RU2407792C1 - METHOD FOR PRODUCING IgAl-PROTEASE FROM SEROGROUP A NEISSERIA MENINGITIDIS AND RELATED IMMUNOGENIC PREPARATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method consists in the serogroup A strain A 208 N. meningitidis culture growth on the modified Franz nutrient medium that is followed with the cetavlon inactivation of a culture fluid to the final concentration 0.1 % and the centrifugal sedimentation of a microbial mass, the IgAl-protease recovery and purification, and the product lyophilisation. The IgAl-protease recovery is enabled by both fractions, namely cetavlon supernatant and cetavlon sediment. The collected sediment is multiply extracted, the sediment extracts and supernatant are concentrated by an ultrafiltration technique. The prepared concentrates are fractionated by absorption chromatography on a Silochrom-80 sorbent column at temperature +8°C compensated with 0.02 M phosphate salt buffer solution, pH 7.0, with IgAl-protease being produced once washing sorbent with said buffer solution. The invention makes it possible to produce a non-polysaccharide vaccine component for protection against serogroup B meningococcal infection.

EFFECT: invention allows to simplify the method for producing IgAl-protease of N meningitidis due to the substitution of multiphase agent inactivation by a one-phase cetavlon processing, the elimination of a gel-penetration chromatography stage, and to improve ecological safety of the process as a whole.

3 cl, 6 dwg, 2 tbl

Description

The invention relates to microbiology, immunochemistry and vaccinology, in particular to the production and purification of IgAl protease, which can be used both as a standalone preparation and for the preparation of a meningococcal multivalent vaccine.

Various methods for producing the IgAl protease of meningococci have been described. Application (WO 9011367, Kilian M. et al., 04.10.1990) describes a method for producing a meningococcal recombinant IgAl protease and its fragments obtained by cloning an IgAl protease gene from a microorganism, especially N. influenzae serotype B and N.meningitidis strain HF13, in E. coli cells. It is assumed that the indicated recombinant enzyme and its fragments can be used to obtain vaccines intended for the prevention of meningitis of all types of meningococcus and gonococcus, however, data on the immunogenic or protective activity of the obtained enzyme preparations are not provided.

The patent (US 7,235,242, Achtman et al., 06/26/2007) also describes the preparation of an IgAl protease fragment which is used as a carrier peptide. The synthesis of an IgAl protease fragment containing from 40 to 200 amino acid residues and including 40 amino acid residues of the sequence SEQ ID NO: 1 is described using an automatic synthesizer. However, when conjugated to a polysaccharide, the IgAl protease provides protection only against serogroup C meningococcus and is not effective against serogroup B meningococcus.

The patent (US 7407653, Plaut et al., 08/05/2008) describes the use of bacterial IgAl proteases for the treatment of diseases associated with IgAl deposition in tissues and organs of the body. Soluble IgAl proteases containing various labels, for example a polyhistidine fragment, are produced by recombinant methods. The enzyme is isolated by a known method using ultrafiltration, ion exchange and metal chelate chromatography. The resulting enzymes can be used to obtain therapeutic agents intended for the treatment of diseases associated with the accumulation of IgA. However, the yield of the active enzyme is not given, although it is indicated that approximately 20 L of culture medium containing recombinant IgAl protease is used for isolation.

A method for isolating an IgAl protease (MS Blake and C. Eastby. Studies on the gonococcal IgAl protease II. Improved methods of enzyme purification and production of monoclonal antibodies to the enzyme is also described. J. Immunol. Meth., 1991, 144, 215 -221), including fractionation of the culture fluid containing IgAl protease, chromatography on phenyl-sepharose, then on an Mono S ion exchange column. The supernatant obtained by centrifuging the culture fluid after biomass growth of N. Meningitides and N. Gonorrhoeae was applied to a phenyl-column sepharose. Fractions with enzymatic activity were pooled and applied onto a Mono S column, and a highly purified IgAl protease was obtained in 0.4 mg yield from 1 L of culture fluid.

