EFFECT OF THE PROPHAGE CTXΦ DELETION UPON PHENOTYPIC PROPERTIES IN STRAINS OF VIBRIO CHOLERAE BIOVAR EL TOR, ASSOCIATED WITH VIRULENCE AND PERSISTENCE

Objective of the study is to evaluate the influence of CTXφ prophage deletion, which carries ctxAB genes, on phenotypical properties associated with pathogenicity or biofilm formation in non-toxigenic mutants. Materials and methods. Utilized have been the clinical strains of Vibrio cholerae biovar El Tor and their spontaneous non-toxigenic mutants that lost CTXφ prophage. Applied have been microbiological and biochemical methods, inoculation of model animals with cells of the strains under study. Results and conclusions. The results of comparative analysis of phenotypic properties in isogenic toxigenic and non-toxigenic strains of Vibrio cholerae biovar El Tor, which lost CTXφ prophage encoding the cholera toxin, are represented. It is established that the deletion of CTXφ prophage leads to the simultaneous change of several phenotypic properties associated with virulence (colonizing ability, production of soluble hemagglutinin/protease and heat labile hemolysin/cytolysin) and biofilm formation (motility, exopolysaccharide biosynthesis) in spontaneous non-toxigenic mutants. It is suggested that the reason for these phenotypic changes in the mutants might be the changes in activity of the related to each other regulatory genes controlling virulence and biofilm formation process in cholera agent

Experimental Substantiation of the Possibility to Use Finite Cell Line CHO-K1 for Determination of Specific Activity of Components of Chemical Cholera Vaccine

Objective was to experimentally substantiate the possibility to use the finite cell line CHO-K1 for measuring specific activity of cholera toxin and component of the vaccine choleragen-anatoxin in the process of chemical cholera vaccine manufacturing. Materials and methods. The studies involved the finite cell line CHO-K. The registration of results of bio-indication method was performed visually with the help of inverted microscope and photometrically - in colorimetric test for the assessment of metabolic activity of the cells at the wave length of 595 nm. Results and discussion. The proposed method allows for determining the toxin-production activity of Vibrio cholerae 569B strain during submerged cultivation in bioreactor and specific activity of choleragen-anatoxin by anatoxin binding measuring using cell cultures. The results correlate with the data obtained using intradermal Craig’s technique, GM1-ELISA and radial passive immune hemolysis (RPIH). Introduction of cell culture method into practice will provide for significant decrease in the volumes of usage of animals at the stages of manufacturing of chemical bivalent cholera vaccine

Usage of nutrient Medium Based on Dry Hydrolysate of Casein in Manufacturing Bivalent Chemical Cholera Vaccine

Objective of the study was to select the standardized substrate containing dry hydrolysate of casein for preparation of nutrient medium utilized for manufacturing bivalent chemical cholera vaccine under submerged cultivation of cholera vibrio strains in fermenters. Materials and methods. We used Vibrio cholerae O1 strains of classical biovar: strain 569B Inaba and strain M-41 Ogawa. Examined were two dry substrates of the medium: enzymatic hydrolysate of casein, Type I Himedia (India) and pancreatic hydrolysate of casein, produced by the State Scientific Center of Applied Microbiology and Biotechnology (Russian Federation). Produced under laboratory conditions at the premises of the RusRAPI “Microbe” medium was used as a control. Submerged cultivation was conducted in bioreactors during (9±1) h with aeration and automatic feeding of glucose and ammonia. Production of protective antigens was measured applying immunochemical and biological methods. Results and discussion. It is demonstrated that submerged cultivation of cholera vibrio production strains on nutrient media under study provides for synthesis of protective antigens the parameters of which comply with the requirements of normative documentation. More standardized and higher indicator values of the target product are ensured by cultivation of producer strains on nutrient medium with a substrate from dry enzymatic hydrolysate of casein, containing (1.5±0.1) g/l of amino nitrogen for the strain V. cholerae M-41 and (2±0.1) g/l – for V. cholerae 569 B. Transition to the use of standardized dry protein components of cultivation media does not lower the quality of the chemical cholera vaccine, but allows for the reduction of cost price and duration of technological process

Anesthesia of laboratory animals in manufacturing of diagnostic and preventive biomedicines

Preparations such as XilaVet, Zoletil 100 as well as Aeranne (Isoflurane) are successfully applied for animal anesthesia in veterinary practice. We assessed a possibility of using parenteral narcosis with Zoletil 100 in combination with muscle relaxant Xila for producer-rabbits involved in manufacturing of natural rabbit serum subsequently deployed for production of diagnostic serum and immunoglobulin preparations. Administration of preparations into auricular vein is easy to do, while animals are sedated immediately allowing for safe fixation on restraining table and causing no additional stress for biomodels. This type of narcosis provides for expected depth of anesthesia and its maintenance until the end of blood-letting procedure. Parameters characterizing the state of cardiovascular system due to anesthetic products remained within the permitted limits. These preparations do not reduce heart beat rate allowing for collecting sufficient blood volumes. Application of inhalation anesthesia with Aeranne in laboratory animals provides for the specified depth of anesthesia and its maintenance until the end of the whole procedure. However, it requires specialized equipment and highly trained personnel with appropriate skills. Usage of Xila as a mono narcosis is not recommended as exhibits weak analgesic effects and strong hypotensive activity by decreasing quantity of collected blood volume. It was found that anesthetics such as Xila, Zoletil 100, Aeranne did not affect specific activity of immune sera in case of total dehematizing procedure. Moreover, antibody titers were not declined throughout entire observation (12 months) period and complied with the requirements of regulatory documentation. In addition, a feasibility of replacing old-fashioned anesthesia method with diethyl ether for a combination of safer contemporary preparations of Zoletil 100 and Xila was demonstrated while manufacturing tableted chemical cholera vaccine in experimental series with suckling rabbits used at diverse stages of raw material verification during surgical interventions. Xila, Zoletil 100, and Aeranne examined by us had no impact on the amount of blood obtained from donor-animals, immunological properties of the sera and ready-to-use diagnostic preparations. Such drugs were safe for all-age animals that comply with the requirements to anesthesia of animal biomodels and producer-animals in manufacturing of immunobiological preparations. Thus, our study allowed to conduct experiments with laboratory animals in a more humane manner

