RU2144369C1 - Live cultural antimeasles vaccine - Google Patents

Abstract

FIELD: medicine, pediatrics. SUBSTANCE: invention relates to development of live cultural antimeasles vaccine for immunization of children. Live cultural antimeasles vaccine containing dried vaccine suspension and stabilizing additions has additionally antibiotic (kanamycin monosulfate or gentamycin sulfate) and aminopeptide and stabilizing addition that consists of the mixture of 50-% sorbitol solution and 25-% gelatin solution or the mixture of stabilizing agent LS-18 and 10-% gelatin solution. Vaccine has also viral suspension of the strain (L-16) or (L16-M) or (M-5) at biological activity 2.51 TCD₅₀/0.5 ml, not less. EFFECT: stable vaccine at storage, lasting immunity in vaccination. 2 cl, 2 ex

Description

 The invention relates to medicine and relates to the creation of a live culture vaccine for immunizing children.

A live culture measles vaccine is known containing a dry viral suspension of a strain of Leningrad-16 (L-16) with biological activity of the virus 10 ^3.5 TCD ₅₀ and a stabilizer sorbitol and gelatose (see the Guide to vaccine and serum. M., "Medicine", 1978 g., pp. 220-223).

 The technical task of the present invention is to expand the range of measles vaccines, the creation of a vaccine stable during storage and provides long-term immunity during vaccination.

 To solve this problem, a live dry culture vaccine (Vaccinum morbillorum culturarum vivum sicum), intended for the active prevention of measles, was obtained.

 The vaccine is a measles virus strain Leningrad-16 (L-16) or (L-16M) or Moscow-5 (M-5) obtained in the culture of cells of embryos of Japanese quail or quail line "Pharaoh".

The measles vaccine contains a dry viral suspension of these strains with a biological activity of at least 2.51 g TCD ₅₀ /0.5 ml; a stabilizer, which is a mixture of 50% sorbitol solution and 25% gelatine solution or a mixture of stabilizer LS-18 and 10% gelatin solution; antibiotic kanamycin monosulfate or gentamicin sulfate up to 20 units. at the dose of the vaccine and aminopeptide.

 The preparation does not contain intact cells. The vaccine is a homogeneous mass of yellow-pink or pink. The finished vaccine in dry form is sealed in ampoules or vials in one, two or five doses in a volume of 0.1 ± 0.012 ml or 0.2 ± 0.02 ml and contains at least 1000 TCD.

 Immediately before use, the vaccine is dissolved in a solution of 0.5 ± 0.1 ml per vaccination dose.

 The dissolved vaccine is a clear or slightly opalescent, pink or colorless liquid that does not contain suspended particles.

 Example 1

 For the production of culture, live, dry, measles vaccine, vaccine strains of measles virus L-16, M-5 and L-16 M are used.

 The vaccine strain of measles virus L-16 was launched in 1960 at the Leningrad Research Institute of Epidemiology and Microbiology named after Pasteur from the blood of a child with measles in a primary culture of kidney cells of newborn guinea pigs (PMS), in which 21 passages passed, after which it was additionally attenuated in the culture of embryo cells of Japanese quail (2 passages).

The vaccine strain of measles virus M-5 was obtained in the laboratory of measles and mumps vaccines of the Moscow Research Institute of Viral Preparations in 1978 from strain L-16 by triple cloning cells of Japanese quail embryos at a temperature of 35 ^o C.

Dry production strains are prepared from the vaccine strain, which are stored in a lyophilized state at a temperature of minus 50 ± 10 ^o C, it has an activity of at least 31 g TCD ₅₀ /0.5 ml.

An intermediate passage with a specific activity of not less than 10 ^5.0 TCD ₅₀ /0.5 ml is obtained from the production strain.

An intermediate passage is used to prepare a series of seed virus, the specific activity of which is not lower than 10 ^5.0 TCD ₅₀ /0.5 ml and which is stored in a frozen state at a temperature of 55 ± 5 ^o C for 18 months.

 Passivation of production strains and obtaining seed viruses from them is carried out in such a way that the number of additional passages does not exceed 10.

To prepare the intermediate passage, a 2-3-day monolayer of quail embryo cell culture is infected with a vaccine strain of measles virus L-16. The multiplicity of infection from 0.001 to 0.02 TCD ₅₀ per cell.

 Viral collections are collected from a cell monolayer with severe specific degeneration, which, after adding 10% of cattle serum (SCRS) to them, is monitored for sterility.