Other methods for producing a meningococcal IgAl protease are described (see, for example, V.I. Surovtsev et al. Bacterial IgAl - production, properties, application prospects // Vestnik RAMS, 2001, No. 5, pp. 39-42). The protease was isolated from Hottinger broth, on which N. meningitidis culture was grown. Cells were removed by centrifugation and the enzyme was isolated by salting out (NH ₄ ) ₂ SO _{4 by} hydrophobic chromatography on phenyl-sepharose, chromatography on an ion-exchange medium and ultrafiltration, followed by freeze drying. The yield of purified enzyme was 1-2 mg from 50 l of culture fluid.

The closest analogue of the first invention of the group is a method for producing meningococcal IgAl protease (see V.I. Surovtsev et al. Obtaining IgAl protease secreted by Neisseria meningitidis cells // Biotechnology, 2006, No. 3, S.62-67 - prototype) . The technology for the production of highly purified meningococcal IgAl protease is described. The method is based on the cultivation of bacteria in a culture fluid and its concentration, the use of chromatography on C-80 silochrome for purification of IgAl protease, hydrophobic chromatography on phenyl-sepharose and gel filtration on CL 6B-200 sepharose. The yield of purified enzyme was 15.0 mg from 50 L of culture fluid. However, this method is complex and uses multi-stage inactivation of the pathogen using an antibiotic, thiolate and EDTA.

Vaccines for the prevention of meningococcal infections are described. So in the patent (RU 2333007 C2, PIZZA, NOVARTIS VEXINES AND DIAGNOSTICS S.R.L. (IT), 09/10/2008) a vaccine is described which, after administration, is able to induce a humoral immune response and protect the body against some highly virulent strains (A4, ET5) N. meningitidis serogroup B. Five protein antigens of the outer membrane are used, in particular: (1) NadA protein; (2) the protein "741"; (3) protein "936"; (4) protein "953" and (5) protein "287".

In another invention (RU 2343159 C2, MASINYANI et al., January 10, 2009), proteins with established amino acid sequences are described, obtained from bacteria mainly of strains A and B, which exhibit antigen properties. Proteins can be used as antigens for the production of specific antibodies and the manufacture of compositions for the treatment, prevention, or diagnosis of infection caused by N. meningitidis. Compositions prepared on the basis of a protein, nucleic acid fragments or antibodies with the addition of a pharmaceutically acceptable carrier are vaccine, diagnostic or pharmaceutical preparations.

The invention (RU 2361609 C2, GIULIANI et al., July 20, 2009) provides a vaccine composition having immunogenic activity against serogroups B and C of N. meningitidis containing an immunogenic amount of N. meningitidis serogroup C oligosaccharide, serogroup B meningococcus outer membrane protein b in the form of protein-lipid vesicles. The specified composition provides high titers of antibodies, determined by ELISA, and bactericidal antibodies, which may find application as a protective vaccine against N. meningitidis serogroups B and C.

However, for the manufacture of the above vaccines, recombinant products are used, the preparation of which requires a complex multi-stage process. At the same time, the use of meningococcal IgAl protease, which has been established to be an immunogenic and protective drug against meningococcal infection of heterologous serogroup B, is not known for the preparation of vaccine and drug components.

The objective of this group of inventions is to improve the method of producing meningococcal IgAl protease and the creation of an immunogenic drug based on it.

A method of producing an IgAl protease from N. meningitidis consists in growing a culture of N. meningitidis serogroup A strain A 208, followed by inactivation of the culture fluid and sedimentation of the microbial mass by centrifugation, isolation of the IgAl protease by purification, including ultrafiltration, absorption column chromatography on silochrome-sorbents 80 and phenyl-sepharose, concentration and freeze drying of the product.

