Avirulent strain of vibrio cholerae km 263 bacteria of serovar inaba of biovar eltor - producer of protective o1 antigen

FIELD: medicine.

SUBSTANCE: avirulent Vibrio cholerae KM 263 strain of biovar eltor of serovar Inaba - a producer of a protective O1 antigen is produced in a simulation experiment of virulent natural V.cholerae M569 Inaba strain by spontaneous loss of "СТХφ" prophage at cholera agent continuance in sterile river water. The strain is characterised by a high production level of the major protective O1 antigen of serovar Inaba.

EFFECT: invention aims at preparing chemical cholera vaccines by formation of antibacterial cholera immunity, and producing purified preparation of the O1 antigen of serovar Inaba for a diagnostic serum preparation .

5 ex

 

The invention relates to biotechnology, namely, to obtain avirulent strains of V. cholerae biovars El tor circulation of serovar Inaba, characterized by high product main protective O1 antigen Inaba, intended for production of chemical vaccines against the causative agent of cholera and obtaining a purified preparation O1 antigen Inaba 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, responsible, respectively, for forming 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 other two Ho is hernych 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 pathogen the current seventh pandemic of cholera (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 KM207 O1 serogroup classical biovars of serovar Inaba producer of protective antigens - the cholera toxin and the toxin-coregulatory pilej adhesion (patent RU 2222595 C1). However, the products of the O1 antigen this strain is unknown.

A known strain of classical biovars of serovar Inaba producing cholera toxin V.cholerae KM (strain previously used in the manufacture of preformed bivalent chemical vaccines) (patent RU 1460075). However, this strain is high is virulent and require increased safety measures when working with it.

Known strains of V.cholerae 569 classical biovars of serovar Inaba producing cholera toxin and O1 antigen of serovar Inaba used in both domestic and foreign medicine as production for the production of chemical vaccine (patent RU 2159128, 20.11.2000; J. Ketley et al. Infect. Immun., 1990, v.141, p.893-899). However, this strain is highly virulent and require large expenditures to ensure biological safety chemical vaccines.

Strains of classical biovars stably inherit embedded in the chromosome phage φ. For strains of V. cholerae biovars El tor circulation there are some literature data loss in certain propaga φ that leads to the formation of a stable avirulent clones (Kulshan T.A. with co-authors, Probl. especially dangerous info. 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 Inaba, effectively producing O1 protective antigen of serovar Inaba, providing the formation of antibacterial immunity in cholera.

The invention consists in obtaining avirulent strains of V.cholerae biovars El tor circulation of serovar Inaba, which is the producer of the primary protective O1 antigen of serovar Inaba.

The inventive strain obtained in the model experiment of the virulent nature of the CSOs strains of V.cholerae M569 Inaba, isolated from a patient in the territory of the Republic of Mordovia (Saransk), in 1974, by spontaneous loss of propaga φ during the long stay of Vibrio cholerae in sterile river water.

The result is avirulent strain of V. cholerae El tor circulation biovars of serovar Inaba, effectively producing a protective O1 antigen Inaba.

Strain V. cholerae M569 (PV) deposited in the Public collections of pathogenic bacteria antiplague research Institute "Microbe" number CM

The basic properties of strains of V.cholerae KM263.

- high level of production O1 antigen Inaba (agglutination titer serum Inaba 1:3200);

the absence of genes in the genome of the cow area propaga φ (ctxA, ctxB, ACE, zot, orfU, ser);

- no choleragenic effect on the 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 in 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 red blood cells, increasing the and alkaline agar with the addition of 50 μg/ml polymyxin b. Agglutinated to title holonymy diagnostic sera and Inaba. 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 KM263 scatter on LB-agar for testing 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 10,000 rpm PCR performed in microtubes with a volume of 600 ál on a programmable thermostat "Terzic". Identification of gene fragments cow area propaga SDF in the chromosome of the studied strain is carried out using the following oligonucleotide primers:

ctxA1(5'-CGGGCAGATTCTAGACCTCCTG-3')

ctxA2(5'-CGATGATCTTGGAGCATTCCCAC-3')

zot1(5'-TCGCTTAACGATGGCGCGTTTT-3')

zor2(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". Products the CR fractionary in 2% agarose gel (BioRad) and register under ultraviolet light. When PCR analysis of strain CM genes ctxA, ctxB, ACE, zot, orfU, vol absent, indicating the absence of propaga φ.

Example 2, confirming the absence of production of cholera toxin strains of V.cholerae KM263 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.17, p.596-601). Growing cells of V. cholerae spend 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 buffer pH 7,2-7,4 add working dilution enzyme conjugates, which are labeled with peroxidase goat diagnostic antibodies against rabbit immunoglobulins. The reaction account for 15 minutes after addition of the substrate and 0.03% hydrogen peroxide in citrate buffer pH 4.0 with 0.1% of ABTS [2,2 azino-di-(3-thylbenzthiazoline sulphonate)]. As a positive pin is Olya 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 KM263, 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 KM263 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 a 4-hour agar culture grown at 37°C. In experiments using 8-10 day rabbit-suckers who enter vnutriserdechno two infecting dose of 1×105and 1×107M.K. observation of the animals are in a period of 48 hours In the intestine of experimental animals infected with strains of V.cholerae KM263 not see any visible changes, which confirms the absence of virulence in the claimed strain.

Example 4. Product definition protective O1 antigen Inaba.

Dried ampoule culture strains of V.cholerae KM263 seeded on agar of Hottinger pH 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, Ogav 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 KM263. 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 KM263 agglutinated serum to O1-antigen titer of 1:3200 and serverspecific serum to the antigen Inaba in titer of 1:3200. Agglutination with serverspecific serum Ogawa 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 proposed strain of V. cholerae KM263 to develop protective biofireplaces antigen Inaba 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 O-antigen used formalistically centres is what broth culture. The content of the antigens O1 and Inaba determine the reaction diffuse precipitation of the agarose gel on Ouchterlony (RDP) with specific agglutinating sera O1 and Inaba. Serum against antigens O1 and Inaba 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 KM263 is avirulent strain producing protective antigen Inaba. 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 to obtain purified preparations of antigen Inaba used for preparation of diagnostic serum.

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



 

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