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Rabbits and mice immunised with chemically extracted O-antigens (O-Ags) of Vibrio cholerae O139 (O-AgB and O-AgD) developed antibodies (Abs) which appeared to be highly specific in ELISA for the relevant antigens and V. cholerae O139 strains without absorption, in contrast to the Abs against the heated O-Ag (O-AgH). An ELISA test based on the use of these Abs was shown to detect V. cholerae O139 strains down to concentrations of (9.4 x 10(4))-(7.5 x 10(5)) vibrios/ml and demonstrated no cross-reaction with other vibrios including representatives of serogroup O22. Native and proteinase K-treated O-AgB, O-AgD, O-AgH, as well as whole-cell lysates of V. cholerae O139 strains of different origin were used in immunoblotting with these Abs. Clear differences in the patterns of zones of specific reaction between chemically extracted and heated O-Ags and between lipopolysaccharide profiles of the V. cholerae O139 strains of different origin were observed. Serogroup-specific protein bands in the native O-AgB and O-AgD preparations were defined. The approach described for obtaining serogroup-specific Abs against vibrios and other bacteria seems to be promising for the development of specific diagnostic tests and further investigation of bacterial antigenic structure.

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The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V. cholerae used for the production of oral chemical cholera vaccine is shown. The established regularities of the synthesis of the protective antigens of V. cholerae in the process of scaled-up cultivation are discussed.

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Strains of V. cholerae serovar 0139 showed a higher intensity of multiplication and lesser nutritive requirements and produced 2-5 times higher amounts of enterotoxin during in-depth culturing than cholera vibrios of groups O1 and non-O1. R-forms of V. cholerae were characterized by the highest production of toxin. Dot-immunoanalysis and immunoblotting with monoclonal antibodies demonstrated the identity of cholera toxin and enterotoxin of V. cholerae serovar O139. The authors come to a conclusion that selective efficacy of the developed vaccines against diarrhea caused by V. cholerae serovar O139 depends on the presence of homologous O-antigen in their composition.

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Conditions for the submerged cultivation of a strain of V. cholerae O139, were worked out. These conditions ensured a high yield of biomass, soluble O-antigen and exoenzymes (proteinase, phospholipase A) into the culture medium, which exceeded their production by strains of serovar O1, respectively, 2, 3, 4 and 8 times. The preparation, isolated from the culture fluid and lyophilized, contained up to 50% of O-antigen and exoenzymes. In experiments on white mice the preparation exhibited low toxicity (LD50 was equal, on the average, to 1.2 mg) and immunogenicity (ED50 was equal to 3-5 micrograms) with respect to V. cholerae O139, which corresponded to the protective potency of commercial vaccine against V. cholerae O1 infection.

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The reactogenicity and immunological effectiveness of different doses of cholera vaccine for oral administration (in tablets), containing choleragen toxoid, Ogawa and Inaba O-antigens and some of Vibrio cholerae exoenzymes, have been tested on 143 children aged 2-17 years. In this investigation the optimum immunization doses of the preparation have been established: two tablets containing 100,000 binding units (BU) of the toxoid and 2,500 units of O-antigen for children 11-14 years; four tablets containing 200,000 BU of the toxoid and 5,000 units of O-antigen for adolescents aged 15-17 years. An essential advantage of the oral vaccine over vaccines intended for parenteral administration lies in its capacity for inducing the accumulation of secretory antibodies (coproantibodies) in practically all vaccinees.

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The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made.

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The material on the development of chemical vaccine, prepared from two newly formed strains (KM-76 Inaba and KM-68 Ogawa) and intended for oral administration, is presented. The conditions for the submerged cultivation of these strains have been established, which makes it possible to increase the production of choleragen 8- to 10-fold and O-antigen 3- to 4-fold in comparison with V. cholerae natural strain 569B. The maximum accumulation of neuraminidase, protease, phospholipase, along with choleragen, has been registered in the logarithmic phase and that of O-antigen, in the stationary phase of growth. The use of strains KM-76 and KM-68 has led to the fourfold increase of the specific activity of the main immunogens, thus permitting the respective increase of the yield of the oral vaccine without changes in its high capacity for the formation of specific antibodies and its low residual toxigenicity.