The patented method is characterized in that a modified Franz nutrient medium is used to grow N. meningitidis serogroup A culture, microorganisms are inactivated by adding cetavlon to the culture fluid to a final concentration of 0.1%. The resulting mixture was separated by centrifugation, and both fractions — the cetavlon supernatant and the cetavlon precipitate — were used to isolate the IgAl protease. The resulting precipitate was extracted many times, the extracts of the precipitate and the supernatant were concentrated by ultrafiltration. The obtained concentrates were fractionated by absorption chromatography on a column with a silochrome-80 sorbent at a temperature of + 8 ° C balanced by 0.02 M phosphate-buffered saline, pH 7.0, whereby the IgAl protease was obtained by washing the sorbent with the indicated buffer solution.

The method may be characterized in that the precipitate is extracted with a buffer solution of 0.05 M Tris-HCl, pH 7.0 with stirring at a temperature of + 8 ° C, then the suspension is centrifuged at 11000 g at a temperature of + 4 ° C.

An immunogenic preparation containing an IgAl protease derived from a culture of N. meningitidis serogroup A, which is a vaccine component for the prevention of infection caused by bacteria of N. meningitidis serogroup B.

The technical result of the first invention of the group is the simplification of the method by replacing the multi-stage inactivation of the pathogen using an antibiotic, merthiolate and EDTA with a single-stage treatment with cetavlon, eliminating the stage of gel permeation chromatography and increasing the environmental safety of this process as a whole.

The technical result of the second invention of the group is to obtain a component of a vaccine of non-polysaccharide nature to protect against infection not only by meningococcus serogroup A, but also from infection by meningococcus serogroup B. This possibility was first established experimentally by the authors of the application and does not follow from the prior art.

The drawings show the cleaning results and characteristics of the obtained meningococcal IgAl protease, as well as the results of microbiological experiments:

figure 1 - chromatography results of the supernatant in the first stage;

figure 2 - chromatography results of the supernatant in the second stage;

figure 3 - the results of protein electrophoresis in PAG samples containing IgAl protease before and after chromatography;

figure 4 - the dynamics of the formation of antibodies to IgAl protease;

figure 5 - the protection of mice immunized with an IgAl protease from infection by meningococcus serogroup B;

Fig.6 - the level of bacteremia in mice immunized with an IgAl protease, 4 hours after infection with serogroup B meningococcus.

The method can be implemented as follows.

The microbial mass of N. meningitidis serogroup A strain A 208 (L.A. Tarasevich Institute for Standardization and Control of Medical and Biological Preparations) is grown in bottles containing Franz's modified semi-synthetic medium (see Frantz ID Growth requirements of the meningococcus. - J. Bacteriol. - 1942. - N6. - P.757-761; US 4123520, Hagopian et al., 10.31.1978; Kuvakina V.I. Meningococcal polysaccharide vaccines of groups A and C // Diss. Doct. Biol. Moscow Research Institute of Epidemiology and Microbiology named after G.N.Gabrichevsky, M., 1989).

The bottles with the culture fluid, which is a suspension of bacteria in Franz’s medium, are incubated at a temperature of 37 ± 1 ° C with constant shaking for 12-16 hours. Then the contents of the bottles are combined and a 10% solution of cetavlon (hexadecyltrimethylammonium bromide, cetavlon) is added to a final concentration of 0.1% in suspension, the suspension is thoroughly mixed and kept at room temperature for 2-3 hours. The resulting mixture is separated by centrifugation to obtain cetavlon supernatant and cetavlon sediment.

The indicated supernatant in an amount of 2.5 L (total protein content of 27500 mg) was concentrated 10 times by ultrafiltration on a Halipor-1 membrane with a nominal cut-off molecular weight of 65 kDa, and then several times washed with water from salts and reagents by diafiltration on the specified membrane. As a result, 35 ml of concentrate with a total protein content of 55 mg and an enzymatic activity of 0.775 thousand units / mg were obtained (see Table 1). Then, the resulting concentrate (35 ml) was dialyzed against the initial buffer solution I (see below) for chromatography on silochrome (2.0 L).

