Firiona cytomegalovirus vaccine and its preparation

 

(57) Abstract:

The invention relates to biotechnology. To obtain televizionnoi vaccine against human cytomegalovirus use strain AD-169, the virus is grown on a monolayer of graduation cells, previously placed in the growth medium. The adsorption of the virus carried out on the cells and then adding runtime support. The release of virus from cells occurs in a single cycle of freezing monolayer and thawing. Purification of the vaccine from the detritus produced when low-speed centrifugation. As cell cultures are used diploid cells human fibroblasts (line M-22) or cell culture green monkey kidney Vero-B. Cytomegalovirus Inuktitut formalin at a concentration of 1: 2000 at 37oC for 3 days at 41oC for 10 days or cobalt-60 for 2 h at 19-21oC. Immunogenicity of the drug is assessed according to the cell index of lymphocyte activity (synthesis of neutralizing antibodies after vaccination of experimental animals). The vaccine used in the form of a suppository, containing in addition to the suspension of cytomegalovirus 20,0 ml (106tissue cytopathogenic doses CMV) polyoxidonium 0.02 g, hyaluronic keys injectable form or in the form of a suppository causes virusspecific immunity in experimental animals. 2 S. and 1 C.p. f-crystals.

The invention relates to medicine, namely to the production of antiviral vaccines, and medications based on them.

A method of obtaining the live, attenuated cytomegalovirus (CMV) vaccine (US 4689225, a 61 K 39/12, 1987), which is used for vaccination of adult patients with organ transplants from donors. However, widespread use of this vaccine has prevented that when the manufacturer uses a live virus, carcinogenesis which cannot be excluded, and possible reactivation of recombination with wild option CMV, persistent in the body of the patient CMV infection.

Subunit cytomegalovirus vaccine, developed earlier by researchers in Europe (EPA 0180288, IPC AND 61 TO 39/245, 1986; EPA 0236145, IPC 12 N 15/100, 1987), and recombinant cytomegalovirus vaccines EPA 0389286, IPC And 61 To 39/245, With 12 N 15/38, 1990) containing the CMV glycoproteins, appeared to be poorly immunogenic and highly expensive and therefore in public health practice were not implemented.

In Russia vaccine for CMV is not yet developed, and foreign developments on the Russian market't do that. Analogues killed mirinoi cytomegalovirus vaccines are vaccines p is CESA according to patent US 4816250 (424/89, published in the Gazette 28.03.89,) infected cells - fibroblasts MRC-5 after incubation process surface-active agent, polypeptide chain cytoplasmic fraction fix and carry out centrifugation at 650 g. The method is quite complex and involves the use of antibodies immobilized on sepharose.

Getting a vaccine against herpes simplex virus at the request of EPA 48201 (MCI: a 61 K 39/245, published in the Gazette 1985) involves the extraction of infected cells with urea at a concentration of 2M-8M, centrifugation or filtration, exposure to ultrasound, the concentration by filtration through special sorbents, processing enzyme DNA Asai and purification by gel - chromatography. It is obvious that such a complex method is hardly applicable to large-scale developments.

Known then selected as the closest analogue of the prototype of the claimed invention, a method of obtaining mirinoi herpes vaccine herpes simplex viruses type 1 and 2 grown on the surface of the stationary microsites when using primary cell culture of rabbit kidneys (Antonov P. et al., "Questions Virology", 1979, 6, S. 665-671). This method is however not polsih amounts of formalin inactivated virus, that is accompanied by burning sensation (due to the presence of formalin).

The aim of the present invention is to provide such a method of obtaining mirinoi cytomegalovirus vaccines, which

- would not require complex equipment design;

- would allow to increase the yield and concentration of viruses;

- would due to the use of domestic immunomodulator to increase the immunogenic activity of the vaccine;

- would provide the opportunity for large-scale get killed cytomegalovirus vaccine.

The solution to this problem is achieved due to the fact that to obtain mirinoi CMV vaccine is used strain AD-169, the virus is grown on a monolayer of diploid cells, previously placed in the growth medium. Adsorption of the virus is performed on the cells and then adding runtime support. The release of virus from cells occurs in a single cycle of freezing monolayer and thawing. Purification of the vaccine from the remnants of the destroyed cells (detritus) is performed by low-speed centrifugation. As cell cultures are used diploid cells human fibroblasts (line M-22) or cell culture green monkey kidney VERO, cos im. L. A. Tarasevich. The Ministry of health of the Russian Federation (Russian Ministry of health) for the production of viral vaccines. Inactivation of CMV is formalin at a concentration of 1:2000 at 37oC for 3 days, and 4o1oC for 10 days. In the stabilizer is used 0.22% human albumin. Use as a commercial immunostimulant drug polyoxidonium (000 Immupharma"), it is necessary to increase the immunogenicity of the vaccine. In particular, the use of polyoxidonium in the way of receiving the vaccine against influenza virus has greatly increased the immunogenicity and dramatically reduce the dose of the drug (RF patent 1580617, priority from 13.05.88,, MKI And 61 To 39/145). The study of the immunogenicity of the drug is evaluated In a cellular indicator of lymphocyte activity (synthesis of neutralizing antibody in experiments vaccination of rats, weighing 100,0-120,0 g).

