RU2637093C1 - Method of obtaining live cultural attenuated vaccine for the prevention of varicella - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of obtaining live cultural attenuated vaccine for the prevention of varicella includes infection with varicella-zoster virus strain of OKA diploid cells LEC-3 with the subsequent reproduction and the release of virus into cultural medium. Thereafter, at least one collection of the virus-containing liquid, the addition of a stabiliser and the preparation of the vaccine are carried out. The group of inventions also relates to the use of a strain of diploid LEC-3 cells for the production of a live cultural attenuated vaccine for the prevention of varicella. The use of diploid cells of strain LEC-3 with high adhesiveness to the surface of growth allows to obtain from 7 to 15 individual virus collections with a high content of extracellular vaccine Varicella zoster virus.

EFFECT: obtaining extracellular virus Varicella zoster by individual collections from infected diploid cells LEC-3 using trisodium citrate provides increased harvest of Varicella zoster virus than in similar cultural models when used as a dispersant of trypsin.

7 cl, 1 tbl, 3 dwg

Description

The invention relates to medicine, namely to medical virology and biotechnology, and can be used to create means of specific prophylaxis, in particular to obtain a mono-vaccine for the prevention of chickenpox in children.

Viruses cause various diseases, such as measles, rubella, mumps, chickenpox, hemorrhagic fever with renal syndrome, Japanese encephalitis B, polio, hepatitis A, hepatitis B, hepatitis C and smallpox, leading to serious consequences for the infected up to fatal outcome.

Chicken pox is a highly contagious disease that affects children, the elderly and immunocompromised patients due to infection with the Varicella zoster virus (VZV). Over 90% of the population develop this disease during the first two decades of their lives. This disease is difficult to treat in people with reduced immunity and in adults. In most cases, VZV becomes latent in the cells of the spinal ganglion.

As a rule, viral diseases can be prevented by introducing vaccines obtained from inactivated or attenuated pathogenic viruses into the body. So, in order to prevent chickenpox diseases, it is planned to carry out universal vaccination of children using, for example, live attenuated chickenpox vaccine virus.

The prior art US patent No. 3985615, published 12.10.1976, which discloses the receipt of the VZV vaccine by repeated attenuation of the viral strain "vOKA" on the cells of the primary embryonic tissue of guinea pigs (GPEC).

The main disadvantage of the proposed method is that the yield of VZV when using GPEC cells is very small.

To increase the yield of VZV, and accordingly, for the production of vaccines, it is customary to use chicken embryos, brain cells of rat rats, and diploid mammalian cells as host cells. Despite the fact that chicken embryos or brain cells of rat rats can be used as host cells to lower production costs, their use is disadvantageous due to their low susceptibility to viruses, complex cleaning methods and side effects resulting from the presence of contaminants in the form of foreign proteins. The use of normal human diploid cells can reduce the side effects caused by foreign proteins, and, therefore, they are more preferable for obtaining viral vaccines.

Typical human diploid cells are the MRC-5 cell strain (ATCC, CCL 171), the WI-38 cell strain (ATCC, CCL 75) and the HEL 299 cell strain (ATCC, CCL 137). The MRC-5 cell strain was obtained from the lung tissue of 14-week-old male embryos (Proc. Symp. Human Diploid Cells, Yugoslavia. Acad. Sci. Arts. Zagreb., Pp. 43-45 (1970); and Nature (London), 227, p. 168-170 (1970)); WI-38 was obtained from lung tissue of 3-month-old female embryos (Exp. Cell Res., 25, p. 585 (1961) and Am.J. Hyg., 75, p. 240 (1962)) and cell strain HEL 299 obtained from pulmonary tissue of male embryos (WD Peterson, Jr. et al., Proc. Soc. Exp. Biol. Med. 128, p. 772 (1968)).

