Avirulent strain of vibrio cholerae km 262 bacteria of serovar ogawa of biovar eltor - producer of protective o1 antigen

FIELD: medicine.

SUBSTANCE: avirulent Vibrio cholerae strain of biovar eltor of serovar Ogawa - a producer of a major protective O1 antigen of serovar Ogawa is produced in a simulation experiment of virulent natural V.cholerae M569 Ogawa strain by spontaneous loss of "СТХφ" prophage at cholera agent continuance in sterile river water. The strain is deposited in the State Collection of pathogenic bacteria "Microbe" Registration No. 262 of the Collection of Miscroorganisms. A feature of the strain is the absence of genes of a core region of "СТХφ" prophage. The strain is avirulent for suckling rabbits, agglutinated by cholera serums O1 and Ogawa in dilution exceeding a diagnostic titre.

EFFECT: use of the invention allows producing a vaccine of O1 protective antigen of serovar Ogawa providing immunity formation in cholera.

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The invention relates to biotechnology, namely, to obtain avirulent strains of V. cholerae biovars El tor circulation of serovar Ogawa, characterized by high product main protective O1 antigen Ogawa, intended for production of chemical vaccines against the causative agent of cholera and obtaining a purified preparation O1 antigen Ogawa for the preparation of diagnostic serum.

The main virulence factors of Vibrio cholerae, which are the major protective antigens include cholera toxin, toxin-coregulatory drank adhesion and O-antigen, respectively responsible for the formation with cholera antitoxic, anticholinesterases and antibacterial immunity. In addition, the O-antigen has serological specificity and is used for serodiagnosis and identification of the causative agent of cholera. A key virulence factor of V. cholerae is cholera exotoxin - thermolabile protein, which causes severe diarrhea is the main clinical symptom of cholera. Biosynthesis of the cholera toxin encode structural genes ctxA and ctxB included in the composition of cow's temperate filamentous phage φ integrated into the chromosome of V. cholerae. Crustal area propaga φ in addition to the ctxAB genes includes genes zot and ACE encoding products of two other x is larnach toxins, genes orfU and ser, the products of which are necessary for the formation of phage particles. All virulent strains of V.cholerae effectively secrete in vitro and/or in vivo cholera toxin. For vaccination when the vaccine both chemical and use live strains of V. cholerae, producing major protective antigens.

At the present time in the manufacture of the licensed bivalent chemical tablets cholera vaccine in the Russian Federation use virulent strains of V. cholerae O1 serogroup classical biovars that caused six previous pandemics Asian cholera. However, this vaccine is missing biofireplaces antigens of the exciter current, seventh cholera pandemic (V.cholerae O1 biovars El tor circulation) and it may not provide a high level of protection against cholera El tor circulation.

Known virulent strain of V. cholerae KM 206 O1 serogroup classical biovars of serovar Ogawa producer of protective antigens - the cholera toxin and the toxin-coregulatory pilej adhesion (patent RU 2222594, 27.01.2004). However, the products of the O1 antigen in this strain is not known.

Known strains of V.cholerae 68 KM classical biovars of serovar Ogawa - producer of protective antigen Ogawa. The strain used in domestic medicine as a production to obtain a bivalent chemical tablets is Oh cholera vaccine choleragen-toxoid + O-antigen (patent RU 2076734, 10.04.1997). This strain is highly virulent that require large expenditures to ensure biological safety chemical vaccines based on this strain.

Known avirulent strain of bacteria V.cholerae biovars El tor circulation of serovar Ogawa KM 26 that does not contain some of the genes in a cow field propaga φ (ctxA, responsible for synthesis of the toxic A-subunit of the cholera toxin, zot is responsible for the synthesis of additional cholera toxin), and tcpA genes (controlling adhesion of Vibrio cholerae) and genes of the global regulator toxR (patent RU 2254371, 20.06.2005). Described by the authors of the strain isolated from the external environment on the territory of Turkmenistan. However, the products of the O1 antigen in this strain is not known.

From literature data it is known that strains of classical biovars stably inherit embedded in the chromosome phage Sdsr and strains of V. cholerae biovars El tor circulation in certain conditions, can lose it, leading to the formation of a stable avirulent clones (Kulshan T.A. coauthors. Probl. especially dangerous INF., 2006, No. 91, p.44-48).

