Investigating the stability of the Properties of Vibrio cholerae strains – Producers of Active Components of the Chemical Cholera Vaccine

One of the key requirements to producer strains used in the manufacturing of immunobiological preparations is their stability, which consists in maintaining the main cultural, morphological, physiological, and productive properties in a series of generations. This paper describes a comprehensive methodological approach to testing strain stability using in vitro techniques.

The purpose of this study was to conduct an integrated analysis of the stability in the strains that produce active components of the chemical cholera vaccine when preparing seed material and at the stage of cultivation.

Materials and methods. Toxigenic strains of Vibrio сholerae 569B of the classical biovar, serovar Inaba and V. сholerae M-41 of the classical biovar, serovar Ogawa were used in the work. Cell morphology was monitored through light and transmission electron microscopy. Atomic force microscopy was applied to measure the main parameters of the bacterial cell. The strains were tested for the presence of ctxA gene in the chromosome using the “GenChol” test system with electrophoretic registration of results. Whole genome sequencing of the strains was performed on the Ion Torrent PGM platform using the Ion 318 Chip Kit and the Ion PGM Hi-Q View Chef 400 Kit. To determine the specific activity of cholera toxin and O-antigen, a DOT immunoassay with a conjugate based on staphylococcal protein A and colloidal gold nanoparticles was applied.

Results and discussion. The stability of the main properties of industrial V. сholerae strains – producers of the active components of the chemical cholera vaccine has been confirmed using microbiological, immunochemical, molecular-genetic methods and microscopic analysis at all stages of cultivation, and the prospects for using the integrated methodological approach experimentally substantiated. Tailoring of these methods will make it possible to control the stability of producer strains, optimize cultivation conditions and, as a result, increase the yield of the necessary antigenic component of the vaccine.

1. WHO: Cholera. (Cited 28 Feb 2022). [Internet]. Available from: https://www.who.int/ru/news-room/fact-sheets/detail/cholera.

2. Onishchenko G.G., Popova A.Yu., Kutyrev V.V., Smirnova N.I., Shcherbakova S.A., Moskvitina E.A., Titova S.V. [Relevant problems of epidemiological surveillance, laboratory diagnostics and prevention of cholera in the Russian Federation]. ZhurnalMikrobiologii, Epidemiologii i Immunobiologii [Journal of Microbiology, Epidemiology and Immunobiology]. 2016; (1):89–101.

3. Borisevich S.V., Darmov I.V., editors. [Guidelines on the Vaccine Prevention of Particularly Dangerous Infections]. Kirov: LLC “Kirov Regional Printing House”; 2011. 152 p.

4. Erokhin P.S., Utkin D.V., Osina N.A., Boiko A.V., Kuznetsov O.S., Kuklev V.E., Bugorkova T.V. [The current state of the study of the cell wall surface ultrastructure of microorganisms under the adverse effects of biotic and abiotic factors using atomic force microscopy]. Izvestiya Saratovskogo Universiteta. Novaya Seriya. Seriya: Khimiya. Biologiya. Ekologiya [News of the Saratov University. New Series. Series Chemistry. Biology. Ecology]. 2016; 16(2):186–9. DOI: 10.18500/1816-9775-2016-16-2-186-189.

5. Durakova O.S., Gromova O.V., Livanova L.F., Avdeeva N.G., Samokhvalova Yu.I., Gaeva A.V., Kireev M.N., Volokh O.A. [Modern approaches to isolation and purification of cholera test toxin]. Bakteriologiya [Bacteriology]. 2018; 3(1):59–62. DOI: 10.20953/2500-1027-2018-1-59-62.

6. Utkin D.V., Kuznetsov O.S., Erokhin P.S., Spitsyn A.N., Volokh O.A., Osina N.A. [Development of methodological approaches to the study of pathogens of particularly dangerous infectious diseases by atomic force microscopy]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2012; (2):62–4. DOI: 10.21055/0370-1069-2012-2(112)-62-64.

7. Durakova O.S., Gromova O.V., Kireev M.N., Vorob’eva S.A., Klokova O.D., Livanova L.F., Belyakova N.I., Volokh O.A. [The use of dot-immunoassay to determine the specific activity of antigens in the production of cholera vaccine]. Vestnik Biotekhnologii i Fiziko-Khimicheskoi Biologii imeni Yu.A. Ovchinnikova [Bulletin of Biotechnology and Physical-Chemical Biology named after Yu.A. Ovchinnikov]. 2018; 14(4): 10–3.