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The safety of experimental chemical cholera monovalent vaccine in tablets, produced by the institute "Microbe" (Saratov, USSR), has been studied. The study has shown that the vaccine, administered to adult rabbits and germ-free suckling rabbits by the enteral route, retains residual toxicity, mainly due to the presence of O-antigen. One or two administrations of 1-2 human doses of this preparation to adult rabbits induce minimal structural changes admissible from the viewpoint of safety. After immunization made in two administrations immunobiological transformation develops more rapidly and is more pronounced than after immunization in a single administration.

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276 volunteers aged 19 years and over were placed under observation in the course of the trial of oral cholera vaccine in tablets, containing choleragen toxoid, O-antigens of serovars Inaba and Ogawa and a number of Vibrio cholerae exoenzymes, for safety, reactogenic properties and immunological effectiveness. The vaccine was found to produce no reactions in a dose of 1-4 tablets; the administration of 3 tablets (300,000 binding units of the toxoid and 10,000 units of O-antigens, serovars Inaba and Ogawa) was shown to induce the most intensive synthesis of both antitoxins and vibriocidal antibodies in the blood sera of volunteers, as well as IgA coproantibodies. The oral vaccine was found to have an advantage over parenteral vaccines due to the absence of reactogenic properties and the formation of local immunity: coproantibodies appeared in 80% and 9% of the vaccinees respectively.

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Immunization with live plague vaccine has been shown to give no protection to thymectomized mice from subcutaneous challenge with Y. pestis virulent strain. Under the action of the vaccine or individual Y. pestis antigens (fraction I) the functional and morphological activation of thymocytes and macrophages is observed, more pronounced in C57BL/6 mice and less pronounced in CBA mice. Y. pestis antigenic preparations (fractions I and II, pesticin) act as T-cell mitogens and are thus capable of inducing the in vitro proliferation of thymocytes. At the same time the in vivo action of fraction II induces a decrease in the level of lymphocytes in the peripheral blood of mice and the destruction of lymphocytes in their thymus and spleen.

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The cultural fluid of Vibrio cholerae strains of serovar Ogawa, grown under the conditions of submerged cultivation, has been shown to contain a large amount of soluble O-antigen which sharply differs from all other concomitant components in its molecular weight. By enriching the commercial chemical cholera vaccine known as Choleragen Toxoid with purified Ogawa O-antigen a new preparation, consisting mainly of cholera toxoid and Ogawa and Inaba O-antigens and capable of producing pronounced immunity to V. cholerae of both serovars, has been obtained.

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Oral cholera vaccine contains 45% of O-antigen (serovars Ogawa and Inaba in equal parts) and at least 9 serologically active proteins; of these, toxoid (about 60% of the total amount of protein) and 5 enzymes have been identified: neuraminidase, proteinase, ribonuclease, phospholipase and ATPase. The safety, absence of reactogenicity and definite immunological effectiveness of the preparation in the primary immunization of volunteers have been shown.

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As a result of the enteral and combined subcutaneous-enteral immunization of adult rabbits with cholerogen toxoid and the specific fraction of chemical cholera vaccine in tablets immunological transformation occurred in the organism and a "protective" barrier consisting of antibody-containing cells was formed in the intestine on days 3-5. The combined method of immunization with cholera vaccine proved to be essentially as effective as enteral immunization in 2 administrations.

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Oral cholera chemical vaccine in the doses tested (2 and 3 tablets) proved to be areactogenic, harmless and immunologically effective in a controlled limited trial in 150 volunteers. By the results of titration of specific antitoxins and vibriocidal antibodies in the blood serum, as well as of coproantibodies a dose of 2 tablets was chosen as the optimal one.

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Enterotoxic properties of neuro-and endotoxins of Sonne dysentery bacillus, of cholerogen, and Bowen's cholera vibrio endotoxin were determined on a model of an isolated loop of the mouse small intestine. In definite doses all the mentioned preparations could induce dilatation of the isolated loop of the mouse small intestine. In case of Sonne bacillus neurotoxin this property correlated with the toxicity of the preparation for the animals and failed to depend on the virulence and biochemical reference of the strain from which it was obtained. Marked variations in the sensitivity of mongrel and linear mice to the mentioned preparations were noted.

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Three fractions isolated from the culture fluid of the 569B cholera vibrio strain were studied. The fractions differed by the extent of purification, and the content of the toxid and the O-antigen. In intraintestinal application to rabbits all of them caused formation of antitoxins and vibriocidal antibodies in the blood serum. The immunizing dose of the preparations in intraintestinal administration exceeded the dose required for subcutaneous application. Fraction I should be used for Producing tablet form of cholera vaccine because it was least toxic and provided the greatest yield.

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