The task of the invention to provide avirulent strains of V.cholerae O1 biovars El tor circulation of serovar Ogawa, producing protective antigen, providing the formation of antibacterial immunity is when cholera.

The invention consists in that the avirulent strain of El tor circulation biovars of V.cholerae serovar Ogawa, which is the producer of the primary protective O1 antigen of serovar Ogawa.

The inventive strain obtained in the model experiment of virulent natural strains of V.cholerae 5/65 Ogawa isolated from a patient in Iran in 1965, by spontaneous loss of propaga φ during the long stay of Vibrio cholerae in sterile river water.

The result is a strain of El tor circulation biovars of serovar Ogawa, devoid of major genes cow area propaga φ, effectively producing a protective antigen Ogawa.

Strain V. cholerae 5/65 (PV) deposited in the Public collections of pathogenic bacteria antiplague research Institute "Microbe" number KM 262.

The basic properties of strains of V.cholerae KM 262:

- high level of production of antigen Ogawa (agglutination titer serum Ogawa 1:1600),

- the absence in the genome of strains of V.cholerae genes cow area propaga φ (ctxA, ctxB, ACE, zot, orfU, ser),

- lack of virulence in a model of rabbit-suckers,

- the lack of production of cholera toxin.

Cultural and morphological characteristics.

Motile, slightly curved sticks that do not form capsules and spores. On solid nutrient media grows in the form of a circular grayish-bluish translucent colonies, while sowing the broth is uniform clouding of the environment. The strain grows on simple environments. Prototroph.

Pathogenic properties - avirulence for rabbit-suckers in the dose of 105, 107M.L./ml

Physiological properties.

Analyzes sheep erythrocytes, agglutinate chicken erythrocytes, grows on alkaline agar with the addition of 50 μg/ml polymyxin b. Agglutinated to title holonymy diagnostic sera and Ogawa. Sensitive to cholerea diagnostic phage El tor circulation. Not ferments lactose and arabinose. To acid without gas ferments mannose, sucrose, mannitol, maltose.

The strain stored in a lyophilized state at least one year.

Example 1, showing the absence of the genes in a cow field propaga φ.

Culture strains of V.cholerae KM 262 scatter on LB-agar to test different genes propaga φ using polymerase chain reaction (PCR) with specific primers. The total DNA preparations produced by the method of boiling the bacterial suspension (107M.K./ml) in distilled water for 30 minutes in a water bath followed by centrifugation at 10000 rpm PCR performed in microtubes with a volume of 600 ál on a programmable thermostat "Terzic". The identification of fragments of genes in the chromosomes of the studied strains were carried out using oligonucleotide primers:

ctA1(5'-CGGGCAGATTCTAGACCTCCTG-3')

ctxA2(5'-CGATGATCTTGGAGCATTCCCAC-3')

zot1(5'-TCGCTTAACGATGGCGCGTTTT-3')

zot2(5'-TCGCTTAACGATGGCGCGTTTT-3')

zot2(5'-AACCCCGTTTCACTTCTACCCA-3')

ace1(5'-TAAGGATGTGCTTATGATGGACACCC-3')

ace2(5'-CGTGATGAATAAAGATACTCATAGG-3')

cep1(5'-CAGAACAATTGCCCCCACCAC-3')

cep2(5'-AAGCACGCTTTCACTCGGGG-3')

orfU1{5'-CAAAATGAGCATGGCGGC-3')

orfU2{5'-CCCATTGTGCAATCGGTGT-3')

synthesized in NPF Liteh" (Russia) on an automated DNA synthesizer "ACM102U". The PCR products fractionary in 2% agarose gel (BioRad) and register under ultraviolet light. PCR analysis of strain CM shows the absence in the genome of genes propaga φ - ctxA, ctxB, ACE, zot, orfU, ser.

Example 2, confirming the absence of production of cholera toxin strains of V.cholerae KM 262 immunoassay method.