The prior art US patent No. 4000256, published 12/28/1976, which describes the preparation of the VZV vaccine by subculturing 10 to 80 times the cell strain of human embryonic fibroblasts WI-38 containing the VZV virus, and US patent No. 5360736, published 01.11.1994, which discloses a method for producing a VZV vaccine on a cell strain MRC-5.

However, the disadvantages of the proposed methods is that the cell strains WI-38 and MRC-5 are repeatedly passaged, thereby reducing their ability to produce VZV, and therefore these methods cannot be used for large-scale production of the VZV vaccine.

Thus, basically the development of the vaccine is aimed at using or creating new strains that can be used to obtain them.

So, in particular, it is known from the prior art (RF patent No. 2146289 C1, published March 10, 2000) a strain of diploid cells of lung fibroblasts of the human embryo LBHEL obtained from lung tissue of a 20-week embryo. The strain is deposited under CCC number 0127 BP. The strain is characterized by rapid growth, high susceptibility to viruses, especially to the VZV virus. The strain grows well on a medium containing 5% cow serum. Based on this strain, a method for producing a VZV vaccine using the vOKA virus strain has been developed.

However, the use of this strain in industrial production is not described and suitability for these purposes is not confirmed.

It should be noted that today there is no domestic method for producing a monocomponent live culture vaccine attenuated for the prevention of chickenpox.

Known vaccines against chickenpox and, accordingly, methods for their preparation: Varillrix, manufactured by GlaxoSmith-Klein Bioolozhikals, Belgium and Okavax, manufactured by the Osaka University Infectious Disease Research Foundation (BI-KEN), Japan, are associated with foreign companies (World Health Organization, WHO Technical Report Series, No. 848, 1994, p. 22-52. Requiremenrs for varicella vaccine (live), Varicella zoster virus vaccine WO 2006094756A2, Glaxosmithkline Biologicals S / A)).

The disadvantages of foreign technology for the preparation of the component of the monovaccine against chickenpox from the Japanese attenuated strain "vOKA" of the VZV virus are as follows:

1. The need to obtain extracellular Varicella zoster virus exclusively from infected cells by multi-stage extraction methods (harvesting cells mechanically; ultrasonic cell disaggregation; differential centrifugation of the cell mass to obtain extracellular virus).

2. The use of infected cells (master seed) of infected cells contained in the cryoprotector and stored at low temperatures (minus 196 ° C).

3. Assessment of the infectivity of the extracellular virus by the method of pseudo-plaque forming units using infected cells as the viral inoculum, which allows only indirectly judge the infectivity of the obtained extracellular virus.

4. Foreign technology allows to obtain a single crop of the extracellular Varicella zoster virus in the process of creating a vaccine containing, in addition to the Varicella zoster virus, residual components of MRC-5 substrate cells, including DNA and proteins, which significantly reduces the quality of the vaccine.

5. This technology creates the high cost of the vaccine preparation.

Therefore, the main task solved by the invention is the development of both a vaccine and an original method for its preparation on the basis of domestic cell strains, taking into account the elimination of the above disadvantages.

The problem is solved, and the technical result is achieved as follows.

A method for producing a live culture attenuated vaccine for the prevention of chickenpox is proposed, which involves infection of chickenpox with the vOKA strain of LEC-3 diploid cells, followed by reproduction and release of viruses into the culture medium, after which at least 7 to 15 virus-containing collections are carried out liquids, with the introduction of a stabilizer in them and obtaining a vaccine.

In this case, an additional determination is made of the infectious activity of individual viral collections of the virus-containing fluid.

In addition, viral collections are combined.

The strain of diploid cells LEC-3 is passaged with 0.4% trisodium citrate (TNC) in an isotonic solution of monovalent salts and glucose (6.0 grams per liter).

Also, the diploid cell strain was cultured in DMEM / F12 growth medium with 0.13% NaHCO ₃ and 10 mM HEPES enriched with growth additives of OPI (oxaloacetate, pyruvate, bovine insulin) and growth-promoting factor 0.08% TNC with 5% ETS, inactivated by heating and diluted equally conditioned medium from growing LEC-3 cells (4-5 days of growth).

