RU2492235C1 - Rubella virus strain "orlov" (u) for obtaining medicinal immuno-biological preparations (mibp) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain was deposited under number V-1 in the collection "SCPM-Obolensk". The strain may be used to produce monovalent rubella vaccine, associated combined vaccines comprising the vaccine strain of rubella virus, as well as for production of diagnostic products.

EFFECT: strain has a high reproductive, hemagglutinating and immunogenic activity.

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Description

The invention relates to the field of medical biotechnology and can be used to obtain a single rubella vaccine and associated vaccines containing the rubella component, as well as to obtain diagnostic drugs.

Currently, the most known vaccine strain of the rubella virus Wistar RA27 / 3 S.Plotkin), which is used both for vaccines and for the development of diagnostic drugs. It was found that vaccines from the Wistar strain RA27 / 3 cause 40% arthralgia in adults (Banatlava J.E., Brown D.W. Rubella / Lancet, 2004, v. 3663 p.1127-1137). It was also established that the Wistar RA27 / 3 strain belongs to rubella viruses of the first genetic line, while for the territory of the Russian Federation, according to WHO, strains of the second genetic line, namely genotype 2C, are considered to be endemic (World Health Organization. Global distribution of measles and rubella genotypes-update / VWkly. Epidemiol. Rec. - 2006. - V.81 (51/52) .- P.474-479)

Also known is the strain of rubella virus "Orlov-D" and a method for producing a vaccine against rubella in the diploid cell line "M-22" (patent No. 2173344, priority from 06/28/1999) However, the strain "Orlov-D" is adapted to fibroblasts human embryo. Diagnostic preparations of high sensitivity obtained on its basis (antigens for enzyme immunoassay and immunofluorescence analysis) can cause false-positive reactions due to the presence of antigens of human origin in the final product

Closest to the claimed vaccine strain of the rubella virus "Orlov-B" (RF patent No. 2081912, priority May 22, 1995). The strain was obtained by repeated passivation in primary culture of rabbit kidney cells (BPC), followed by cloning by limiting dilution and selection on the basis of a decrease in reproductive activity at a temperature of 40 ° C. Cultural-morphological, physico-biological characteristics, antigenic properties of the strain are disclosed in the patent description. However, this strain does not have a sufficiently high immunogenicity Strongly.

The invention is aimed at creating a universal strain suitable for use in the production of rubella vaccines and diagnostic preparations for the diagnosis of rubella in the Russian Federation

EFFECT: increased reproductive, hemagglutinating and immunogenic activity of a strain without reversing pathogenic properties, as well as expanding the field of its application.

To achieve the specified technical result, the authors made changes to the technology of cultivation of the strain. As a producer of MIBP, the rubella virus strain Orlov-B was used after ten years of storage. The authors changed the conditions of infection of producer cells and repeatedly passaged the virus under modified conditions.

The vaccine strain "Orlov-B" obtained by the suspension method of infection of the cell culture of the AUC. Under such infection conditions, all viral particles have the opportunity for adsorption on the surface of cell membranes, regardless of their reproductive activity, since the infectious material is not removed. The consequence of this is to obtain a population of viral particles that differ in reproductive properties. To obtain a homogeneous population with a given property of high reproductive activity, the suspension method is replaced by infection of the cells with a formed monolayer of cells with an adsorption time of 20 minutes. Under these conditions, viral particles with the highest reproductive activity have an advantage for attachment to cell receptors. In the heterogeneous population of the initial “wild” rubella virus, the ability of a certain proportion of viral particles to actively reproduce in the host cells ensures the realization of the pathogenic properties of the virus. To obtain the strain “Orlov” (U), the producer strain “Orlov-B” was used, attenuated by repeated passaging in another system of host cells (PPC culture). This allows you to count on the high immunogenic activity of the obtained strain without reversing the pathogenic properties.

As a result, the Orlov (U) strain was obtained with altered properties: high reproductive, hemagglutinating, and immunogenic activity.

Strain "Orlov" (U) passed laboratory tests for authenticity, specific activity and the absence of contaminating agents.