The products of the cholera toxin is determined using an immunoassay method GM1 ELISA (Svennerholm A.M. et al. J. clin. Environ., 1983, v.l7, p.596-601). Growing cells of V. cholerae were carried out in AKI broth at 37°C for 4 h without aeration and subsequent 16 h under conditions of intensive aeration (Iwanaga M, Yamamoto K, Higa et al., J. Environ. Immunol., 1986, v.30, p.1075-1083). After the time of cultivation samples centrifuged at 10,000 rpm for 10 minutes. The supernatant incubated for two hours at 37°C in the wells of the blade, then block nonspecific sorption of inert protein (bovine serum albumin). Spend incubation with rabbit anti-toxic cholera serum, diluted 1:200 for 1 h at 37°C. After washing the wells 0.05 M phosphate b is from, pH of 7.2 to 7.4, add working dilution enzyme conjugates, which are a peroxidase labeled goat diagnostic antibodies against rabbit immunoglobulins. The reaction consider 15 minutes after substrate addition: 0.03% of hydrogen peroxide in citrate buffer, pH 4.0, 0.1% ABTS [2,2-Azino-bis-(3-ethylbenzthiazolinesulfonic)]. As a positive control and to determine the sensitivity of the method using purified product cholera toxin obtained from the reference high-toxigenic V.cholerae strains 569B. The sensitivity of the method GM1 ELISA is in this series of experiments, 1 μg/ml the Results obtained when determining the products the cholera toxin strains of V.cholerae 262 KM, were negative, indicating the absence of the production of this protein.

Example 3, confirming the absence of virulence in a model of rabbit-suckers.

The virulence of strains of V.cholerae KM 262 is determined by intracolonic infected rabbits-suckers on N.K.Dutta and M.K.Habbu (Brit. J. PharmacoL, 1955, v.256 (23), p.12252-12256). For infection use 4-hour agar culture grown at 37°C. In experiments using 8-10-day-old rabbit-suckers who enter vnutriserdechno two infecting dose: 1×105and 1×107M.K. observation of the animals are in a period of 48 hours In animals infected with strain V.choleras KM 262, is not observed nick is their visible changes in the intestine of experimental animals, that confirms the absence of virulence among strains KM 262.

Example 4. Product definition protective antigen Ogawa.

Dried ampoule culture strains of V.cholerae KM 262 seeded on agar of Hottinger, pH of 7.6, and incubated 18 h at 37°C. the Grown culture scatter on agar of Hottinger, pH 7,6, and used to study the antigenic properties using the expanded agglutination reaction with holonymy sera O1, Inaba, Ogawa and RO. Prepare a suspension of a strain of V. cholerae, corresponding to 5 units of the industry standard sample turbidity (CCA 42-28-P), equivalent to 1×109M.K./ml Diagnostic serum twice diluted with 0.3%solution of sodium chloride in a volume of 0.5 ml, respectively, the value of a diagnostic titer and add 0.5 ml of the suspension of the studied strain 262 KM. The tubes are thoroughly shaken, incubated at 37°C for 2 h and another 18-20 hours at a temperature of 20-22°C. the Studied strain KM 262 agglutinated serum to the O1 antigen (reciprocal titer is 3200) and serverspecific serum to the antigen Ogawa (reciprocal titer is 1600). Agglutination with serverspecific serum Inaba and RO is missing.

Example 5. The use of strain in the production of experimental batches of chemical cholera vaccine.

To confirm the ability of the claimed piece is MMA V. cholerae KM 262 to produce protective antigen Ogawa conduct in-depth cultivation of strain within 7-10 hours on the fermenter with automatic maintenance systems of cultivation parameters with food (glucose 40%) and pH correction (ammonia). The volume of nutrient medium 14 is l, the cultivation temperature of 37°C. the culture medium is casein broth with 1% peptone (pH of 7.6 to 8.0). To obtain preparations Of antigen used formalistically centrifugal broth culture. The content of the antigens O1 and Ogawa determine the reaction diffuse precipitation of the agarose gel on Ouchterlony (RDP) with specific agglutinating sera O1 and Ogawa. Serum against antigens O1 and Ogawa form a line of precipitation in the RDP with formalisation centrifugation in three experimental batches of the drug in dilutions of 1:16 and 1:32, respectively, which is comparable with the data in cultivation of virulent strains of V. cholerae, currently used in the production of chemical anti-vaccine.

Thus, the claimed strain of Vibrio cholerae KM 262 is avirulent strain producing protective antigen Ogawa. The strain can be used in production for chemical cholera vaccines, the production of which is based on the cultivation of avirulent strains, as well as the La obtain purified preparations of antigen Ogawa, used for preparation of diagnostic serum.

Avirulent bacterial strain Vibrio cholerae biovars El tor circulation of serovar Ogawa, deposited in the Public collections of pathogenic bacteria "Microbe" number KM, producing protective O1 antigen.



 

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