Peculiarities of the Formation of Antimeningococcus Immunity in Mice Immunized with Fragments of N. meningitidis IgA1 Protease.

We studied immunogenicity of two recombinant proteins FR.9 and FR.11-3 created on the basis of fragments of the primary structure of N. meningitidis IgA1 protease with different molecular weights containing different sets of T and B epitopes. The proteins actively protect animals infected with live virulent culture of meningococci, serogroups A, B, and C. Analysis of CD4+, CD8+, and CD19+ lymphocyte populations in mouse blood showed predominant contribution of different cell populations to the formation of immune response to different proteins. Injection of FR.11-3 protein to animals did no affect the immunoregulatory index, hence, this protein can be used for creation of immunologically safe vaccine preparation.

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

[Protective properties of recombinant IgA1 protease from meningococcus].

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

[Isolation and determination of activity of IgA1 protease from Neisseria meningitidis].

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

[Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B].

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306-332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.

Mechanisms of defense formation against meningococcal infection in mice immunized with synthetic peptides.

Immunization of mice with synthetic peptide fragments of conservative sites of meningococcal outer membrane proteins led to defense formation against infection with virulent serogroup A and B Meningococci. The role of cellular immunity in the formation of defense against meningococcal infection after immunization with the peptides and the possibility of stimulating lymphocyte population with these peptides were demonstrated.

Protective properties of synthetic peptides of outer meningococcal membrane.

Peptide fragments of conservative sites of PorA, OpaB, and NspA proteins of the outer membrane of serogroup B meningococci were synthesized. These peptides caused a pronounced protective effect in immunized mice infected with virulent homologous and heterologous strains of serogroups B and A meningococci. The protective effect appreciably increased, if the studied peptides were associated in a polycomponent preparation, which can be used in the construction of meningococcal bivalent B+A vaccine.

[Induction of the anti-meningitis immunity with synthetic peptides. III. Immunoactive synthetic fragments of NspA protein from Neisseria meningitidis]. / Induktsiia protivomeningitnogo immuniteta s pomoshch'iu sinteticheskikh peptidov. III. Immunoaktivnye sinteticheskie fragmenty belka NspA iz Neisseria meningitidis.

Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Induction of antimeningitis immunity by synthetic peptides. II. Immunoactive synthetic fragments of OpaB protein from Neisseria meningitidis]. / Induktsiia protivomeningitnogo immuniteta s pomoshch'iu sinteticheskikh peptidov II. Immunoaktivnye sinteticheskie fermenty belka OpaB iz Neisseria meningitidis.

Mice of various lines were immunized by 11 synthetic peptides that correspond to the sequences of fragments of the OpaB protein from the outer membrane of Neisseria meningitidis involving the known human T-helper epitopes and all the potential mouse T-helper epitopes calculated for the protein. The mice were immunized with the free peptides without their conjugation with a protein carrier. Most of the peptides were found to induce in mice the production of antipeptide antibodies. The mice protection against the experimental infection by a virulent strain of N. meningitidis of the B serotype was studied, and two peptides were shown to exert the most pronounced protective effect.

[Induction of anti-meningitis immunity using the synthetic peptides. I. The immunoreactive synthetic fragments of porin A from Neisseria meningitidis]. / Induktsiia protivomeningitnogo immuniteta s pomoshch'iu sinteticheskikhpeptidov. I. Immunoaktivnye sinteticheskie fragmenty belka porina A iz Neisseria meningitidis.

Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane of Neisseria meningitidis strain B:15:P1.7,16 were synthesize. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection from an experimental infection with N. meningitidis were identified.

[The isotypic characteristics of antibodies in persons inoculated with a serogroup-A meningococcal polysaccharide vaccine]. / Izotipicheskaia kharakteristika antitel u privitykh meningokokkovoi polisakharidnoi vaktsinoi serogruppy A.

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.

[The relationship between the immunological efficacy of a dried meningococcal group-A polysaccharide vaccine and the molecular parameters of the group-A polysaccharide]. / Vzaimosviaz' immunologicheskoi éffektivnosti sukhoi polisakharidnoi meningokokkovoi gruppy A vaktsiny i molekuliarnykh parametrov gruppovogo A-polisakharida.