The resulting concentrate in an amount of 35 ml was further fractionated by two-stage column chromatography. In the first stage, this concentrate was applied to a column with a volume of 70 ml (30 × 130 mm) filled with a silochrome-80 sorbent and balanced with 0.02 M FSB, pH 7.0 (phosphate-buffered saline, initial buffer solution I). The column was washed with initial buffer solution I, then with 0.2 M sodium phosphate buffer, pH 8.2, and finally with 0.2 M glycine / NaOH / 0.3 M NaCl buffer, pH 9.2, elution rate 2 5 ml / min. Chromatography was performed at a temperature of + 8 ° C, fractions with a volume of 10 ml were collected, protein content and IgAl protease activity were determined in fractions in arbitrary units per mg of protein by enzyme-linked immunosorbent assay (RU 2310853, Kozlov et al., 20.11.2007). The results are presented in figure 1 and in table 1.

Fraction 1 obtained after the first purification step and containing an active IgAl protease (volume 35 ml, protein content 0.385 mg, enzymatic activity 47.3 thousand units / mg, protein yield 0.001%, purification 61) was purified by hydrophobic chromatography (second purification step) on a column (10 × 90 mm) with 5 ml phenyl-sepharose balanced by a buffer solution of 0.05 M Tris-HCl, ppH 9.2, containing 0.7 M ammonium sulfate (initial buffer solution II) . The specified fraction was concentrated by ultrafiltration to a volume of 2-3 ml, ammonium sulfate was added to it to a concentration of 0.7 M, and the resulting solution was applied to a column. The column was washed with the initial buffer solution and then with 0.05 M sodium acetate buffer solution, pH 6.0, until absorbance at λ = 280 nm. The IgAl protease was eluted by washing the column with demineralized water. The enzymatic activity of IgAl protease with high specificity was found only in fractions 1-3 (protein content of 0.012 mg, protein yield of 0.0004%, enzymatic activity of 640 thousand units / mg, purification degree of 824). The results are presented in figure 2 and in table 2.

The results of chromatography on silochrome (the first stage of purification) indicate a change in the properties of the enzyme compared with the properties described in the prototype article (V.I. Surovtsev et al. // Biotechnology, 2006, No. 3. - P.62-67), due to the presence of cetavlon, a surfactant, in the starting material. The isolated enzyme is not adsorbed on silochrome and elutes in the initial buffer solution, while a significant part of the impurities is adsorbed on silochrome and eluted in these solutions.

The experimental data presented show that an enzyme with a sufficiently high degree of purification, but with a low yield of IgAl protease, was obtained from the indicated amount of culture fluid (2.5 L). However, it is obvious to a person skilled in the art that the yield of the product can be improved by using more efficient ultrafiltration units, which can reduce the residence time of the enzyme in dissolved form.

A 103 g cetavlon precipitate obtained from 36 L of culture fluid was suspended in 2 L of 0.05 M Tris-HCl buffer, pH 7 and stirred overnight at + 8 ° C, then the suspension was centrifuged at 11000 g at + 4 ° C. The resulting supernatant was concentrated on a membrane and diafiltered as described above. The concentrate (40 ml, 10.9 thousand units / mg) was dialyzed against 2 L of 0.02 M PBS, pH 7, overnight at a temperature of + 8 ° C, then the enzyme was purified by two-stage column chromatography as described above for cetavlon supernatant. The precipitate was extracted a total of five times and an enzyme was isolated from each extract as described above. The results are shown in table 2.