Strain "Orlov" (U) was deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" for the purposes of the national patent procedure. Registration number V-1 dated January 18, 2011 No. 169a.

Taxonomic position of the Orlov strain (U): Togaviridae family, genus Rubivirus.

Strain "Eagles" (U) is characterized by the following features.

Cultural and morphological characters. By electron microscopy, the population is homogeneous spherical particles with a diameter of 50-70 nm. A floating density in the sucrose gradient of 1.18-1.20 g / cm ³ . Propagated with the development of cyto-destructive action to a titer of 5-6 lg TCD ₅₀ / 0.5 ml in cell cultures of animals and humans. It is poorly propagated in primary tissue cultures of chicken and quail fibroblasts. When using roller or suspension methods of culturing on BHK-21 cells, the output of viral biomass is 7-8 lg TCC ₅₀ / 0.5 ml. Does not breed at temperatures below 20 ° C and above 40 ° C. The optimum cultivation temperature in tissue cultures is + 35 ° C.

Physiological and biochemical characteristics. Optimum pH = 7.4. Inactivated for 30 min at 56 ° C. Sensitive to ether and acetone.

Antigenic properties. The in vitro hemagglutinating activity is 16-32 GAE. Agglutinates the red blood cells of pigeons, 1-day-old chickens, a person with a blood group of 0.

Toxicity. The strain is not toxic when administered to guinea pigs and mice at a dose of 1000 TCD ₅₀ / 0.5 ml / 1 human inoculation dose.

The strain does not have residual neurovirulence and contagiousness.

Immunogenic properties. With a single intramuscular injection of the drug from the Orlov strain (U) to rhesus monkeys, rhesus monkeys exhibit 100% seroconversion with inverse antibody titer values in RTGA from 640 to 1280 GAE.

Example 1. A method of obtaining a strain of "Eagles" (U).

Growth medium 199 containing 10% serum of cow embryos (ES) is removed from a 1-liter mattress with a monolayer of tissue culture PPC; the monolayer is washed 6 times from serum with Hanks pH 7.4, after which 5 ml of 199 medium containing the virus are added rubella piece "Orlov-V" in an infectious dose of 0.01 TCD ₅₀ / cell. and left at room temperature for 20 minutes, shaking the mattress every 2-3 minutes to irrigate the virus-containing liquid (VSL) of the entire surface of the cell monolayer. In 20 minutes. The VSL is removed, infected cells are poured into 150 ml of support medium 199 and incubated in an incubator at a temperature of 34 ° C for 2 days to select viral particles with high adsorption and reproduction activity. After 2 days, without waiting for the CPP characteristic of the rubella virus, VSL is removed from the mattress and used for subsequent infection of the monolayer of a new batch of cells of PPC, which is carried out according to the scheme described above. This infection procedure is repeated four times. With the last, fourth infection of PPC cells with the virus, the incubation time at 34 ° C is extended to 18 days. Every 2-3 days, as cytopathogenic changes in the cell monolayer characteristic of the rubella virus increase (observed under a microscope), the virus-containing liquid is poured into vials and stored under a rubber stopper at (-20) ° C for subsequent use, and added to the mattress 150 ml of fresh support medium 199 and cultured under the same conditions. In this example, 4-5 useful virus removals are carried out (virus activity of at least 4 lg TCC ₅₀ / 0.5 ml).

All operations are performed under sterile conditions. The output of the virus at different at different working stages is presented in table 1.

As can be seen from table 1 and the description of the patent of the Russian Federation No. 2081912, the new strain "Orlov" (U) provides a greater yield of infectious virus (0.4-1.0 TCD ₅₀ / 0.5 ml) than the prototype strain "Orlov -AT".

Virus-containing material from individual viral collections is combined into a combined viral collection (OVS), a stabilizer (gelatin with sucrose based on a final concentration of 1 vol%) is added. The resulting semi-finished vaccine is poured into 0.5 ml ampoules and subjected to freeze drying. In this case, 500 ml of the semi-finished product is stored in liquid form at (-20) ° C for laboratory controls.

After checking for specificity, sterility and the absence of extraneous agents, the drug from the Orlov strain (U) was tested for the absence of residual neurovirulence and immunogenic activity in rhesus monkeys. The comparison drug was a prototype - the vaccine strain Orlov-B.