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.

[Immune bacteriolysis reaction in the assessment of immunological effectiveness of serogroup B meningococcal vaccine]. / Reaktsiia immunnogo bakterioliza dlia otsenki immunnologicheskoi éffektivnosti meningokokkovoi vaktsiny serogruppy B.

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).

[A system for the mass combined vaccination of the adult population against influenza, viral hepatitis, typhoid, meningitis and diphtheria]. / Sistema massovoi kompleksnoi vaktsinatsii vzroslogo naselennia protiv grippa, virusnogo gepatita, briushnogo tifa, meningita i difterii.

A safe, moderately reactogenic and immunologically effective scheme of complex (combined) immunization against meningitis A, diphtheria, typhoid fever, viral hepatitis A and influenza has been developed as the result of experimental and clinico-immunological studies. Depending on the epidemiological situation, the newly developed scheme can be used in two variants. According to the first variant of this scheme, the following preparations are injected subcutaneously into three different sites: a mixture of group A meningococcal vaccine and diphtheria toxoid, typhoid vaccine and influenza vaccine. The second variant of the scheme differs from its first variant in using intramuscular injection of normal human immunoglobulin instead of injection of influenza vaccine. Moreover, in practical realization these variants may be altered by excluding vaccines, unnecessary under present conditions. The newly developed scheme of vaccinal prophylaxis is recommended for practical use.

[The effect of detergents on the immunological activity of the antigens of Neisseria meningitidis serogroup B[]. / Vliianie detergentov na immunologicheskuiu aktivnost' antigenov Neisseria meningitidis serogruppy B.

The complex study of the influence of detergents of different classes and aluminum hydroxide, a traditional adjuvant, on the immunological activity of individual N. meningitidis antigens (outer membrane proteins, polysaccharide B) and the complex preparation containing all these antigens revealed that changes in the antigenic and immunogenic properties of the antigens under study depended on the degree of their purification and the character of modifying substances. Aluminum hydroxide proved to be the most active adjuvant: it stimulated immune response to both outer membrane proteins and antigens of the protein-polysaccharide complex, while decreasing the antigenicity of outer membrane proteins and polysaccharide. Detergents increased the antigenicity of outer membrane proteins, both purified and, to a lesser extent, contained in the complex; still the immune response only to the purified preparation could be stimulated.

[The immunological status indices of monkeys inoculated with a meningococcal B vaccine]. / Pokazateli immunologicheskogo statusa obez'ian, privitykh meningokokkovoi B-vaktsinoi.

The method for the determination of bacterial antibodies to group B meningococci was worked out. The method was used for the determination of antibodies to group B meningococcal vaccine produced in the USSR. The dynamic study of antibodies to protein, polysaccharide and lipopolysaccharide antigens of group B meningococci was made by the method of the enzyme immunoassay (EIA), and the safety of the vaccine was studied by the determination of autoantibodies active against brain tissue antigens. The data thus obtained were indicative of the immunological activity of group B protein-polysaccharide vaccines, manifested by the capacity for stimulating bactericidal antibodies whose level increased 8- to 10-fold after the immunization of monkeys in 2 and 3 injections. Similarity in the dynamics of the formation of bacteriolysins and antibodies to protein antigen, as determined in EIA, was noted. The vaccine was found to stimulate no cytotoxic anticerebral antibodies in the glia migration test, which was indicative of the safety of group B meningococcal vaccine.

[The reactogenicity and antigenic activity of a meningococcal group B vaccine made from a natural complex of the specific polysaccharide and outer membrane proteins]. / Reactogennost' i antigennaia aktivnost' vaktsiny meningokokkovoi gruppy B iz prirodnogo kompleksa spetsificheskogo polisakharida i belkov naruzhnoi membrany.

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 sorption of a protein-polysaccharide complex isolated from Neisseria meningitidis serogroup B on aluminum hydroxide gels and the immunological activity of the sorbed preparations]. / Sorbtsiia belkovo-polisakharidnogo kompleksa, vydelennogo iz Neisseria meningitidis serogruppy B, na geli gidroksida aliuminiia i immunologicheskaia aktivnost' sorbirovannykh preparatov.

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.