The method of manufacture of an inactivated vaccine against rabies

 

(57) Abstract:

The invention is intended for the manufacture of means of specific prophylaxis of rabies all kinds of farm and domestic animals. Fixed rabies virus (WB) were cultured in suspension culture cells KSS-21 at 35-37C for 96-144 hours after the reproduction of the WB in vaccinated suspension make first as stabilizer polyacrylic acid (PAC) or paracril (F) to a concentration of 0.7-1.0%. Then make as inactivant dimer etilenimina (EIA) to a concentration of 0.1% to 0.3%. The mixture is incubated at 35-37C for 20-24 hours Received the vaccine used both in liquid and dry form. To obtain a dry preparation of liquid vaccine is dried by sublimation. The invention increases the specific security, immunogenic activity, stability and safety of the vaccine, and also reduces its reactogenicity and allergenicity. 7 C.p. f-crystals, 2 tab.

The invention relates to the field of veterinary Virology and biotechnology and can be used in the manufacture of means of specific prophylaxis of rabies all kinds of farm and domestic animals.

Rabies in animals refers to C is utilized to humans from animals. Therefore, the fight against it is not only economic, but a social problem, the successful solution of which depends largely on the quality of rabies vaccines used prophylactically. Problems with the creation of an effective vaccine against rabies is primarily due to the difficulties associated with operating high-quality biomass of rabies virus (WB).

A known method of manufacture of an inactivated vaccine against rabies in animals, including reproduction of fixed WB, strain "sheep" VGNKI, in brain tissue of sheep, making vaccinated suspension of 1 % solution of phenol as inactivant, the incubation mixture at 22oC for 48 hours, its connection with the environment-drying, packing of the drug and its drying. Wednesday drying contains 90 ml of distilled water, 10 g of sucrose and 1.5 g gelatin (1).

The disadvantages of this method are low immunogenic derived vaccine, the high toxicity of phenol and significant content in the product of residual virus that contributes to the manifestation of postvaccinal complications in vaccinated animals, especially in cattle and ornamental breeds of dogs.the oneness and allergenicity of the vaccine. This method permits the release of non-sterile product.

A known method of manufacture of an inactivated vaccine against rabies using inactivate hydroxylamine (2).

The disadvantage of this method is that used hydroxylamine not inactivate the microflora present in the viral suspension.

A known method of manufacture of an inactivated vaccine against rabies in animals, including reproduction of fixed WB, strain "sheep" VGNKI, in brain tissue of sheep, making vaccinated suspension 20-21% solution of ethanol as inactivant, the incubation mixture at 30-31oC for 12-14 days, its connection with the environment-drying, packing of the drug and its drying. The environment includes drying 22.5 g of sucrose and 3 g of gelatin in 100 ml of distilled water (3).

The disadvantages of this method are low immunogenic activity of the drug received, the high complexity of its manufacture and open the target product. In addition, contained in a final product of high concentration of sucrose and gelatin increased reactogenicity and allergenicity of the vaccine.

The known method izgotovlenie cells KSS-21, introduction in vaccinated suspension as inactivate beta propiolactone to the concentration of 0,02-0,03%, the incubation mixture at 4-6oC for 2-4 hours followed by the addition of aluminium hydroxide and saponin as adjuvant (4).

The disadvantages of this method are the instability derived vaccine, because it is produced in liquid form, the high cost of the product, as prepared from the concentrated antigen, the presence of viral suspension contaminating viruses and germs trapped with serum, cells KSS and from the environment, as the use of beta-propiolactone to inactivate the infectivity of the WB does not ensure sterility of viral suspension.

The closest present invention, the set of essential characteristics is a method of manufacturing inactivated vaccine against rabies in animals, including reproduction of fixed WB in cell culture KSS-21, the introduction vaccinated suspension as inactivate beta propiolactone to the concentration of 0,02-0,03%, the incubation mixture at 4-6oC for 2-4 hours, adding stabilizer, adjuvant and lyophilization of the desired product. The stabilizer content 6).

The disadvantages of the prototype method:

1) low immunogenic activity derived vaccine;

2) the use of beta-propiolactone mode of inactivation of the infectivity of the WB does not ensure sterility virus suspension from contaminating agents caught in her serum, cells KSS and the environment;

3) contained in a final product of high concentration of peptone, sucrose, gelatin and saponin increased reactogenicity and allergenicity of the vaccine.