It was noted above that in a subcutaneous formalin inactivated vaccines often have undesirable side reactions (burning, redness and even swelling). For this reason, not once attempted to find a different pathway of administration other than injection, for example, in the proposal EPA 640347(MCI a 61 K 39/00, published the proposal EPA 242205 (MCI a 61 K 39/00, published 06.10.93) proposed vaccine composition, including oil and saponin and used orally in the form of an emulsion. In the work of the Imperial family barinskiy (Russian medical news, 1999 , T. IV, 3, S. 64-66) proposed intradermal method of introducing herpes vaccine. Vaccine for intranasal described in patents US 4800078 (CL 424/86 published 29.01.89,; US 5124148, CL 424/86 published 23.06.92). Use of the vaccine in the form of spray offered in veterinary medicine to combat the plague dogs (patent US 3420934, CL 424/89, 1968). In our proposed invention televizionnaya vaccine against human cytomegalovirus proposed for use in two dosage forms: (a) the liquid form to 0.3 ml in ampoules for intradermal injection; b) in the form of candles (suppository), containing in addition to the suspension of cytomegalovirus 20,0 ml (106tissue cytopathogenic doses CMV) Immunostimulants: polyoxidonium and hyaluronic acid, as well as confectionery fat, waxes and surfactants:

- polyoxidonium 0.02 g

hyaluronic acid 1.0 g

- suppozitornyj basis, which includes confectionery fat, waxes and emulsifiers - up to 100.0 g of the masses.

The invention is illustrated by the following examples of its Osipovich cells human fibroblasts (line M-22) or to the cell culture green monkey kidney VERO-B. Named culture, for example, M-22 with seed concentration of 250 cells/ml, incubated in mattresses at a temperature of 37o1oC for 48 hours to form a tight monolayer of cells. Then make infective material CMV (strain AD-169) in a dose of 0.1 TCD50/the cell and carry out the adsorption of the virus. The infected culture is incubated for 6-7 days to complete destruction of the monolayer of cells in the cytopathic effect of the virus. After that the contents of the mattresses once subjected to freezing at -20o1oC and thawing at room temperature for a full release of cytomegalovirus from the cells and their transition into the culture fluid. Low-speed centrifugation (1500 rpm, 10 min at 4o1o(C) separating the remains of the destroyed cells (detritus), and then at in the culture fluid inactivate formalin at a concentration of 1: 2000, first at the 36oloC for 72 hours and then at 4o1oC for 10 days. In the suspension of cytomegalovirus entered immune polyoxidonium (Polyoxidonium), a commercial product for injection in a dose of 0.1 mg/ml Received a vaccine able to induce in rats synthesis of antibodies that neutralize CMV t 50% of those infected with the virus vials monolayer above sensitive cells.

Example 2 (comparative). Proceed as described in example 1.

Next to formalin inactivated suspension of cytomegalovirus in the amount of 20.0 ml, which corresponds to 106tissue cytopathic doses of CMV add polyoxidonium 0.02 g, hyaluronic acid 1.0 g, suppozitornyj basis, which includes confectionery fat, paraffin, emulsifying agent to the total weight of 100.0 g Of the obtained mass produced in a known manner candles with the specified ratio of ingredients. Each candle has a mass of from 1.1 g to 2.0 g suppository contains up to 500 µg/g of protein, has a melting point of not more than 37oC, PH from 6.8 to 7.2, specifically safe and able to induce the synthesis of antibodies in experimental animals, neutralizing the virus neutralization index for the initial suspension of cytomegalovirus not less than 2 lg TCD50/ml.

Thus, the proposed method for mirinoi cytomegalovirus vaccine is original, allows you to increase the immunogenicity and thereby reduce the dose of the vaccine, and does not require the introduction of relatively large amounts of the drug in injectable form. The proposed vaccine in injectable form or in the form of a suppository can cause virousspecificakih immunodeficiency is Further purified by low-speed centrifugation, the suspension of cytomegalovirus inactivate irradiation. As the source gamma emitter used cobalt-60 at a dose of 15-20 GSR for 2 hours at a temperature of 19-21oC. In the inactivated by irradiation of a suspension of cytomegalovirus injected immune polyoxidonium in a commercial product for injection at a dose of 1 mg/ml Received inactivated vaccine is able to induce in rats synthesis of antibodies, neutralizing cytomeglovirus when the neutralization index 3 lg TCD 50/ml

1. Firiona cytomegalovirus vaccine containing inactivated suspension of cytomegalovirus, characterized in that it further comprises polyoxidonium, hyaluronic acid and suppozitornyj the basis of the following ratio of ingredients:

Inactivated suspension of cytomegalovirus - 20,0 ml

Polyoxidonium - 0.02 g

Hyaluronic acid is 1.0 g

Suppozitornyj the basis of Up to 100 g

2. Firiona cytomegalovirus vaccine for p. 1, characterized in that suppozitornyj base contains confectionery fat, wax and emulsifier.

3. The method of obtaining mirinoi cytomegalovirus vaccines, including the cultivation of sensitive virus in human cells or animals, the introduction of infective material cytomegalovirus strain AD-169 and the current by freezing - repulsion, Department of damaged cells by low-speed centrifugation, the inactivation of the virus in the culture fluid, characterized in that as a sensitive cells using diploid human cell culture M-22 or cells of green monkey kidney Vero-B, cultivate them at a temperature of 36-38oC for 48 h, the incubation of infected cells is carried out for 6-7 days, and the inactivation of the virus in the culture fluid conducting formalin at a concentration of 1:2000 for 72 h at a temperature of 35-37oWith, and then at 3-5oWith in 10 days or cobalt-60 for 2 hours at a temperature of 19-21oWith cobalt-60 add in the amount of 15-20 GSR, then in the resulting suspension sequentially injected polyoxidonium, hyaluronic acid and suppozitornyj basis.

 

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