Until the end of the control, individual viral collections are stored at a temperature of 4 ± 2 ^o C. Sterile viral collections with a titer of at least 3.51 g TCD ₅₀ /0.5 ml are combined in one container, poured into vials and stored at a temperature of (-55 ± 5) ^o C.

 The resulting intermediate passage, each individually, is monitored for sterility and specific activity.

From a 9-10 day old quail embryo, a cell culture is prepared and a two to three day old monolayer is infected with an intermediate passage. The multiplicity of infection from 0.001 to 0.01 TCD ₅₀ per cell.

The cell suspension with the virus is poured into containers and incubated at a temperature of (35 ± 1) ^o C in a roller unit with a rotation speed of 28 ± 1 rpm. After 3 days, roller bottles with a well-formed monolayer are subjected to 6-fold washing with Hanks solution pH 7.4 ± 2.

 The nutrient medium is removed from the roller bottles, 120 to 150 ml of saline solution is introduced, placed in the roller unit and rotated for at least 3 minutes at a rotation speed of 10 ± 1 rpm. The operation of washing the monolayer is repeated 6 times.

After washing the monolayer, up to 300 ml of support medium 199 is added to the roller bottles with the addition of kanamycin monosulfate or gentamicin sulfate up to 100 units / ml, 5% aminopeptide and continue to incubate in the roller at (35 ± 1) ^o C.

 Starting from 4-6 days after the cultivation of infected cells and with the appearance and increase of specific degeneration, the whole medium is removed every day and replaced with a new one. Virus-containing liquid is poured into vials of various capacities (individual viral fees). Fresh medium is added to the roll bottles in a volume equal to the volume of the drained virus-containing liquid. The collection of virus-containing fluid and its replacement with fresh support medium is carried out repeatedly until at least 50% of the cells are stored on the surface of the walls.

Individual viral collections are stored at (4 ± 2) ^o C.

All sterile individual viral collections in vials with a specific activity of at least 4.51 g TCD ₅₀ /0.5 ml after cell precipitation (from 5 to 14 days) are placed in a layer of dry ice to freeze the sediment.

 The supernatant from the vials is combined into a bottle. 25% gelatose and 50% sorbitol are added to the combined viral collection in proportions: 200 ml of gelatose and 100 ml of sorbitol are added per 700 ml of viral collection (17.5% dry virus suspension). The volume of viral fluid in a 5.0 L bottle should be 2.5 L with an aminopeptide content of 2.5 - 3.5%.

 In a bottle with viral liquid, gelatose is first introduced under vacuum and then sorbitol. When using the stabilizer LS-18 and gelatin 10%, LS-18 and gelatin 10% are added to the combined viral collection in ratios: for 500 ml of viral collection (16.5% dry virus suspension), 400 ml of LS-18 and 100 ml of gelatin are added 10 % of.

The liquid semi-finished product is stored up to 25 days at (4 ± 2) ^o C and not more than 1 year at a temperature of (40 ± 2) ^o C.

The liquid semi-finished product stored at (-55 ± 5) ^o C is thawed at a temperature of (4 ± 2) ^o C 24-48 hours before the spill and mixed thoroughly with the liquid semi-finished product stored at (4 ± 2) ^o C.

 The liquid prefabricated vaccine is poured into (0.1 ± 0.02) ml or (0.2 ± 0.02) ml into sterile ampoules or vials.

The semi-finished vaccine containing sorbitol and gelatose is poured at a temperature of (4 ± 2) ^o C, and with a stabilizer LS-18 at room temperature.

Vaccine ampoules are stored at (6 ± 2) ^o C for no more than 5 hours.

Next, freezing of the vaccine poured into ampoules is carried out at a temperature of -30 ^° C. and freeze-drying.

 In this case, a series of vaccines with a stabilizer of sorbitol-gelatose at the end of the freezing process, the chamber is vacuumized to a residual pressure of not higher than 10 Pa (75 μm Hg). Drying is carried out without circulation of the heat carrier. Duration 2 hours.

The temperature of the drug at the sublimation stage should not exceed -40 ^o C. The total duration of the drying process is 40-44 hours.

Drying of the pre-frozen vaccine with the stabilizer LS-18 is carried out at a pressure of no higher than 10 Pa (75 μm Hg) without circulation of the coolant, at a preparation temperature of sublimation not higher than -40 ^o C, the drying process takes 46-54 hours.