The task of the invention was to develop a method of manufacture of inactivated vaccines against rabies, providing a unified production specifically safe, highly immunogenic, stable, harmless, free from contaminating agents vaccines both in liquid and dry form. The use of the drug should ensure effective rabies prevention of all kinds of farm and domestic animals.

The technical result from the use of the invention is to improve a specific security, immunogenic activity, stability and safety of the vaccine, as well as in the reduction of its reengagement and allergenicity.

Specified Nakov:

1) a method of manufacture of an inactivated vaccine against rabies;

2) reproduction of a fixed WB in cell culture;

3) as cell culture using suspension culture cells KSS-21;

4) upon completion of the reproduction of the virus in vaccinated suspension first make a stabilizer, and then inactivant;

5) in the stabilizer is used polyacrylic acid (PAC) or its salt;

6) as a salt PAK use paracril (F);

7) PAK or FA bring in vaccinated suspension to a concentration of 0.7-1.0%;

8) as inactivate virus using dimer etilenimina (EIA);

9) the EIA contribute in vaccinated suspension to a concentration of 0.1-0.3%;

10) a mixture of vaccinated suspension, PAK or FA with the EIA incubated at 35-37oC for 20-24 hours;

11) received the drug is used in liquid form;

12) the resulting preparation is dried by sublimation.

The present invention includes the following set of essential features that provide technical result, in all cases, which sought legal protection:

1) a method of manufacture of an inactivated vaccine against rabies;

Yu suspension first make a stabilizer, and then inactivant;

4) use a PAK or a salt thereof as a stabilizer;

5) the use of EIA as inactivant WB.

The proposed method is also characterized by other symptoms, expressing the specific forms of its implementation:

1) as cell culture using suspension culture cells KSS-21;

2) as a salt PAK use paracril (F);

3) the stabilizer PAK or FA bring in vaccinated suspension to a concentration of 0.7-1.0%;

4) the EIA contribute in vaccinated suspension to a concentration of 0.1-0.3%;

5) the mixture is incubated at 35-37oC for 20-24 hours;

6) received the drug is used in liquid form;

7) the resulting preparation is dried by sublimation.

The achievement of the technical result from use of the present invention can be explained as follows.

Increasing the specific safety of the drug obtained by the proposed method is achieved by inactivation of the WB and contaminating agents mixture PAK or FA with the dei.

Increasing the immunogenic activity of the drug obtained by the proposed method is achieved due to the presence in the vaccine mixture PAK or FA with the dei.

Reduced reactogenicity and allergenicity dry product obtained by the proposed method is achieved due to the exclusion of saponin and environment drying, including peptone, sucrose and gelatin and liquid vaccine due to the exclusion of aluminium hydroxide and saponin. PAK FA are not antigens.

Features of the invention, describing the proposed method and the matching with the characteristics of the prototype, including a generic term that reflects the assignment are:

1) a method of manufacture of an inactivated vaccine against rabies;

2) reproduction of a fixed WB in cell culture;

3) making inactivant in vaccinated suspension to inactivate the obtained virus;

4) incubation of the mixture;

5) incorporation of the stabilizer.

Compared with the method of the prototype of the salient features of the invention are:

1) upon completion of the reproduction of the virus in vaccinated suspension first make a stabilizer, and then inactivant;

2) use a PAK or a salt thereof as a stabilizer;

3) the use of EIA as inactivant WB.

The proposed method is also characterized other distinctive when the organizational culture cells KSS-21;

2) as a salt PAK use FA;

3) PAK or FA bring in vaccinated suspension to a concentration of 0.7-1.0%;

4) the EIA contribute in vaccinated suspension to a concentration of 0.1-0.3%;

5) the mixture is incubated at 35-37oC for 20-24 hours;

6) received the drug is used in liquid form;

7) the resulting preparation is dried by sublimation.

Conducted by the applicant's analysis of the prior art, including the search for patents and scientific and technical information sources, and identify sources that contain information about the analogues of the proposed method allowed us to establish that the applicant had not found the source, which is characterized by signs, identical to all the essential features of the proposed method. The definition from the list of identified unique prototype, as the most similar set of features analogue, has allowed to establish the essential towards perceived by the applicant to the technical result of the distinctive features in the proposed method, set forth in the claims.

Therefore, the proposed method meets the condition of patentability "novelty."

