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Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.

Methods for the evaluation of the molecular parameters of B polysaccharide (B-PS) in meningococcal protein-polysaccharide vaccine of group B are proposed. The comparison of two proposed methods, the passive hemagglutination inhibition test and rocket immunoelectrophoresis (RIEP), has shown that the latter method has the highest degree of correlation with the chemical method of the detection of B-PS, which is often hindered by lactose added as a bulking agent. RIEP may be recommended for the standardization and control of the commercial preparations of group B meningococcal vaccine. B-PS contained in the vaccine is known to be in a highly polymeric state: its yield is 66.4 +/- 1.7% to Kd = 0.25 and 85.3 +/- 1.2% to Kd = 0.5. B-PS contained in the prepared vaccine has been found to be highly stable. Outer membrane proteins of meningococci, forming a noncovalent complex with capsular B-PS, seem to stabilize its structure and prevent the depolymerization of molecules.

New data concerning the influence of cultivation conditions on the synthesis aimed at producing immunologically active antigens of group B Neisseria meningitides were obtained. The combination of conditions (deficiency in ions of iron, pH of the medium, growth phase of the culture) capable of essentially increasing (9- to 28-fold) the content of iron-dependent protein with a mol. wt. of 75-76 kD, was selected. At the same time the content of other immunologically active proteins with mol. wt. of 92-94 kD, 100-102 kD and 141-144 kD perceptibly increased (1.5 to 2.2-fold). Group B meningococcal vaccine prepared on the basis of native protein-polysaccharide complex with a higher content of the above-mentioned species-specific proteins may be expected to have a wider spectrum of immunological action.

In this work the results of the study of specific antibodies (Ab), isotypes IgM, IgG, IgA, types kappa and lambda, in 235 serum samples from 27 adults immunized with group A meningococcal polysaccharide vaccine (AMPV) in a single injection of 50 microg and from 20 control subjects are presented. The study was made by the method of sandwich EIA. The study revealed that in a month after the injection of the vaccine the intensive synthesis of IgA, IgG and IgA Ab and their subsequent circulation for 2 years were observed; 3 years after immunization (the term of observation) the prevalence of IgG and IgA antibodies was registered. Prior to immunization the ratio of kappa and lambda Ab was 1.7. In a month after immunization the maximum ratio of 3.2 was achieved and in all subsequent terms of examination this ratio remained higher than prior to immunization (3.1 -- 2.3). As revealed in this study, the injection of AMPV induced the intensive synthesis of antibodies of types kappa and lambda during the first year after immunization, then the production of type lambda Ab decreased by the second year and in 2 and 3 years after immunization the circulation of type kappa Ab prevailed.

This work deals with the problem of relationship between the molecular parameters of group A meningococcal polysaccharide and its immunological effectiveness for laboratory animals and humans. The depolymerization of group A polysaccharide contained in the vaccine leads to a decrease in its capacity of inducing the production of hemagglutinating (19S and 7S) and bactericidal IgA antibodies in humans, as well as inducing an increase in the number of cells producing IgA antibodies in the spleen of immunized mice and the appearance of circulating IgA antibodies in their sera. As shown in this investigation, fully developed immune response to group A meningococcal vaccine may be achieved in humans only if the content of group A high-molecular polysaccharide in the vaccine is not less than 70%. Mice have been recommended as an experimental model for the prognostication of the effectiveness of meningococcal polysaccharide vaccines and for their control in the process of manufacture instead of currently used titration of bacteriolysins in the sera of immunized humans.

The immunological efficacy of serogroup B meningococcal protein-polysaccharide vaccine was studied in newly developed immune bacteriolysis test. Serogroup B meningococcus strains had different sensitivities to the bactericidal effects of immune sera. A single injection of the vaccine caused an induction of bactericidal antibodies to meningococcus vaccinal strain. Three weeks after vaccination 77.7% of the vaccines developed an appreciable increase of the titers of bactericidal antibodies to the homologous strain of meningococcus. The concentration of bactericidal antibodies to strain 2394 differing from the homologous strain by its serotype did not increase. In the controls no changes in the titers of bactericidal antibodies to both strains of meningococcus were observed, the level of antibodies being the same as that before vaccination in the group of subjects immunized with B vaccine; this proves the specificity of the immunizing effect to serogroup B meningococcus vaccinal strain (No 125).

