RU2407788C1 - Newly recovered bordetella pertussis bacteria strain - pertussis toxin producer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is prepared a most recent Bordetella pertussis bacteria strain No. 162-01 newly recovered from a patient and deposited in Federal State Institution of Science 'L.A. Tarasevich State Research Institution of Standardisation and Control of Medical and Biological Preparations' under No. 287.The strain is adapted for synthetic vaccine strain culture medium. Its culture morphological properties, molecular-genetic characteristic and producing ability are studied.

EFFECT: strain under the invention shows high productive property and can be used for producing pertussis vaccines and diagnostic products.

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Description

The invention relates to medicine, namely to medical microbiology and can be used to obtain pertussis vaccines and diagnostic pertussis drugs.

The known bacterial strain B.pertussis GISK No. 203 - producer of pertussis toxin used in the production of pertussis vaccines to obtain purified antigenic complexes and pertussis toxin (SU 1761795 A1, C12N 1/20, A61K 39/10, 1992).

A disadvantage of the known strain is low toxigenic activity.

The problem of obtaining highly effective pertussis vaccines remains relevant, since the widespread immunization of the population against pertussis infection has led to the adaptation of pertussis microbes to new conditions and to changes in the genetic apparatus of B. pertussis strains circulating among the population. These changes have affected the effectiveness of vaccines used to prevent pertussis.

The objective of the invention is to obtain a new highly productive freshly isolated toxigenic strain of Bordetella pertussis, which can be used for the production of pertussis vaccines and diagnostic preparations.

To solve this problem, a freshly isolated strain of bacteria Bordetella pertussis GISK them. L.A. Tarasevich No. 287 - producer of pertussis toxin.

The essence of the invention lies in the fact that to obtain pertussis toxin used for the production of pertussis vaccine and diagnostic drugs, use a new freshly isolated strain of Bordetella pertussis deposited in the Federal State Institution of Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevicha under number 287 - producer of pertussis toxin. The strain is cultured in a liquid synthetic nutrient medium with the addition of anion exchange resin under static conditions for 72-120 hours at a temperature of (36 ± 1) ° C. The concentration of bacteria at the end of cultivation is 50 ± 5 MOU / ml, the leukocytostimulating activity of the supernatant is 15.0 ± 2.0 leukocytostimulating units (LSE) in 0.1 ml, the amount of pertussis toxin determined by the enzyme immunoassay was 68.4 ± 12 , 2 ng / ME.

Molecular genetic studies of vaccine and freshly isolated B.pertussis strains showed that the gene of the subunit A of pertussis toxin, ptxA, vaccine strains is characterized by the allelic variant ptxA2 (AJ245367) - strains 267, 305, or the variant ptxA4 (AJ245368) - strains 475 and 203 while freshly isolated strains carry the ptxA1 allele (AJ245366).

All this indicates the need to use freshly isolated strains for the manufacture of pertussis prophylactic and diagnostic drugs.

Obtaining new freshly isolated strains of B.pertussis with increased toxigenic activity for the production of pertussis vaccine is one of the important conditions for the improvement of these drugs.

The freshly isolated toxigenic B.pertussis strain obtained from a sick child in Moscow in 2001 was adapted to a synthetic medium for culturing vaccine strains.

The strain has the following characteristics.

Morphological and cultural properties. Gram-negative coccobacteria - typical, ovoid-shaped small sticks, in smears are located separately or in pairs; on Bordeaux-Zhangui medium with the addition of 20-25% defibrinated human blood, the colonies are small, with a diameter of 0.5-1.0 mm, convex, round, the edges are even, shiny (with a characteristic "pearl" shine), translucent, grayish in color a thin (1.5-2 mm) layer of the medium is surrounded by an unsharply limited hemolysis zone. The culture is agglutinated by adsorbed type-specific sera to agglutinogens 1 and 3 to 1: 1600 and 1: 800, respectively.

Molecular genetic characteristics of the strain. The pertussis toxin subunit A gene is characterized as an allelic variant of ptxA1 (AJ 245366).

Example. For the cultivation of Bordetella pertussis use a liquid synthetic medium containing amino acids (cysteine, glutamic acid, proline), salts (K ₂ HPO ₄ , KH ₂ PO ₄ , KCl, MgSO ₄ , CaCl ₂ , NaCl) and vitamins (ascorbic and nicotinic acids) .

Lyophilized strain No. 287 is restored from a dry state on Petri dishes with Borde-Zhangu medium with 20-25% of human blood. Cups are incubated at a temperature of (36 ± 1) ° C for 48-72 hours. Check the morphology and purity of the culture by microscopy of smears stained by Gram. Then, 10-15 typical colonies are transferred from the plate to tubes with Borde-Zhangu medium or with casein-carbon agar and incubated for 48-72 hours. After 2-3 passages on solid media, culture washes are used as seed for growing the strain in a synthetic liquid. The first passage in a liquid medium is carried out in Erlenmeyer flasks (100 ml of medium + anion exchange resin) on a shuttle apparatus at a temperature of (36 ± 1) ° С for 16-18 hours. After checking the morphological properties, the B.pertussis culture is transferred from the flasks into mattresses with a liquid synthetic medium into which an anion-exchange resin is added. Cultivation is carried out under static conditions for 72-120 hours at a temperature of (36 ± 1) ° C.

At the end of cultivation, the concentration of bacteria is 50 ± 5 International Optical Units (MOE), the leukocytosis-stimulating activity of the supernatant is (15.0 ± 2.0) Lsed. in 0.1 ml, the amount of pertussis toxin when determined by enzyme immunoassay - (68.4 ± 12.2) ng / MOE.

A comparative study of the toxigenic properties of the proposed and known B.pertussis strains showed that the proposed strain had higher toxigenic activity (Table 1) than the known strain.

The results showed that the leukocytosis-stimulating activity of the strain was twice that of the known strain, and the amount of pertussis toxin produced by the claimed strain was five times that of the known strain. The differences are statistically significant. This indicates the high productivity of the freshly isolated toxigenic strain of B.pertussis in comparison with the known vaccine strain.

A pertussis vaccine was obtained from the claimed strain with pronounced protective activity: 10.8 international protective units (MU) were contained in 1 ml (20 MOU / ml).

Thus, a new freshly isolated toxigenic strain of Bordetella pertussis was obtained, deposited in the Federal State Institution of Scientific Research Institute for Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevicha under number 287, which has highly productive qualities, which can be used for the production of pertussis vaccines and diagnostic preparations.

Tab. one Toxigenic activity of B.pertussis strains No. strains Leukocytosis-stimulating activity (LSED / 0.1 ml) No. of pertussis toxin ng / MOE Number of experiments M ± m Credibility Number of experiments M ± m Credibility 203 10 7.6 ± 2.3 (P <0.001) 10 14.6 ± 3.1 (P <0.001) 287 eighteen 15.0 ± 2.0 eighteen 68.4 ± 12.2

Claims (1)

Freshly isolated strain of bacteria Bordetella pertussis GISK them. L.A. Tarasevich No. 287 - producer of pertussis toxin.