Another aspect of the invention is the use of a LEC-3 diploid cell strain for the production of a live culture attenuated vaccine for the prevention of chickenpox.

Another aspect of the invention is a live culture attenuated chickenpox vaccine obtained by the above method.

The technical results of the domestic technology for the production of chickenpox monovaccine component are as follows:

1. Obtaining the extracellular virus Varicella zoster by repeated individual collections from once infected diploid cells LEC-3.

2. Use as a seed virus (master seed) virus-containing fluid (VSL), and not infected cells.

3. Evaluation of the infectivity of the extracellular virus is carried out by the method of hemadsorption on cell cultures sensitive to the Varicella zoster virus, which allows a more accurate and unambiguous assessment of the infectious activity of the extracellular virus.

4. The accumulation of the vaccine strain VZV (strain "vOKA") in infected diploid cells of LEC-3 using TNCs increases by 0.75-1.5 lg of infectious doses higher than in similar culture models when using trypsin as a dispersant.

5. Due to the increased natural adhesion of LEC-3 diploid cells, passaged with TNC, the number of extracellular virus-containing plums from the growth surface of infected cells increases from 7 to 15, which significantly reduces the cost of the final target product - an antiviral vaccine.

Brief description of the LEC-3 line.

The method of obtaining this line was developed in the laboratory of cell cultures ENIIVI N.P. Glinsky et al. In 1980. Number of generations and passages: no more than 20 passages passed before the start of the analysis. A monolayer is formed on 4-5 days from the moment of landing. Morphological characteristics: fibroblast-like cells with clear boundaries. Karyological characteristic: according to the structure of the karyotype, the cells correspond to the human karyotype. The modal class contains 87% of cells with a normal diploid set of human chromosomes. Species affiliation: human, confirmed by karyological and isoenzymatic method. Information about contamination: bacteria, fungi, mycoplasmas not detected. It is stored in a growth medium + 10% glycerol in liquid nitrogen. When defrosting, 70% of the cells are restored. Sensitivity to viruses: the culture is highly sensitive to polioviruses of types 1,2,3, Coxsackie B3, ECHO 3, 6, 11, 13, 19, 20, 24, 28, PC viruses, herpes simplex virus. Given the possibility of long-term maintenance of human lung embryo cell strains (LEC-3) while maintaining the basic parameters inherent in a human diploid cell culture, the optimal simplified scheme for producing such cultures is given below. LEC cells formed a dense monolayer of fibroblast-like cells on 3-4 days of growth. Subculturing was performed as monolayers were formed with a sieving coefficient of 1: 2. The cultivation medium has a pH in the range of 7.2-7.4. The lifespan of a diploid strain of LEC cells is 40-50 passages. The phase of active cell growth begins from passage 4-5 and lasts until passage 35-40. The formation of a continuous cell monolayer at this phase of the development of culture occurred on 3-4 days. The sieving coefficient of cells in this period is 1: 2. The aging phase began, as a rule, after 35-40 passage. To create a stock of these cultures, cells had to be accumulated and frozen at -196 ° C in the early stages until passage 10-12. Cell strains frozen during the period of active growth, using the optimal regime and conditions of conservation, have high viability - up to 90-92%, and their biological properties do not differ from the original culture. For the cultivation of diploid cells, the use of nutrient media of constant composition is recommended. The optimal medium for LEC cells is Eagle or MEM Eagle medium supplemented with serum from cattle. Antibiotics are not used, at a pH of 7.2-7.4. Data on the isolation and accumulation of viruses and the rate of development of CPD on diploid cells of LEC showed that the most appropriate is the use of diploid cells for virological studies from 5 to 30 passages (A. Malygin, Assessment of the metabolism of intact and virus-infected cells by dynamic speckle interferometry , abstract of diss. candidate of technical sciences, Ekaterinburg, Tomsk, 2015).