Group B meningococcal vaccine consisting of the natural complex of specific polysaccharide and outer membrane protein (OMP) has been shown to be moderately reactogenic, safe with respect to the effect of undermining tolerance to human brain tissue antigens and to produce no allergization of humans. The vaccine under study possesses antigenic activity: (a) immunization with this vaccine ensures the fourfold rise of the level of antibodies to the group-specific polysaccharide of group B meningococcus in about 80% of persons with the initially low level of antibodies, this percentage being retained during the whole period of observation, i. e. 85 days; (b) the vaccine enhances the level of antibodies to meningococcal OMP, determined in the enzyme immunoassay and the passive hemagglutination test; (c) these data are indicative of the expediency of immunizing the risk groups of persons with the initially low level of antibodies.

The protein-polysaccharide complex, isolated from group B N. meningitidis, is a variant of vaccine for the prophylaxis of group B N. meningitidis infection. In this investigation the influence of the complex of the physical properties of aluminium hydroxide gels, the amount of gel, pH and the duration of sorption on the process of sorption has been studied. Aluminium hydroxide has been shown to produce a stimulating effect on the response of mice to the polysaccharide and protein contained in the complex after immunization made in two injections. Gels with a smaller particle size have been found to possess greater adjuvant activity, as well as greater absorbing activity. The immunological activity of the complex, adsorbed ex tempore, has proved to be no different from that of the complex adsorbed in an hour.

In this work the diagnostic value of group B meningococcal erythrocyte diagnosticum was determined. 585 blood serum samples taken from adult donors were studied: 220 samples from practically healthy persons and 365 samples from 144 patients with meningococcal infection and purulent bacterial meningitis of nonmeningococcal etiology. Group B meningococcal erythrocyte diagnosticum was found to possess serological activity and to reveal the growth of specific antibodies in the sera of patients with meningococcal infection, serologically confirmed by the isolation of group B meningococcal culture, in 100% of cases on weeks 2-3 of the disease. Diagnostic characteristics--specificity and sensitivity--for group B erythrocyte diagnosticum were, respectively, 90.2% and 63.5%. The study revealed that antibodies to several group-specific meningococcal polysaccharides in blood sera can be simultaneously determined in the passive hemagglutination test with a set of erythrocyte diagnostica, which should be taken into consideration in the clinical interpretation of serological results.

The comparative study of two group B meningococcal vaccines manufactured in the USSR and in Cuba was made. The vaccine manufactured in the USSR contained the noncovalent compound of group B Neisseria meningitidis polysaccharide and outer membrane protein, and the Cuban vaccine contained group B N. meningitidis outer membrane proteins and group C N. meningitidis polysaccharide. The data obtained in this study indicated that both vaccines possessed immunological potency evaluated according to their capacity to stimulate the formation of bactericidal antibodies, whose level was found to increase eightfold after the immunization of monkeys in two injections. Besides, group B meningococcal vaccines did not induce the suppression of nonspecific protective activity characteristics of the body and did not stimulate the formation of autoantibodies to brain and liver tissues, which was indicative of the safety of these vaccines.

The protective activity of the sera of mice immunized with the preparations of native and detoxified N. meningitidis lipopolysaccharide (LPS), group A, as well as with monoclonal antibodies to N. meningitidis antigens, groups A and B, was studied on the mucin model of meningococcal infection. The study showed that the maximum level of anti-LPS antibodies in mice was observed on day 7 after the injection of LPS. Immune sera obtained from mice were capable of protecting the animals from fetal meningococcemia induced by N. meningitidis strains of homologous and heterologous groups. As shown by the results of this study, the alkaline treatment of N. meningitidis native LPS did not decrease the protective properties of antibodies. The monoclonal antibodies under study were found to possess high preventive activity in mice challenged with N. meningitidis, groups A and B. Anti-LPS monoclonal antibodies showed greater protective activity than antipolysaccharide monoclonal antibodies.

