5), contains in vaccination dose:

- avirulent and purified antigenic
the material of the virus And22No. 5500.8 µg 146S-component
- avirulent and purified antigenic
the material of FMDV Asia 1 Shamir)1.5 mcg 146S-component
- gel aluminum hydroxide1.2 ml
- saponin2.5 mg
- support the environmentto 2 ml

Example 14.

Trivalent vaccine against foot and mouth disease types A, O and Asia-1 obtained by the above method (example 5), contains in vaccination dose:

- avirulent and purified antigenic
the material of the virus And22No. 5500.8 µg 146S-component
- avirulent and purified antigenic
the material of FMDV O1No. 16181.5 mcg 146S-component
- avirulent and purified antigenic
the material of FMDV Asia 1 Shamir)1.5 mcg 146S-component
- gel aluminum hydroxide1,4 ml
- saponin2.0 mg
- support the environmentto 2 ml

Manufactured vaccines have passed the test methods recommended by the OIE (OIE), which have established their full compliance with the OIE requirements for vaccines against FMD.

Thus, the claimed method of making a vaccine against FMD allows you to increase the amount of antigenic material 146S component in the cultivation of the virus of foot and mouth disease, and received in this way the vaccine against foot and mouth disease is harmless, avirulent and immunogenic.

Table 1
COMPARISON of INDICATORS of FMD VIRUS AT DIFFERENT stages of ITS CULTIVATION
Type of virusIndicators of FMD virus on reaching 95-98% of dead cellsIndicators of FMD virus in its cultivation in the dead cells
146S. com-t (μg/cm3)RACafter 2 hoursafter 4 hafter 6 hoursin 8 hours after 10 hours
146SRAC146SRAC146SRAC146SRAC146SRAC
And22No. 5500,54-3-10,72×4-11,12×4-3-11,63×4-21,63×4-3-11,53×4-3-1
0,82×4-20,92×4-20,92×4-20,82×4-20,92×4-20,72×4-2
0,32-±0,54-21,02×4-21,33×4-11,63×4-3-11,53×4-3-1
1,22×4-3-11,63×4-3-11,83×4-22,33×4-3-13,34×4-23,24×4-2
1,52×4-3-11,83×4-21,73×4-21,83× 4-21,63×4-21,43×4-2
0,82×4-11,23×4-11,93×4-3-12,34×4-13,34×4-3-13,34×4-3-1
O-Manisa0,82×4-21,22×4-3-12,03×4-3-12,74×4-23,24×4-3-13,24×4-3-1
1,22×4-3-11,83×4-12,64×4-13,24×4-3-13,24×4-3-13,04×4-3-1
0,82×4-11,32×4-22,23×4-3-13,14×4-23,14×4-22,94×4-3-1
0,43-10,92×4-11,62×4-3-11,62×4-3-11,42×4-3-10,92×4-3-1
Asia-1 Shamir) 0,92×4-11,62×4-3-12,03×4-3-12,13×4-3-12,03×4-3-12,03×4-3-1
1,12×4-22,13×4-23,64×4-24,85×4-24,85×4-24,35×4-2
1,52×4-3-1233×4-23,1 1 4×4-13,14×4-22,94×4-22,94×4-2

1. A method of manufacturing a vaccine against FMD, including the cultivation of viral antigen in suspension culture cells KSS-21 at a temperature of 36-37°, purification of virus suspension from the ballast impurities, inactivation, concentration of the obtained antigen of FMDV and the connection of the concentrate antigen with adjuvant, characterized in that, starting 6-8 h of cultivation viral antigen, every 2 h to determine the percentage of dead cells when it reaches the level of 95% and above take further cultivation within 2-8 hours depending on the virus type.

2. A method of manufacturing a vaccine against FMD p is 1, characterized in that after completion of the cultivation process of viral antigen is added chloroform to a final concentration of 0.7-0.9%.

3. A method of manufacturing FMD vaccine according to claim 1, characterized in that the purification of viral suspension is carried out by separation.

4. A method of manufacturing FMD vaccine according to claim 1, characterized in that as a viral antigen using the FMD virus type a, and/or the FMD virus type O, and/or the FMD virus type Asia-1.

5. A method of manufacturing FMD vaccine according to claim 4, characterized in that the quality of FMDV type And use the strain And22No. 550.

6. A method of manufacturing FMD vaccine according to claim 4, characterized in that the quality of FMDV type Of use strain O1Manisa or strain O1No. 1618.