Due to the presence of the stabilizer LS-18, the vaccine acquires thermal stability and has a biological activity of at least 2.5 units. TCD ₅₀ /0.5 ml.

The finished vaccine is stored at (6 ± 2) ^o C and relative humidity not more than 60%.

 Shelf life 15 months from the date of lyophilization.

 Example 2

Vaccine test
The specific activity of the vaccines was determined as follows. Virus titer was determined by the cytopathic effect of the virus on cell sensitivity (Vero cell culture). All field trials of the vaccine were carried out as part of the planned vaccine prophylaxis on the basis of children's clinics. Four children's clinics of the Central District of Moscow, children's clinics of the Moscow Region (Ivanteevka, Odintsovo, Zhukovsky, Reutov), as well as children's clinics of the Mogilev, Gomel, Nikolaev and Murmansk regions took part in the work. During the test, 448 children were vaccinated with a domestic live vaccine from the strain Leningrad-16 and 220 children with live measles vaccines of foreign production.

 Children from 12 months to 4 years of age who had no contraindications, who had not been vaccinated against measles before, and who had no measles, were vaccinated. The requirements for the selection of children for inclusion in the experimental and control groups (age and state of health) were identical, i.e. by the number, age, and history of children, all groups were representative. All groups of children were formed by random sampling.

 In each clinic, all children were thoroughly examined by pediatricians before vaccination. The inspection report was recorded on the child’s individual card. Daily clinical monitoring of vaccinated was carried out for 21 days from the moment of vaccination by specially instructed personnel of polyclinics (pediatricians and patronage nurses). In addition, after a special conversation, parents received a diary for monitoring deviations in the child’s health status. After the end of the observation period, the diaries were supplemented and corrected by the employees of the clinics and, with the personal signature of the attending physician, were centrally transported to the GISK named after Tarasovich. The reactogenicity analysis of live measles vaccines was carried out to record information. During all clinical trials, the epidemiological situation among the observed contingents was favorable.

Assessment of reactogenic properties
When reactogenicity was taken into account in the observation diaries, both local and general reactions were recorded within 21 days after vaccination. Local reactions mean hyperemia, tightening, pain, burning at the injection site. When recording general reactions, temperature, rash, catarrhal phenomena, disorders of the gastrointestinal tract, etc. were taken into account. Temperature reactions were conditionally divided into three groups: weak (37.1 - 37.5), medium (37.6 -38.5) and strong (38.6 and higher). The highest temperature for the entire observation period determined to which intensity group it should be assigned.

 Particular attention, given the timing of the development of the vaccination process, was given to reactions between 4-5 and 18 days from the moment of vaccination. Therefore, according to the timing of occurrence, the general reactions were conditionally divided by us into “early” (from 1 to 4 days), “true” (from 5 to 18 days) and “belated” (starting from 19 days after vaccination).

 During the studies, all deviations in the children's health status were recorded within 21 days after vaccination, however, in the analysis, the relationship of reactions with vaccination was evaluated individually.

 As a result of the studies, it was revealed that the live measles vaccine of the new seed series is characterized by 100% seroconversion of the vaccinated and, practically, areactogenicity, causing mild vaccine reactions in only 4.2% of the vaccinated.

Claims (1)

1. Live culture measles vaccine containing a dry viral suspension and stabilizing additives, characterized in that the vaccine additionally contains an antibiotic and aminopeptide, and as a stabilizing additive a mixture of 50% sorbitol solution and 25% gelatose solution or a mixture of stabilizer LS-18 and 10% gelatin solution, while containing a viral suspension of the strain Leningrad-16 (L-16), or (L16-M), or Moscow-5 (M-5) with a biological activity of at least 2.5 lg TCD ₅₀ / 0.5 ml in the following ratio of components, vol.%:
Dry viral suspension of strain (L-16), or (L16-M), or (M-5) with a biological activity of at least 2.5 lg TCD ₅₀ / 0.5 ml - 16.5 - 17.5
Antibiotic - Up to 20 units. in a dose of a vaccine
Aminopeptide - 2.5 - 3.5
A mixture of 50% sorbitol solution and 25% gelatose solution - 30
or
A mixture of stabilizer LS-18 and 10% gelatin solution - 50
2. Live culture measles vaccine according to claim 1, characterized in that it contains kanamycin monosulfate or gentamicin sulfate as an antibiotic.