Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.

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.

Four recombinant proteins, MA 28 –P 1004 LEH 6 , ME 135 –H 328 LEH 6 , MW 329 –H 622 LEH 6 and MH 835 –P 1004 LEH 6 , were prepared based on the genomic sequence of IgA1 protease from Neisseria meningitidis serogroup B strain H44/76. The immunogenic and protective properties of these proteins were studied in a mouse model. The predicted T- and B-epitopes located in the N-terminal part of amino acid sequence of this enzyme are very important for the formation of effective protection against meningococci of the three main epidemic serogroups A, B, and C. The small-sized recombinant protein having the sequence ME 135 –H 328 LEH 6 (molecular weight 23367 Da) appears to be as protective against meningococci of the tested serogroups as the high molecular MA 28 –P 1004 LEH 6 (molecular weight 109019 Da), the latter being a large-sized analog of full-length IgA1 protease. These proteins can be promising candidates for a polyvalent meningococcal vaccine.

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.

Recombinant proteins (M1K2–N963-LEH6, MA28–N963-LEH6 and ME135–H328-LEH6) have been created on the basis of the genome sequence of IgA1 protease of N. meningitidis serogroup B strain H44/76. It is revealed that, similarly to the native enzyme isolated earlier from N. meningitidis serogroup A strain A208, these proteins induce formation of animal protection against the infection with the virulent strain of meningococcus serogroup B. It is shown that these compounds are promising as a basis for a polyvalent anti-meningococcal vaccine.

A new approach to estimation of IgA subclass levels and IgA1/IgA2 ratio using enzymatically active and inactive forms of Neisseria meningitidis IgA1 protease was developed.

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form has shown that the active form of IgA1 protease exhibited species-and type-specificity for mouse and human immunoglobulins. A mutant form, lacking enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C; it protected mice from lethal infection by live virulent cultures of heterologous serogroups of meningococcus. The results obtained in this study suggest that IgA1 protease may be considered as a perspective preparation at the stages of devel-opment of a polyvalent vaccine for protection of human against meningococcal infections of various etiology.

Microbial capsular polysaccharides for many years provided a highly practical public health vaccines for preventing meningococcal, pneumococcal and Haemophilus influenza infection, and typhoid fever. Their application in the form of conjugates with protein carriers eliminate the gap in protection against these infections in children under one year. Extremely promising turned out offered us a new generation of vaccines, which have synthetic peptides conjugated to a meningococcal polysaccharide. Thus, new approaches to the solution of the problem of meningococcal disease vaccination serogroup B were open. In recent years, Russian researchers first suggested to use IgA1 protease (one of the major virulence factors of microbes and almost identical for mentioned below infections) for prevention of such diseases as meningococcal of all serogroups, pneumococcus and hemophilia infections. Patented processes for producing of the vaccine define domestic priority of its production and use.

Microbial capsular polysaccharides for many years provided a highly practical public health vaccines for preventing meningococcal, pneumococcal and Haemophilus influenza infection, and typhoid fever. Their application in the form of conjugates with protein carriers eliminate the gap in protection against these infections in children under one year. Extremely promising turned out offered us a new generation of vaccines, which have synthetic peptides conjugated to a meningococcal polysaccharide. Thus, new approaches to the solution of the problem of meningococcal disease vaccination serogroup B were open. In recent years, Russian researchers first suggested to use IgA1 protease (one of the major virulence factors of microbes and almost identical for mentioned below infections) for prevention of such diseases as meningococcal of all serogroups, pneumococcus and hemophilia infections. Patented processes for producing of the vaccine define domestic priority of its production and use.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain ????58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup ??, ?? and ?? were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. ??? 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, ??? 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. ??? 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. ???5. C. 553-556.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain МС58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup А, В and С were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. № 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, № 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. № 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. №5. C. 553-556.

A method of the isolation and purification of IgA1 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 precipitate 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 developed. 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 µg. It was shown that IgA1 protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.

Chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. Chitosan is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. Here we produced new water-soluble low-molecular weight chitosan (LMW-Chi) lipid derivatives and compared their ability to stimulate humoral response with the effect of unmodified LMW-Chi or its oligosaccharide derivatives. LMW-Chi effectively penetrated into macrophage-like, lymphoid and epithelial cells. It also stimulated in mice IgG production to model proteins delivered either by subcutaneous or intranasal routes. Adjuvant effect of chitosan derivatives was comparable to or lower than that of unmodified LMW-Chi. Thus, it is possible that adjuvant effect is induced by unmodified glucosamine units of chitosan.

α2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosomacruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. сruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for α2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.