The features of individual fragments of IgA1 protease of Neisseria meningitidis serogroup B during the formation of immunity to bacterial infections in animals and humans were studied. The antibodies to the immunogenic regions of the studied proteins are also detected in mice infected with some bacterial pathogens and in humans with bacterial meningitis. A region of IgA1 protease was identified that is not capable of producing antibodies during immunization of animals, but that detects homologous antibodies in the blood of humans and animals recovered from bacterial infections. It has been suggested that this fragment plays a regulatory role in the process of immunogenesis.

The proteolytic enzyme enteropeptidase (EC 3.4.21.9) is a glycoprotein consisting of a heavy and a light polypeptide chain. The light chain of the enzyme is serine protease whose recombinant variants are used in biotechnology for the cleavage of peptide bonds in hybrid proteins downstream of the specific recognition site DDDDK- or DDDDR-. We have developed a pilot method for producing the recombinant human enteropeptidase light chain containing a cascade of two affine tags. The expression plasmid vector рL-HEP-H6-CBD encoding the hybrid protein was constructed, which contains the sequence of the human enteropeptidase light chain and the metal- and chitin-binding domains. The vector was transformed into the Escherichia coli BL21(DE3) strain, and, after the expression of the hybrid protein HEP-H6-CBD, a relatively simple scheme of renaturation and purification of the enzyme was developed. After the immobilization of the purified preparation on a chitin carrier, the enzyme retained the activity of specific cleavage of substrates for at least six cycles.

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.

The immunogenic and protective activities of recombinant IgA1 serine protease obtained on the base of the genome DNA of N. meningitidis serogroup B strain H44/76 were studied. A several recombinant proteins of different molecular weights that are based on the full-length primary structure of the enzyme, taking into account the distribution of B- and T-epitopes, also were studied. In experiments on laboratory animals it was shown that a number of tested preparations demonstrate the immunogenic and protective activity to protect mice from lethal challenge with virulent strains of meningococcus serogroups A, B and C, thereby exhibiting polyvaccine properties. The protective role of antibodies against the IgA1 protease was shown when mice were infected by meningococccus serogroup B. The increase in antibodies to the meningococcal IgA1 protease into the blood of rabbits infected with different serotypes of pneumococci has been detected, indicating potential ability of the meningococcal IgA1 protease to generate protection against microbes the virulence of which is caused by IgA1protease.

The immunogenic and protective activities of recombinant IgA1 serine protease obtained on the base of the genome DNA of N. meningitidis serogroup B strain H44/76 were studied. A several recombinant proteins of different molecular weights that are based on the full-length primary structure of the enzyme, taking into account the distribution of B- and T-epitopes, also were studied. In experiments on laboratory animals it was shown that a number of tested preparations demonstrate the immunogenic and protective activity to protect mice from lethal challenge with virulent strains of meningococcus serogroups A, B and C, thereby exhibiting polyvaccine properties. The protective role of antibodies against the IgA1 protease was shown when mice were infected by meningococccus serogroup B. The increase in antibodies to the meningococcal IgA1 protease into the blood of rabbits infected with different serotypes of pneumococci has been detected, indicating potential ability of the meningococcal IgA1 protease to generate protection against microbes the virulence of which is caused by IgA1protease.

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.

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 protein responsible for the protective and reactivating activities of two active fractions (AF1 and AF2) of the cells of Propionibacterium freudenreichii subsp. shermanii was isolated. The active fraction AF1 was obtained by fractional precipitation of the cell-free extract of propionic acid bacteria between 20 and 40% ammonium sulfate saturation, whereas fraction AF2 was precipitated between 60 and 80% saturation. Further fractionation of AF1 and AM by gel filtration on Sephacryl 8-200 and by ion-exchange chromatography on DEAE-Sepharose yielded seven active subfractions, as revealed by testing for their protective activity on UV-inactivated cells of Escherichia coli. Analysis of subfraction AF2-2.5 by SDS-electrophoresis and HPLC showed that it contained an apparently homogeneous protein with a molecular mass of 44 +/- 2 kDa. The concentrational dependence of the protective activity of this protein was derived. Peptides of subfractions AF2-2.1 and AF2-2.2 with molecular masses lower than 15 kDa also exhibited protective activity.