The claimed method is as follows:

Individual viral collection (IVS) of the vaccine strain "vOKA" obtained by reproduction of viruses in a monolayer culture of diploid cells LEC-3. To increase the reproduction of the above strains of the VZV virus, LEC-3 diploid cells are grown in DMEM / F12 growth medium with 0.13% NAHCO ₃ and 10 mM HEPES enriched with growth additives (oxaloacetate, pyruvate, insulin - OPI) and growth-promoting factor trisodium citrate (TNC ) (0.001% -0.01%) with 5% fetal calf serum (ETS), inactivated by heating, 1-2 mm glutamine, 40 μg / ml gentamicin. The culture of diploid cells LEC-3 is passaged with a 0.4% solution of TNC with glucose (6.0 grams / liter). Upon reaching a dense monolayer of diploid cells, as a rule, on 4-5 days of growth, the growth medium is removed, the cell monolayer is carefully washed 3 times with a phosphate-buffer solution containing Ca and Mg ^{2+ ions} , and the LEC-3 cells are added to the monolayer virus-containing fluid (VSL) of chickenpox with a multiplicity of infection of 0.02. The contact of viruses with cells lasts 2 hours at a temperature of 30 ° C, while every 15 minutes, culture bottles with infected cells are shaken to evenly distribute the VSL. After two hours, without removing the non-adsorbed virus, serum-free DMEM is added to the culture bottles, with 1-2 mM glutamine and 40 μg / ml gentamicin in a volume 2 times smaller than the growth medium. Individual viral collections are collected at certain time intervals (3-6 days). Infectious activity of viral collections is evaluated on cell cultures sensitive to the Varicella zoster virus. The most sensitive cell cultures to the VZV virus are diploid cells from the dermal-muscle tissue of the M-27 human embryos and diploid cells from the tissue of the FEMC-1 and FEMC-2 guinea pig embryos (fibroblast-like). Cell lines are grown on 2 24-well plates; a seed dose of 70-100 × 10 ³ cells per well in DMEM / F12 medium with 5% ETS, 1-2 mM glutamine and 40 μg / ml gentamicin. On the next or after 2 days of growth from a monolayer that has not reached a high density, the growth medium from the wells is removed from the plate, the cell monolayer in the wells is washed twice with phosphate-buffered saline (PBS) and 0 is added to the wells of each of the 2 tablets. 1 ml of ten-fold dilutions of VSZH VZV (from 10 ^-1 to 10 ^-10 ) in DMEM with 2% ETS.

In the control uninfected wells contribute 0.1 ml of DMEM with 2% ETS.

The infectious activity of the VZV virus is evaluated on the 7th day from the moment of infection in the hemi-adsorption reaction with 0.25% suspension of guinea pig erythrocytes, or chicken erythrocytes or human erythrocytes of group “0” and the result is expressed in hemi-absorbing units in 0.1 ml (GADE _{50/0 1} ml).

Red blood cell production. In guinea pigs, blood was taken from the heart with a syringe with a capacity of 5.0 ml and washed three times with 0.9% sodium chloride solution, followed by centrifugation for 10 min at 1500 rpm. The precipitate was diluted with 0.9% sodium chloride solution to 0.25% concentration.