Determination of residual neurovirulence. To control the absence of residual neurovirulence, 10 rubus-rhesus monkeys rubus-resistant monkeys were divided into 2 groups of 5 individuals each.

One group of animals was infected with the drug from the Orlov strain (U), and the other from the Orlov-B strain. Infection was carried out intracerebrally. Before the experiment, the animals were immersed in deep anesthesia, which was achieved by intramuscular injection of 10% hexenal solution at the rate of 1.0 ml per 1.0 kg of animal weight.

Virus-containing material was injected in a volume of 0.25 ml into the visual tubercle of each hemisphere of the brain. The intracerebral dose corresponded to 10 vaccination doses for humans and amounted to 10,000 SCC ₅₀ / 0.5 ml.

Clinical monitoring of the monkeys was carried out for 30 days, during which no clinical symptoms of damage to the central nervous system were recorded.

Morphological and histological studies of tissues and organs of monkeys.

The brain and spinal cord were fixed in 10% neutral formalin. Blocks of fixed nerve tissue were embedded in paraffin and serial sections were prepared with a thickness of 8-10 microns, which were stained with thionine according to the Nissl method. The anterior central gyrus, the posterior central gyrus of the right hemisphere of the brain, the optic tubercle, the medulla oblongata were subject to research; cervical and lumbar thickening of the spinal cord. CNS structures were identified by microscopic examination of the stereotactic atlas of the brain of monkeys (Spinder R.S., Lee C. Stereotaxic Atlas of Monkey Brain Macaca Mulatta / AJniversity of Chicago Press, Chicago, USA - 1961. - 287 p.). The test was considered valid if the formation of virus-specific antibodies was recorded in at least 80% of infected monkeys 28 days after infection.

Morphological study. At the dissection of the meninges, as well as the tissue of the brain and spinal cord from the surface and in the section, as well as during examination of the serous cavities, no pathological changes were found in any of the animals in each of the two groups. During histological examination in the tissues of the brain and spinal cord of each experimental animal, pathological changes in neurons and glial cells in the studied areas were not detected in any case. Nerve cells had clear contours, a light vesicular nucleus with a large centrally located nucleolus, and distinct lumps of Nisslev substance in the cytoplasm.

Thus, the rubella virus strains Orlov (U) and Orlov-B are not neurovirulent because: the infected monkeys did not have clinical signs of damage to the uneven system during the entire observation period; a histological examination of the brain tissue of infected monkeys showed no damage to neurons and glia outside the zone of inoculation trauma; when examining the blood serum of infected animals in rtga in 100% of cases, antibodies to rubella virus were recorded, which confirms the fact of infection. Therefore, the strain "Eagles" (U) retained the sign of the absence of neurovirulence inherent in the prototype.

In an immunogenicity test, 20 rubella-rhesus monkeys rubella were divided into two groups of 10 animals each. One group of animals was inoculated with the drug from the Orlov strain, the other from the Orlov-B strain. Immunogenic activity was recorded 28 days after a single intramuscular injection of 0.5 ml of drugs from the studied strains to the monkeys. The vaccination dose corresponded to 1 vaccination dose for humans - at least 1000 TCD50. Determination of antibody titers in the blood serum of immunized monkeys was performed in rtga. All blood serums were encrypted and examined simultaneously. The results are presented in table 2. As can be seen from the table, seroconversion is 100% in the group of animals vaccinated with the known strain of rubella virus "Orlov-B", and the proposed strain of "Orlov". However, antibody titers in animals vaccinated with the proposed strain were determined in the range of 1: 320-1: 1280, whereas in animals vaccinated with the prototype strain, antibody titers accumulated to a level of 1:80 and higher, but did not exceed the value of 1: 320. The geometric mean antibody titer was 1: 559 for the proposed strain versus 1: 215 for the prototype strain, that is, the immunogenic activity of the proposed strain was significantly higher than that of the prototype strain.

