Background/Objectives: IgA1 protease is one of the virulence factors of Neisseria meningitidis, Haemophilus influenzae and other pathogens causing bacterial meningitis. The aim of this research is to create recombinant proteins based on fragments of the mature IgA1 protease A28–P1004 from N. meningitidis serogroup B strain H44/76. These proteins are potential components of an antimeningococcal vaccine for protection against infections caused by pathogenic strains of N. meningitidis and other bacteria producing serine-type IgA1 proteases. Methods: To obtain promising antigens for creating a vaccine, we designed and obtained several recombinant proteins. These proteins consisted of single or directly connected fragments selected from various regions of the IgA1 protease A28–P1004. The choice of these fragments was based on our calculated data on the distribution of linear and conformational B-cell epitopes and MHC-II T-cell epitopes in the structure of IgA1 protease, taking into account the physicochemical properties of potential compounds and the results of a comparative analysis of the spatial structures of the original IgA1 protease and potential recombinant proteins. We studied the immunogenic and protective effects of the obtained proteins on the BALB/c mice against meningococci of serogroups A, B and C. Results: Proteins MA28–P1004-LEH6, MW140–K833-LEH6, MW329–P1004-LEH6, M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6 and M(W140–Q299)-(Y866–P1004)-LEH6 have shown the following antibody titers, 103/titer: 11 ± 1, 6 ± 2, 6 ± 1, 9 ± 1 and 22 ± 3, respectively. Also, the last two proteins have shown the best average degree of protection from N. meningitidis serogroups A, B and C, %: 62 ± 6, 63 ± 5, 67 ± 4 respectively for M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6 and 70 ± 5, 66 ± 6, 83 ± 3 respectively for M(W140–Q299)-(Y866–P1004)-LEH6. Conclusions: We selected two recombinant proteins consisting of two (M(W140–Q299)-(Y866–P1004)-LEH6) or three (M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6) linked fragments of IgA1 protease A28–P1004 as candidate active component for an antimeningococcal vaccine.

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 mature serine-type IgA1 protease from Neisseria meningitidis serogroup B strain H44/76 (IgA1pr1_28–1004) is considered here as the basis for creating a candidate vaccine against meningococcal meningitis. In this work, we examine the primary structure similarity of IgA1 proteases from various strains of a number of Gram-negative bacteria (N. meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae) in order to find a structural groundwork for creating a broad-spectrum vaccine based on fragments of this enzyme. BLAST has shown high similarity between the primary structure of IgA1pr1_28–1004 and hypothetical sequences of mature IgA1 proteases from N. meningitidis (in 1060 out of 1061 examined strains), N. gonorrhoeae (in all 602 examined strains) and H. influenzae (in no less than 137 out of 521 examined strains). For these enzymes, common regions of sequence correspond to IgA1pr1_28–1004 fragments 28–84, 146–193, 253–539, 567–628, 639–795 and 811–1004, with identity of at least 85%. We believe that these fragments can be used in the development of a vaccine to prevent diseases caused by pathogenic strains of N. meningitidis and N. gonorrhoeae as well as a significant number of strains of H. influenzae.

The review covers the study of the protective properties of IgA1 protease and the possibility of creating a vaccine preparation for the prevention of bacterial meningitis of various origins on its basis. Bacterial meningitis belongs to the group of socially dangerous diseases and is characterized by a severe course, numerous complications and high mortality. The approaches used at present in world practice to create antimicrobial vaccines are based on a narrow targeting against a specific pathogen. The development of a monocomponent vaccine against a wide range of bacterial pathogens with a common virulence factor is still relevant. IgA1 protease, a protein that is one of the main virulence factors of a number of gram-negative and gram-positive bacteria, can serve as such an antigen. Bacterial IgA1 protease is uniquely specific for immunoglobulins A1 (IgA1), cleaving peptide bonds in the hinge regions of the IgA1 in humans and other higher primates. Bacteria, getting on the mucous membrane, destroy IgA1, which acts as the first barrier to protect the body from infections. Neutralization of IgA1 protease at this stage can become an obstacle to the development of infection, hindering the adhesion of a number of pathogens that produce this protein. The data available in the literature on the mechanism of antibacterial protection are scattered and ambiguous. The review considers the literature data and the results of our own experiments on the protective activity of IgA1 protease. We have shown that the recombinant meningococcal IgA1 protease and some of its fragments protect mice from infection with a live virulent culture not only of meningococci of the main epidemic serogroups (A, B, C, and W135), but also of some of the most common virulent pneumococcal serotypes. The data obtained indicate the possibility of creating a monocomponent vaccine against these and, possibly, other bacterial infections. Currently, significant progress has been made in studying the structure and functions of secreted proteins in the bacteria Neisseria meningitidis and Haemophilus influenzae. In this review we describe protein translocation systems of N. meningitidis, which are related to the secretion of proteins in these bacteria, and also present modern data on the functions of these proteins. Analysis of experimental data on the structure of IgA1 protease of N. meningitidis and the formation of immunity during vaccination is of key importance in the development of prophylactic preparations.

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.

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.

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.