It was found that as a raw material for the production of IgAl protease can also be used ready-made intermediates - cetavlon supernatant and sediment obtained after inactivation of the microbial culture in the production of polysaccharides of meningococcal serogroups A and C (RU 2283135 C1, Avakov and Alliluyev, 09/10/2006 ) The use of a supernatant is all the more advisable since it is a waste product at the Bacterial Production Plant named after G.N.Gabrichevsky, in no way used and merged into the garbage. The advantage of the precipitate is that it can be stored frozen at a low temperature of -80 ° C for a long time and used both for the production of polysaccharide meningococcal vaccines and purified IgAl protease. In addition, approximately 24% of the IgAl protease activity is calculated as the total IgAl protease activity in the starting culture fluid.

IgAl protease preparations obtained during purification were analyzed by polyacrylamide gel electrophoresis in Na dodecyl sulfate (EF in SDS page - SDS, reducing conditions, manifestation by silver - see Fig. 3), and enzymatic activity was determined (see table 1, 2) by enzyme immunoassay of an IgAl protease.

Figure 3 presents the results of protein electrophoresis in PAG samples containing IgAl protease before and after chromatography. Cetavlon supernatant: lane 1 - source, 2 - fr. 1; 3 - fr. 3 (phenyl-sepharose). Cetavlon sediment: 4 - initial extract, 5 - fr. 1; 6 - fr. 2; 7 - fr. 3 (silochrome); 8 - fr. 1-1; 9 - fr. 1-2; 10 - fr. 1-3 (phenyl-sepharose). Lane 11 is a control mixture of HMW marker proteins with molecular weights of 250, 150, 100, 75, 50, 37, 25, 30, 15, and 10 kDa.

In the purified IgAl protease preparation (lane 3), a zone with a molecular weight of about 120 kDa is observed, which corresponds to published data on the molecular weight of the intact enzyme [see prototype, as well as Taimurazov MG Candidate dissertation. biol. Sciences., State Scientific Center for Applied Microbiology and Biotechnology, Obolensk, 2006], as well as a certain amount of impurities at the level of 50 kDa.

The results of electrophoresis indicate that the obtained preparation of the present invention contains IgAl protease with a molecular mass of 120 kDa and low molecular weight impurities in much smaller quantities. The presence of such impurities can be explained by the fact that, according to published data (see, for example, WO 9011367), the IgAl protease can be cleaved into lower molecular fragments during the purification process, which also have enzymatic activity.

The immunogenic and protective activity of the obtained IgAl protease was evaluated in Balb / C mice. To evaluate these parameters, animals were immunized with an IgAl protease twice intravenously at an interval of 45 days. For the first immunization of mice at a dose of 10 μg / mouse, a preparation was used obtained after the second extraction of cetavlon sediment and chromatography on C-80 silochrome, dialysis and freeze drying (100 μg IgAl protease, with enzymatic activity of 3 · 10 ⁵ units / mg) . For mice reimmunization, a preparation obtained after two-stage chromatography (195 μg IgAl protease with activity of 5.36 × 10 ⁵ units / mg) in the same dose was used.

An indicator of the immunogenicity of the obtained IgAl protease was the increase in specific antibodies determined by ELISA in the blood of mice on various days after immunization.

On the 12th day after reimmunization, mice were infected with a live virulent culture of meningococcus serogroup B strain H44 / 47.

The protective activity of IgAl protease was evaluated by the level of bacteremia in immunized animals 4 hours after infection with serogroup B meningococcus, as well as by the number of animals surviving on the 5th day after infection, compared with the same parameters for control non-immunized mice.

Figure 4 shows a diagram of the immunogenic activity of IgAl protease. On the abscissa, the numbers indicate the period after immunization: 1 - 6th day; 2 - 13th day; 3 - 20th day; 4 - 28th day; 5 - 35th day and 6 - 41st day after the first immunization, respectively; 7 - 12th day after re-immunization; 8 - control group. The ordinate axis is the optical density of the sera at a dilution of 1:80 at λ = 492 nm. It is seen that an increase in specific antibodies is observed on the 13th day after a single immunization and reaches its maximum value by the 35th day. The maximum level of antibodies was observed on the 12th day after reimmunization.