Conducting a haemadsorption reaction. On the 6-7th day from the moment of infection, the culture fluid is drained from the plate with infected and control (uninfected) cell cultures, the FBI is washed twice with a monolayer and 0.5 ml of 0.25% suspension of guinea pig erythrocytes is added to each well. The plates are incubated for 30 minutes at a temperature of from 3 ° to 5 ° C, and then 30 minutes at a temperature of from 20 ° to 22 ° C. A suspension of red blood cells is removed from the plate and the monolayer of cells is washed 3 times with the FBI and examined under a light microscope (10 ^x eyepiece, 20 ^x objective). The virus titer in the GADA _{50 / 0.1} ml is defined as the largest dilution that causes a hemi-absorbing effect in 50% of infected cell cultures in the absence of a hemi-absorbing effect in control uninfected cell cultures. The virus titer is calculated according to the Kerber formula:

lg GADE ₅₀ = Ld (S-0.5),

where L is the lg of the smallest dilution used in titration;

d is the interval in lg between two successive dilutions;

S is the sum of the proportions of the positive test units (i.e., wells with cell culture, where the hemadsorbing effect was manifested) in each dilution.

The results can be illustrated by the following example.

lg GADE ₅₀ = -1- (8-0.5) = - 8.5

Thus, the infectious activity of the virus-containing material (virus titer) is 10 ^-8.5 GADE ₅₀ in 0.1 ml, i.e. the titer of the virus is 8.5 lg GADE _{50 / 0.1} ml.

Also, these results are illustrated in Fig. 1, where hemadsorption on LEC-1 (A) cells is reflected on the 7th day from the moment of infection when the virus was diluted 10 ^-6 , B - control of uninfected LEC-3 cells.

Further, the hemabsorbent activity of VZV viruses is confirmed in the immunofluorescence reaction with specific antiviral immunoglobulins.

Formulation of the reaction of indirect immunofluorescence:

The second 24-well plate with ten-fold dilutions of VSW VZV was transferred to minus 20 ° С overnight, then thawed and a preparation was prepared from infected cells on defatted glass slides from each well, dried at room temperature, and fixed with 3% paraformaldehyde (Sigma) , USA) for 20-30 min at room temperature. After fixation, it is washed with tap water, dried at room temperature and 30-50 μl of immune antiretroviral serum in a dilution of 1: 100 is applied to each spot. The preparations are placed in a humid chamber and incubated at 36.5 ° C for 45 minutes. After incubation with immune serum, the preparations are washed with tap water, dried at room temperature, and an FITC conjugate is applied to each spot — this is protein A labeled with FITC (Sigma, USA) at a 1:50 dilution. The preparations are again placed in a moist chamber and incubated for 45 min at 36.5 ° C. After incubation with A-FITZ protein, the preparations are washed, dried and the number of infectious foci is counted using an Optica luminescent microscope (Italy) at a wavelength of 520 nm. The VSZH titer VZV is determined according to the same scheme as for the hemadsorption reaction. Indirect immunofluorescence of LEC-3 cells infected with VZV is visually shown in Fig. 2 (picture on the left) and control of uninfected LEC-3 cells (picture on the right).

In the table. 1 presents the results of the assessment of the infectious activity of 15 individual viral collections obtained from infected LEC-3 cells grown in 175 cm ² culture bottles and infected with the attenuated vOKA strain.

As you can see from the table below, 15 individual virus collections were made from infected LEC-3 cells, and the titers of the Varicella zoster virus ranged from 7.0-8.5 lg GADE _50/01 ml.

In fig. 3 presents the results of a study of the sera of BALB / c SPF mice immunized subcutaneously with a virus-containing fluid obtained from individual viral collection (N 15). Mice were injected subcutaneously with 0.5 ml of Varicella zoster BCG, serum from mice was collected from the tail vein at various time intervals, starting from 2 weeks and for 8 months (observation period). Immune sera were tested in ELISA in a solid-phase variant of the immune analysis, where a lysate of infected M-27 cells was adsorbed on the solid phase. The immune complex was detected by anti-mouse peroxidase conjugate in working dilution (Sigma).

Moreover, in fig. 3, the abscissa axis indicates the timing of the study of sera from the time of administration of the drug, along the ordinate axis, the OD ₄₅₀ values. Values are evaluated on a Thermo Scientific Varioskan Flash photometer, serum dilutions are indicated on the right in colored squares.