The contagiousness of the Orlov strain was evaluated after 28 days of close contact (being in the same cage) of monkeys immunized and not immune to rubella. As can be seen from table 3, the strain "Orlov" has lost contagiousness, since rubella-specific antihemagglutinins were not determined in the blood serum of unvaccinated animals.

Example 2. Obtaining rubella diagnosticum for rtga. Two batches of PPC cells were used for infection with the Orlov-B prototype strain and the proposed Orlov strain (U). The accumulation of virus-containing material was carried out in liter mattresses with a monolayer of PPC cells, as described in Example 1, infecting one cell pair with the Orlov strain (U), and the other with the Orlov-B strain. After 18 days of incubation at 34 ° C, mattresses with infected cell culture were 3 times frozen in a mixture of dry ice with alcohol, followed by thawing at a temperature of 35 ° C. The culture medium together with the cells was poured into vials, centrifuged at 2000 rpm for 20 min, and individual samples of the supernatant were titrated in the RGA using red blood cells of one-day-old chickens to determine hemagglutinin titers. Virus-containing material with a titer of at least 4 GAE in 0.05 ml was combined and placed in a flat-bottomed flask, to which tween-80 previously diluted 10 times with distilled water was added to a final concentration of 2% and placed on a magnetic stirrer for stirring for 15 min at + 4 ° C. Then ether was added to the mixture in a volume equal to ½ the volume of the virus-containing liquid and stirred further for 15 minutes under the same conditions. The resulting suspension was centrifuged at 2000 rpm for 30 minutes. After centrifugation, a layer of liquid under a layer of ethereal jelly was carefully aspirated with a pipette, transferred to open containers to remove ether residues, and sodium merthiolate was added to the resulting material to a final concentration of 1: 10000. Thus obtained semi-finished diagnosticum was subjected to freeze drying. The hemagglutinin titer in the diagnosticum obtained by the described method was: for the drug from the proposed strain "Orlov" (U) - 128-256 GAE. In the preparation from the Orlov-B strain, the hemagglutinating activity remained at the level of 4-8 GAE. Strain "Orlov-B" is represented by a population with a predominance of particles defective in terms of hemagglutinating activity, which excludes the possibility of its use for the industrial production of hemagglutinating antigen, in contrast to the proposed strain "Orlov" (U).

As can be seen from the above examples, the use of the proposed strain instead of the prototype allows you to save the safety of use (lack of residual neurovirulence and contagiousness) and, at the same time, increase the immunogenicity of the rubella vaccine. And also allows you to expand the scope of the prototype, namely to use as a production strain for obtaining rubella antigenic diagnosticum.

Table 1 Titles of the strain "Eagles" (U) for example 1 Virus Removal Number one 2 3 four 5 6 Virus titer in lg TCD ₅₀ / 0.5 ml 4.8 5.7 6.5 6.4 4,5 3.9

table 2 The results of a comparative test of the immunogenicity of vaccines from the proposed and known strains of rubella virus No. The strain used for immunization Antibody titers in rtga one Orlov (U) 1: 320 2 Orlov (U) 1: 320 3 Orlov (U) 1: 640 four Orlov (U) 1: 640 5 Orlov (U) 1: 1280 6 Orlov (U) 1: 320 7 Orlov (U) 1: 640 8 Orlov (U) 1: 1280 9 Orlov (U) 1: 640 10 Orlov (U) 1: 320 eleven Orlov-V 1: 160 12 Orlov-V 1: 320 13 Orlov-V 1: 160 fourteen Orlov-V 1: 320 fifteen Orlov-V 1: 320 16 Orlov-V 1: 320 17 Orlov-V 1: 160 eighteen Orlov-V 1: 160 19 Orlov-V 1: 320 twenty Orlov-V 1:80

Table 3 Antibody titers in the blood serum of monkeys immunized and not immunized with the Orlov strain (U) Cell number The strain used for immunization Antibody titers in rtga four Orlov (U) 1: 640 four Contact <1:10 5 Orlov (U) 1: 1280 5 Contact <1:10 8 Orlov (U) 1: 640 8 Contact <1:10

Claims (1)

Rubella virus strain, Togaviridae family, genus Rubivirus, GKPM-Obolensk collection, registration number V-1 for receiving medical immunobiological preparations.