To verify compliance predlahayuschyh solutions to identify the signs, included in the characterizing part of the claims. In the search result set as follows.

Know the use of PAK as a stimulator of the immune system (7, 8).

Know the use of FA (incomplete iron salt of polyacrylic acid) as hemostatic, an analgesic, and also drug with bactericidal and bacteriostatic activity against several gram-positive and gram-negative microbes (9, 10).

The authors of the invention installed new, not previously known properties of PAK and its salts FA to increase virucidal and bactericidal activity of EIA when they are added to the virus suspension before making it inactivant and simultaneously serve as a stimulant immunogenicity and vaccine stabilizers when it is drying or storage in liquid form. Sequential introduction PAK or FA and the EIA virus suspension was allowed to obtain a sterile suspension of inactivated WB. In this case, inactivation of microflora provides three times lower concentration of EIA. Using each drug alone or the order of their entry in the viral suspension was unable to achieve its sterility and stability.

Astarta in the drying process. Safety and immunogenicity of liquid vaccine tested for 12 months at a temperature of 4-8oC with a positive result.

It is also known the use of EIA for the inactivation of the FMD virus (II).

To inactivate the WB EIA used by the authors for the first time. The EIA inactivates WB first order reactions. However, studies on the inactivation of contaminating microflora in the samples of the suspension WB using the EIA showed that at 37oC for 24 hours, the concentration of EIA up to 0.3% is not always ensured sterility specially cleaned suspension. The question of obtaining sterile suspension WB was solved using PAC or F when added to the virus suspension before inclusion in the EIA. From the use of PAK FA received the same results.

The search results show that the proposed method is not apparent to the expert in the obvious way from the prior art, as set out in the description section (not identified solutions that have the signs consistent with the distinctive features of the proposed method), and revealed no effect provided the essential features of the proposed method transforms for detereminately level."

Example 1.

For the manufacture of vaccines against rabies using a fixed WB strain Shchelkovo-51", reproduced in suspension culture transplantable cells KSS-21 in the environment of the Needle containing 5-10% serum and 0.25% hemodialysate. The cultivation of the virus are within 96-144 hours at a temperature of 35-37oC, receiving the viral suspension with a titer of infectivity 5,5-6,5 lg LD50/ml. The accumulation of WB in the cultural medium and the cells KSS-21 occurs without signs of cytopathic effect of the virus. Upon completion of the reproduction of the virus in vaccinated suspension make first as stabilizer PAK m m ~ 1.000.000 or FA to a concentration of 0.7-1.0%, and then as inactivate the dei to a concentration of 0.1% to 0.3%. The mixture is incubated at 37oC for 20-24 hours. The resulting vaccine is filled into 10 ml vials for freeze-drying or implementing in liquid form. In liquid form, the vaccine has the following ratio of components,%:

PAK or FA - 14-20

THE EIA - 0,5-1,5

vaccinated suspension - rest.

For the preparation of dry preparation of liquid vaccine is dried by sublimation. The finished vaccine control on physical properties, sterility, immunogene the method of determining the value of the50and in the dynamics of reducing the titre of infectivity using mice weighing 10-12 g

TO50is expressed as % concentration of dei, which reduces the infectivity of the WB to the level 1 LD50/0,03ml for 24 hours at a temperature of 37oC50calculated according to the formula Cerberus-ashmarina

K50= lgDm-lgd(Li-0,5),

where Dmthe concentration of EIA in the sample suspension, providing avirulence WB;

d - the ratio of the tested concentrations of EIA;

L - the ratio of the number of living mice to the number of infected each sample viral suspensions;

i is the number of sample suspension.

The results of the experiments are shown in table 1.

From the data in table 1 it follows that the avirulence suspensions provided the concentration of dei below 0,009-0,012%. The estimated concentration of EIA equal 0,004%, reduced the infectivity WB to one LD50/0,03Jr.

Example 3.

It is known that the oligomers etilenimina belongs to the EIA, inactivate all viruses by the reaction of the first order. To verify this fact took the dynamics of inactivation WB, using the 0.2% concentration of dei at 37oC. Test suspension, taken after an hour of inactivation, had an infectivity titer < a 0.5 lg LD5 50 times (0,2 : 0,004 = 50) the rate of inactivation increased ~ 50 times, i.e. after 28 min of inactivation of the infectivity of the WB in suspension should be about one-LD50/0,03Jr.

Example 4.