Cultures of Neisseria meningitidis Bc5S No. 125 with continuous and synchronous cell fission have been obtained. The synchronization index is 0.59-0.63. The synthesis of protein at different periods of the synchronized cycle occurs at constant speed. The synthesis of RNA and DNA is periodical, the maximum accumulation of DNA occurring at the period prior to cell fission. The amount of polysaccharide in the whole culture and the cells is considerably greater than in the supernatant fluid. At the period of fission the content of polysaccharide per 1 billion cells decreases. The indices characterizing the increase of biomass, the synthesis of macromolecular compositions and polysaccharide, the titers of the reaction of agglutination remain unchanged during the whole period of cultivation, which is indicative of the stability of the continuous and synchronous process. The use of this process holds considerable promise for obtaining N. meningitidis synchronized cultures in large amounts, for the study of the biological properties of meningococci and for obtaining immunoprophylactic preparations.

The immunogenic potency, toxicity, homologous and heterologous protective activity of lipopolysaccharide preparations obtained from serogroup A N. meningitidis (LPS A) were studied in animal experiments. These preparations were shown to possess very high protective activity. The alkaline treatment of native LPS A decreased the toxicity of the preparation almost 20 times and did not affect its immunogenic potency. Detoxified LPS A was capable of protecting mice from fatal meningococcemia resulting from infection with N. meningitidis strains, serogroups A, B and C; the adsorption of the preparation on aluminium hydroxide did not affect its protective activity. In view of the properties of detoxified LPS A revealed in this investigation, it may be regarded as a possible vaccinal preparation.

Chromatographic properties of meningococcal polysaccharides and vaccines of serogroups A and C prepared at the G. N. Gabrichevskiĭ Moscow Research Institute of Epidemiology and Microbiology were studied during determination of their molecular parameters by gel filtration on sepharose 4B. It was shown that the character of the polysaccharide elution was not affected by the composition of the eluent and pH changes within the ranges of 6.5-8.5. The ionic strength of the solvent should be at least 0.2 mol/l. Immunochemical methods such as countercurrent and rocket immunoelectrophoresis were standardized and recommended for quantitative evaluation of the content of meningococcal polysaccharides of groups A and C in the eluates. Rocket immunoelectrophoresis was shown to be more accurate and reproducible.

The immunological effectiveness of dried group A meningococcal polysaccharide vaccine, developed at the Gabrichevsky Research Institute of Epidemiology and Microbiology, Moscow, for children aged 5-14 years was studied. The intensiveness of the immune response of children to 0.5 ml of the vaccine introduced in a single injection was evaluated by a rise in the level of agglutinating antibodies to group A meningococcal polysaccharide in the sera of the vaccinees 3-4 weeks after immunization with the following optimum doses: 25 micrograms for children aged 5-8 years, 50 micrograms for children aged 9-13 years and 75 micrograms for children aged 14 years and over. The vaccine was shown to be highly immunogenic. Antibodies to group A meningococcal polysaccharide were identified as IgM. These antibodies in a titer of 1:40 and higher could be detected in 90% of the vaccinated children in the younger age group, 7 months after immunization.

The optimum conditions for the isolation and purification of the specific polysaccharide of group B meningococci have been developed. The advantages of the use of synthetic culture media for growing the initial bacterial culture have been demonstrated. The purified polysaccharides have been found to contain about 70% of sialic acid and less than 1% of protein and nucleic acid admixtures. The molecular parameters of group B polysaccharide depended on the growth phase of the bacterial culture. The most valuable culture was obtained at the exponential phase of growth. High serological activity and specificity of the polysaccharide in the passive hemagglutination test recommend it for studies on the development of diagnostic and prophylactic preparations.

The influence of changes in the physico-chemical parameters of serogroup A meningococcal polysaccharide on its immunogenicity for mice was studied by means of passive local hemolysis in gel and the passive hemagglutination test. The polysaccharide was depolymerized by heating at 100 degrees C for 5, 30 and 120 minutes; during this process the progressing decrease of the molecular weight and the content of O-acetyl groups in the preparation could be observed. Mice showed high sensitivity to changes in the above-mentioned physico-chemical parameters, which was manifested by a sharp drop in the intensity of the immune response of the animals to the heated samples of the antigen. The role of the parameters under study, i. e. the molecular weight of the antigen and the presence of O-acetyl groups, in the manifestations of the immunogenicity of polysaccharide A is discussed.