7. A method of manufacturing FMD vaccine according to claim 4, characterized in that the quality of FMD virus type Asia-1 use strain Shamir.

8. Vaccine against FMD, obtained according to any one of claims 1 to 7, containing antigenic material of FMD virus type a, and/or FMD virus type O, and/or FMD virus type Asia-1 in an effective amount, gel aluminium hydroxide, saponin and supportive environment.

9. Vaccine against FMD of claim 8, obtained according to any one of claims 1 to 5, characterized in that as avirulent and purified antigenic material of the virus PWD the RA type And contains avirulent and purified antigenic material strain of FMD virus And 22No. 550 in the amount of not less than 0.8 µg 146S component in vaccination dose.

10. Vaccine against FMD of claim 8, obtained according to any one of claims 1 to 4, to 6, characterized in that as avirulent and purified antigenic material of FMD virus type O contains avirulent and purified antigenic material strain of FMDV O1Manisa in the amount of not less than 1.6 µg 146S component in vaccination dose.

11. Vaccine against FMD of claim 8, obtained according to any one of claims 1 to 4, to 6, characterized in that as avirulent and purified antigenic material of FMD virus type O contains avirulent and purified antigenic material strain of FMDV O1No. 1618, in the amount not less than 1.5 µg 146S component in vaccination dose.

12. Vaccine against FMD of claim 8, obtained according to any one of claims 1 to 4, 7, characterized in that as avirulent and purified antigenic material of FMD virus type Asia contains avirulent and purified antigenic material in the form of a strain of FMDV Asia 1 Shamir) in an amount of not less than 1.5 µg 146S component in vaccination dose.

13. Vaccine against FMD of claim 8, obtained according to any one of claims 1 to 7, characterized in that the gel contains aluminium hydroxide, saponin and supportive environment at the next number in the vaccination dose:

gel g is kookie aluminum 0,5-1,4 ml
saponin1.5-2.5 mg
support the environmentto 2 ml



 

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The invention relates to veterinary Virology and biotechnology

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15 cl, 7 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to a polyepitope vaccine protein applicable for immunisation against murrain virus, nucleic acid coding this protein, recombinant plasmid pA7248-AMV-H-PE for production of said protein in plants and to a vaccine preparation for prevention and protection against infection caused by murrain virus. The polyepitope vaccine protein has the amino acid sequence SEQ ID NO: 1 which contains the amino acid sequence of B-cell epitope of VP4 protein from 21-th to 40-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 135-th to 160-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 200-th to 213-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 2C protein from 68-th to 76-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 3D protein from 1-st to 115-th amino acid starting from N-terminal, and the amino acid sequence of T-cell epitope of 3D protein from 421-st to 460-th amino acid of murrain virus serotype O/ Taiwan/99. Nucleic acid coding such protein has the nucleotide sequence SEQ ID NO: 2. Recombinant plasmid pA7248-AMV-H-PE has a physical map presented in dwg. 5.

EFFECT: invention provides creating polyepitope vaccine protein which is applicable for immunisation against murrain virus.

5 cl, 7 dwg, 2 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: application of aethonium as the adjuvant for production of sorbed foot-mouth disease vaccine is proposed, where aethonium is used as a 10% aqueous solution which is introduced into the composition of foot-mouth disease vaccines in an amount of 750 mcg per 1 cm3 of the preparation.

EFFECT: invention extends the list of adjuvants for production of foot-mouth disease vaccines.

4 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: method of production of vaccine against foot and mouth disease comprises culturing virus antigen in suspension culture of cells BHK-21 at a temperature of 36-37°C, purification of the viral suspension from ballast impurities, inactivation, concentration of the obtained foot and mouth disease virus antigen, and connection of the antigen concentrate with an adjuvant. Purification of the virus suspension from ballast impurities is carried out by adding derived polyguanidines at a final concentration of 0.005-0.01%, or the mixture of chloroform and derivatives of polyguanidines taken in weight ratios of (40-160):1, respectively, at a final concentration of 0.4-0.8. As derivatives of polyguanidines dihydrochloride 1,12 diguanidinohexane or dihydrochloride bis (3-guanidinopropyl) piperazine, or dihydrochloride 3,6-dioxaoctane-1,8-diguanidine, or dihydrochloride 4,9-dioxadodecane-1,2-dibiguanide.

EFFECT: improvement of purification level of virus suspension from ballast impurities and increase in immunogenicity of the target product.

2 cl, 1 tbl, 16 ex

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