Studies on the inactivation of contaminating microflora in the samples of the suspension WB using the EIA showed that at 37oC for 24 hours, the concentration of EIA up to 0.3% is not always ensured sterility specially cleaned suspension. The sterility check was carried out according to GOST 28085-89. The question of obtaining sterile suspension WB was solved using PAC or F when added to the virus suspension to the optimum concentration of 0.7-1.0% prior to the introduction into the dei. In this sequence of entry in the viral suspension of the above drugs inactivation microflora provided three times lower concentration of dei.

Example 5.

Differences in the number of EIA, providing avirulence suspension WB (0,009-0,012%) and inactivation of microflora in the virus suspension (0,2-0,3%) required research on the effect of high concentrations of EIA for immunogenic activity of liquid and dry vaccine against rabies.

It is established that the immunogenic activity of these drugs is not decreased by increasing the concentration of dei from 0 to 0.5% (within which it is possible to inactivate the infectivity of WB, ensuring a high degree of specific security vaccines.

PAK FA, in addition to increasing the activity of EIA are immune stimulants. However, they have the same activity. In the manufacture of dry vaccine PAK and F form a solid stroma, preventing loss of product during drying by sublimation.

Persistence immunogenic liquid vaccine tested in the past 12 months at 4-8oC with a positive result.

The results of the control immunogenic in mice liquid and dry vaccines against rabies, made by the proposed method are shown in table 2.

From the table 2 data suggest that FA and PAK, being a stimulant bactericides the EIA, significantly enhance the immunogenicity of both liquid and dry vaccine.

The economic effectiveness of the proposed method will be determined by the following factors:

1) the absence of a final product of biological contaminant agents, capable of reproduction under favorable conditions;

2) the absence of additional organic or inorganic impurities in the form of peptone, gelatin and sucrose Oh and dry vaccine.

Thus, the presented data suggest the implementation of the use of the proposed method the following cumulative conditions:

the method embodying the invention, intended for use in veterinary Virology and biotechnology;

for the proposed method as it is described in the independent claim, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and techniques;

the method embodying the invention in its implementation, provides achievement perceived by the applicant of the technical result: increased specific security immunogenic activity, stability and safety of the vaccine, as well as reducing its reactogenicity and allergenicity.

Therefore, the present invention meets the condition of patentability "industrial applicability".

The sources of information.

1. Auth. mon. USSR N 117464, A 61 K 39/205, 21.11.58,

2. Vagabov P. M. Development and experimental study of dry rabies vaccine, inactivated by hydroxylamine. Kida. dis. M., 1969, 44-60.

ical and veterinary chemotherapeutic drugs. M., 1963, 28-38.

5. Pat. RF N 955577, A 61 K 39/205, 03.03.81, (prototype).

6. Temporary regulation on production and control of inactivated dry culture rabies vaccine. Approved by the LIPS of the Ministry of agriculture of the USSR 16.09.76,

7. Petrov R. C., P. M. Khaitov R.M and other Immunogenetic and artificial antigens. M, Medicine, 1983, 255 S.

8. Chemical encyclopedia. M, BDT, 1992, 3, 602 (PAK).

9. Register of medicines of Russia. M Enfermagem, 1993, 876-877 (F).

10. Auth. mon. USSR N 698622, A 61 K 33/26, 22.02.74, (F).

11. Pat. RF N 594771, A 61 K 39/12, C 12 N 7/04; 07.05.73,

12. GOST 28085-89. Biological preparations. Bacteriological control of sterility.

13. Syurin C. N., Belousova R. W. and other Dynamics of viral diseases of animals. The Handbook. M, Agropromizdat, 1991, 255-270.

14. Ivanov B. C. prospects for improving the technology culture of inactivated rabies vaccines. Viral disease C. farm animals. Vladimir, 1995, 203.

15. Ivanov B. C., Skichko N. D. Development and implementation in industrial production and veterinary practice Russian culture inactivated vaccines against rabies. Kursk bio and agro industry Series N 1551437, C 12 N 7/00, 30.08.79,

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1. The method of manufacture of an inactivated vaccine against rabies in animals, including reproduction of fixed rabies virus in cell culture, making inactivant in vaccinated suspension, incubation of the mixture and introducing stabilizer, characterized in that upon completion of the reproduction of the virus in vaccinated suspension first make a stabilizer, and then inactivant, the stabilizer is used as polyacrylic acid or its salt, and as inactivant - dimer etilenimina.