Hardware-Controlled Method of Desalting Antigen Components of Cholera Chemical Vaccine

Experimentally substantiated is the possibility to apply tangential ultrafiltration for desalting antigen components of the tableted bivalent chemical cholera vaccine. Specified are the technological parameters of the process. It is demonstrated that the properties of choleragen-anatoxin (produced by Vibrio cholerae strain 569B Inaba) and O-antigens (produced from V. cholerae 569B Inaba and M-41 Ogawa strains) obtained using the designed methodology are as efficient as the ones manufactured using certified procedure and satisfy regulatory requirements. Experimentally substantiated technology for the desalting of antigen components of chemical cholera vaccine provides for the reduction of the time elapsed up to 5-6 hours from the original 3 to 4 days. It also allows for the manufacturing under controlled conditions. This hard-ware controlled method of desalting has been implemented into the vaccine production practice

Improvement of Technology of Cholera Toxin B-Subunit Production

Consideration is given to implementation of state-of-the-art filtration technologies for up-scaled manufacturing of cholera toxin B-subunit, produced by recombinant Vibrio cholerae non O1 KM93 strain. Selected are micro- and ultra-filtration membranes to be incorporated into manufacturing method. Investigated are the properties of cholera toxin B-subunit, obtained applying the pilot technology. The engineered method for up-scaled manufacturing of cholera toxin B-subunit makes the procedure easier-to-maintain due to tangential micro- and ultra-filtration, performed at the stage of purification and concentration. It excludes labor-consuming chromatographic purification, while retaining B-subunit properties. The studies undertaken make it possible to manufacture cholera toxin B-subunit with the same characteristics as in the case of the pilot technology, but under production conditions, and use it as a component for chemical cholera vaccine

Properties of Experimental and Standard Preparations of the Protective O Antigen of <I>Vibrio cholerae</I> M41 Ogawa

Investigated are immunochemical, chemical and biochemical properties of the O-antigen preparations obtained from Vibrio cholerae M41 classical biovar, serotype Ogawa strain using standard manufacturing and improved technologies of their concentrating. The preparations have been concentrated by ultrafiltration in the mode of tangential liquid flow with membranes of various cut-off levels for a particular molar weight. ELISA has revealed equal O-antigen load in all of these preparations. Studies of specific fractures by means of chromatographic and electrophoretic methods have demonstrated their properties to be alike. Chemical composition analysis has also identified coincidence in the load of components under specification. Thus, it has been proved that application of the improved V. cholerae protective antigen concentrating technology for manufacturing cholera chemical bivalent palletized vaccine is justified

Methods and Technologies of Cholera Vibrio Cultivation (Scientific Review)

Displayed is the review of domestic and foreign literature sources devoted to matters of cholera vibrio cultivation. Discussed is the information concerning the following methods utilized in the technological process of vibrio growth stimulation: batch cultivation, fed-batch, fermentation and dialysis, periodic and continuous cultivation. Analyzed is the impact of such parameters as dissolved oxygen concentration, count of carbon nutrition source, medium pH, temperature rate , concentration and physiological condition of inoculate, duration of technological process, affecting the growth of this microorganism and synthesis of its antigens. Consequently, literature data analysis has contributed to the selection of proper method for cholera vibrio cultivation and consideration of the factors mentioned above for the development of manufacture technology applied to the production of medical immunoglobulin preparations for diagnostics and prophylaxis of cholera

Assessment of Stability of Chemical Cholera Vaccine in a New Primary Packaging

The bivalent chemical cholera vaccine is the only drug for the prevention of cholera registered in the Russian Federation. The vaccine has been produced in glass bottles containing 210 tablets. At the same time, modern trends dictate the need to produce the drug in varying dispensing and more practical packaging for the convenience of the consumer.The aim of the work was to study the stability of the properties of the immunobiological medicinal product “Bivalent chemical cholera vaccine” with modified filling and in new primary packaging.Materials and methods. When studying the quality of bivalent chemical cholera vaccine batches, physicochemical parameters, formaldehyde content, specific activity and safety, abnormal toxicity, immunogenicity, and microbiological purity were assessed. Stability in terms of “specific activity” was evaluated using dot immunoassay.Results and discussion. As a result of this work, the use of several dispensing options and new primary packaging of cholera vaccine has been experimentally substantiated. The stability of the finished vaccine preparation has been established in the “accelerated aging” test and during long-term storage. The possibility of using dot immunoassay with a conjugate based on staphylococcal protein A, labeled with colloidal gold, to monitor the stability of cholera vaccine has been experimentally demonstrated