The diagram of figure 5 presents the level of bacteremia (in%) in immunized mice (shaded bar) compared with control, non-immunized animals (black bar). The bacteremia level in the group of immunized mice was reduced by 35% (65 CFU) compared with control non-immunized mice (100 CFU).

The diagram of Fig. 6 shows the protection of mice from infection by serogroup B meningococcus. The ordinates show the number of surviving mice (in%): immunized mice — shaded column, control — black. When mice were infected with meningococcus at a dose of 25 · 10 ⁴ microbial cells in the control group, only one animal out of 10 (10%) survived, while in the group immunized with IgA-protease, 9 mice survived (90%).

The obtained results indicate the high efficiency of the IgAl protease preparation isolated from serogroup A meningococcus culture and capable of protecting animals from infection by the living meningococcal culture of the heterologous strain - serogroup B meningococcus. In addition, the obtained preparation can be used to protect homologous serogroup A from meningococcus.

Thus, the experimental data presented show the promise of a patented method for preparing the active component of a multivalent vaccine against meningococcal infection based on IgAl protease for the prevention of the main epidemiologically significant pathogens of meningococcal infection. In addition, the resulting substrate can be used as an enzyme for experimental purposes.

Table 1 CHARACTERISTICS OF CETAWLON SUPERNANT CLEANING PRODUCTS AT DIFFERENT STAGES Stages of cleaning Volume ml Protein IgAl protease activity mg % Specific activity, thousand units / mg The degree of purification * Source supernatant 2500 27500 one hundred Supernatant Concentrate 35 55 0.2 0.775 1,0 Chromatography on silochrome C-80 Fraction 1 pH 7.0 35 0.385 0.001 47.3 61 Fraction 2 pH 8.0 45 0.540 0.002 0 Fraction 3 pH 9.0 one 0.012 0.0001 0 Chromatography on phenyl-sepharose fraction 1 Concentrate fraction 1 before chromatography 2.5 0.385 0.0014 47.3 61 Fraction 1-1 pH 9.2 5 0.2 0,0007 0 Fraction 1-2 pH 6.0 6 0.1 0,0004 0 Fraction 1-3 H ₂ O 3 0.012 0,0004 638.9 824 * the ratio of the specific activity of the purified enzyme to the specific activity of the starting concentrate

Claims (3)

1. A method of producing an IgAl protease from N. meningitidis, which consists in growing a culture of N. meningitidis serogroup A strain A 208, followed by inactivation of the culture fluid and sedimentation of the microbial mass by centrifugation, isolation of the IgAl protease by purification, including ultrafiltration, absorption column chromatography on silochrom-80 sorbents and phenyl-sepharose, concentration and freeze drying of the product,
characterized in that
for the culture of N. meningitidis serogroup A, a modified Franz culture medium is used,
the inactivation of microorganisms is carried out by adding cetavlon to the culture fluid to a final concentration of 0.1%,
after separation of the resulting mixture by centrifugation, both fractions — the cetavlon supernatant and the cetavlon precipitate — are used to isolate the IgAl protease,
the precipitate obtained is extracted many times, the precipitate extracts and the supernatant are concentrated by ultrafiltration,
The obtained concentrates are fractionated by absorption chromatography on a column with a silochrom-80 sorbent at a temperature of + 8 ° С balanced with 0.02 M phosphate-buffered saline, pH 7.0, whereby the IgAl protease is obtained by washing the sorbent with the indicated buffer solution. 2. The method according to claim 1, characterized in that the precipitate is extracted with a buffer solution of 0.05 M Tris-HCl, pH 7.0 with stirring at a temperature of + 8 ° C, then the suspension is centrifuged at 11000 g at a temperature of + 4 ° C. 3. A preparation containing an IgAl protease obtained from a culture of N. meningitidis serogroup A by the method according to any one of claims 1 and 2, which is a vaccine component for the prevention of infection caused by bacteria of N. meningitidis serogroup B.

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