As can be seen from the diagram in fig. 3, the attenuated viral strain elicits a high immune response over an 8 month observation period.

After determining the infectious activity of the individual viral collections, they are combined, a stabilizer is added, 0.5 ml are poured into vials and lyophilized.

The stabilizer composition:

A mixture of 0, 100 ml of a solution of LS-18 * and 0.025 ml of a 10% solution of gelatin.

Note: * LS-18 aqueous solution contains sucrose - 250 mg, lactose - 50 mg, glutamic acid sodium - 37.5 mg, glycine - 25 mg, L-proline - 25 mg, Hanks dry mix with phenol red - 7, 15 mg, water for injection up to 1 ml.

Before lyophilization, the vaccine, poured into 2 ml vials, is frozen to minus 70 ° C.

Freeze-drying is carried out according to the instructions - "Instructions for freeze-drying" according to the scheme:

1. Launch a sublimation unit and verify the operation of the main functional systems;

2. Loading cassettes with frozen vaccine in a sublimation unit;

3. Freeze-drying.

Cassettes with frozen vaccine are placed on sublimator shelves pre-cooled to a temperature of 0 + 2 ° C. After installing the cassette, the material is kept for 2 hours at a shelf temperature of 0 + 2 ° C. Then the shelves are cooled to - 52 ° C - 55 ° C and incubated for at least 8 hours.

The sublimator chamber is evacuated to a pressure of 75-80 μbar (microbar). Sublimation Stage. Include heating shelves. The temperature of the shelves is adjusted to minus 20 ° C in 5 hours, which is maintained for the next 45 hours. The temperature of the shelves was adjusted within 1 hour to -15 ° C and held for 12 hours. The temperature of the material during sublimation in the range from 40 ° C to -32 ° C. After 63 hours from the start of sublimation, the temperature of the shelves is increased at a rate of 5 ° C / hour to the temperature of the shelves + 25 ° C - + 27 ° C. Stage of completion. When the material temperature reaches + 25 ° С - + 27 ° С, it is maintained for 15-18 hours. Vacuum can be brought up to 4-60 μbar (microbar).

After completion of the drying phase, the bottles are capped under vacuum. Vacuum sealed vials are wrapped in aluminum caps.

Sealed vials are additionally viewed for seaming quality. Put in plastic pallets and send to the refrigerator.

Thus, the claimed technical solution allows to simplify the technology for producing a monovaccine for the prevention of chickenpox for children, to improve their quality and reduce the cost of the final target product.

Claims (7)

1. A method of producing a live culture attenuated vaccine for the prevention of chickenpox, comprising infection of the chickenpox virus with the strain “OKA” of LEC-3 diploid cells with subsequent reproduction and release of viruses into the culture medium, after which at least one virus-containing liquid is collected, introducing a stabilizer into it and obtaining a vaccine. 2. The method according to p. 1, characterized in that it further conducts the determination of the infectious activity of individual viral collections of the virus-containing fluid. 3. The method according to p. 1, characterized in that when preparing the vaccine additionally carry out the combination of viral fees. 4. The method according to p. 1, characterized in that the strain of diploid cells LEC-3 is passaged with 0.4% trisodium citrate (TNC) in an isotonic solution of monovalent salts and glucose (6.0 grams per liter). 5. The method according to p. 1, characterized in that the diploid cell strain is cultured in a DMEM / F12 growth medium with 0.13% NaHCO ₃ and 10 mM HEPES enriched with growth additives of OPI (oxaloacetate, pyruvate, bovine insulin) and growth-promoting factor 0 , 08% TNC with 5% ETS, inactivated by heating and diluted equally with conditioned medium from growing LEC-3 cells, 4-5 days of growth. 6. The use of a strain of diploid cells LEC-3 to obtain a live culture attenuated vaccine for the prevention of chickenpox. 7. Live culture attenuated vaccine for the prevention of chickenpox, obtained by the method according to any one of paragraphs. 1-5.

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