2. The method according to p. 1, characterized in that culture cells using suspension culture transplantable cells KSS-21.

3. The method according to p. 1, characterized in that as salts of polyacrylic acid used paracril.

5. The method according to p. 1, characterized in that the dimer etilenimina contribute in vaccinated suspension to a concentration of 0.1% to 0.3%.

6. The method according to PP.1-5, characterized in that the mixture is incubated at 35-37oC for 20-24 h

7. The method according to PP.1-6, characterized in that the resulting product is used in liquid form.

8. The method according to PP.1-6, characterized in that the resulting preparation is dried by sublimation.

 

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SUBSTANCE: the present innovation deals with biotechnology of viral preparations. One should apply RB-71 strain of rabies virus which should be introduced into culture reservoir (0.1-0.01 MLD50/cell) simultaneously with VNK-21 at initial cell concentration being 0.5-0.5 mln cells/ml to be grown in suspension. Cultivation should be performed in suspension medium at 37 C for 5-6 d at constant mixing and maintaining pH value of 7.2-7.4. The obtained viral raw material at infectious 6.5-6.8 lg MLD50/ml and antigenic activity being 1:90-270 should be inactivated with 1,8,36-diendomethylene-1,3,6,8-tetriasecyclodecane 0.01% at 37 C for 3 d. Ready-to-use vaccine preparation meets the requirements of the standard for veterinary preparations being of 1.5-2.0 IU activity.

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1 ex, 1 tbl

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EFFECT: higher efficiency of manufacturing.

2 ex, 1 tbl

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EFFECT: high sensitivity; accelerated toxicity drop; low cost.

5 tbl

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SUBSTANCE: method involves infection of transplantable cells VNK-21 with virus as measured for 0.01-0.001 MMLD 50/cell, multiplied for 36-70 h in suspended state and inoculated on microcarriers in the dose (1.2-2.5) x 105 cells in 0.3-0.6 ml of nutrient medium per each 1 cm2 of microcarriers. Culturing cells on microcarriers is carried out in bioreactor for 5-6 days and virus-containing cultural liquid is collected every day. Prepared vaccine possesses the high immunogenic activity and method for its preparing shows the high output.

EFFECT: improved preparing method of vaccine.

1 tbl

FIELD: veterinary virology.

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EFFECT: higher efficiency.

2 cl, 2 ex

FIELD: veterinary science, virology, biotechnology.

SUBSTANCE: the suggested canine rabies vaccine contains a plasmid that contains a nucleic acid that codes rabies virus protein G. The vaccine provides complete protection against rabies for the period of 1 yr after a single injection of the vaccine suggested. It has been, also, suggested the method for vaccination with the help of such a vaccine and a kit that includes this vaccine.

EFFECT: higher efficiency of vaccination.

24 cl, 10 dwg, 19 ex

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SUBSTANCE: method involves cultivation of CSF virus LK-VNIIVViM strain in a cell culture, titration and lyophilisation. Culture made of swine embryo kidney cells (RK-15), IGLA nutritional medium and CSF serum is used. Protection medium for lyophilisation is made of peptone and lactose, 5% each, 2% of gelatinose and 5% of CSF serum. The invention can be applied in biological industry and veterinary.

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1 tbl, 2 ex

FIELD: medicine, pharmacology.

SUBSTANCE: invention concerns biotechnology, namely manufacture of medical biological preparations. It is developed a virus-safe method of obtaining of a heterological antirabic immunoglobulin from a high-avid heterological antirabic Serum using a capril-alcohol method with peptic treatment which allows obtaining a high cleaning heterological antirabic immunoglobulin for intravenous and intramuscular administration. Thus as initial raw materials use a high-avid antirabic Serum obtained from immunization of animals-producers by the cleared virus of furiousness, grown up on culture of cells Vero or PSH. The content of γ- globulene fraction in a preparation makes not less than 90%, molecular weight distribution: units no more than 1.0%, diabodies and monomers less than 10.0%, F(ab)2 - fragments not less than 84.0%, Fab - and Fc - fragments less than 5.0%, low anticomplementary activity, specific activity not less than 150 ME/ml. An osmolarity of a ready preparation is within 250-350 mosm/kg.

EFFECT: obtaining of high cleaning heterological antirabic immunoglobulin, stable at storage and transportation.

10 cl, 3 ex, 3 tbl

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