RU2311197C1 - Method for preparing protective protein-containing fraction of microorganism - Google Patents

Abstract

FIELD: medicine, microbiology, preparative biochemistry, proteins.

SUBSTANCE: invention relates to vaccine preparations and to a method for preparing protective antigens based on secreting protein-containing fractions of microorganisms. Method involves culturing microorganisms on liquid nutrient media, separation of cultural medium from cells, purification of medium and concentrating by ultrafiltration with the removal threshold of protein of molecular mass 30 kDa and column chromatograpy by gel-filtration on Sephadex G-75 followed by purification on DEAE-Sepharose in gradient of concentration of NACl yielding fractions of molecular mass 30-35 kDa containing protein in the concentration 4-5 mg/ml. Method provides preparing immune preparation eliciting prophylactic and curative properties.

EFFECT: improved preparing method.

2 cl, 8 tbl, 5 dwg

Description

The invention relates to medicine, namely to vaccine preparations, and relates to a method for producing protective antigens based on secreted protein-containing fractions of bacteria, which can be used to obtain prophylactic and therapeutic immunopreparations.

The problem of bacterial infections over the years is one of the most important problems of medical science and healthcare. For the prevention of these diseases, in addition to toxoids, a large number of vaccines are used, which are designed on the basis of microbial cell antigens. These vaccines are characterized by varying degrees of protective activity and strict serotypic specificity.

A known method of obtaining protective prophylactic drugs (toxoids), consisting of protein-containing fractions (toxins) secreted by toxin-forming bacteria; further processing of the culture medium containing the toxin is carried out by neutralizing the toxin with formalin, concentration and purification of ballast substances.

(Anatoxins. A Guide to the Vaccine and Serum Cases, edited by P.N. Burgasov, 1978, pp. 103-157.)

The disadvantage of this method is the inability to obtain protective secreted drugs when using non-toxigenic bacteria strains.

The technical result to which this invention is directed is to create a universal method for producing bacterial antigens from any kind of bacteria based on secreted protein-containing fractions of various types of bacteria characterized by high intraspecific cross-protective activity.

To achieve the technical result in a method for obtaining a protective protein-containing fraction of bacteria, including culturing bacteria on liquid nutrient media, separating the culture medium from bacteria, according to the invention, the culture medium containing secreted protein-containing fractions is purified and concentrated by ultrafiltration with a protein cut-off threshold of 30 kDa and column chromatography using gel filtration on Sephadex G-75 and subsequent purification on DEAE-Sepharose in a gradient of NaCl d about obtaining fractions with a molecular weight of 30-50 kDa and a protein content of 4-5 mg / ml.

To obtain protective antigens based on secreted protein-containing fractions, non-toxigenic strains of gram-negative or gram-positive microorganisms are used.

The invention is illustrated by the following examples.

Example 1. A method of obtaining a protective secreted protein-containing antigens of Klebsiella K2, comprising the following stages:

a) the cultivation of Klebsiella K2 in a liquid semi-synthetic nutrient medium.

b) inactivation of microbial mass;

c) separation of the supernatant, centrifugation;

d) concentration and purification of the supernatant by column chromatography;

d) fractionation by molecular weight using ultrafiltration with a cutoff threshold of protein 30 kDa.

The strain K. pneumoniae K2 (B5055) from the International Standard Klebsiella Collection, which is in a lyophilized state in ampoules, is seeded in 50 ml of 0.2% sugar broth for 4 hours, then on a semi-synthetic liquid medium of the following composition:

KN ₂ RO ₄ - 4.50 g / l

K ₂ NRA ₄ - 5.00 g / l

(NH ₄ ) ₂ SO ₄ -1.00 g / l

MgSO ₄ × 7H ₂ O - 0.30 g / l

Sodium Citrate - 0.50 g / L

Glucose - 10 g / l

Aminopeptide -103.0 ml

The cultivation of K. pneumoniae strains is carried out in a fermenter, for example, Ankum firm with continuous stirring and air supply in a volume of 10 l at pH 7.8 t = 37 ° C for 5-6 hours. The concentration of microorganisms is (8.0 ± 1.0) · 10 ⁹ microbial cells / ml. The microbial suspension is inactivated with sodium azide. The completeness of inactivation of microbial cells is controlled by plating on nutrient agar. Above the sedimentary liquid, they are separated from microbial cells by centrifugation at 6,000 g for 30 minutes at 4 ° C, as with all subsequent procedures. Ammonium sulfate was added to the resulting supernatant to 80% saturation according to the method of N. Koide et al. The precipitate was separated by centrifugation at 10,000 g for 30 minutes, dissolved in 50 ml of phosphate buffer pH 7.4, ultrafiltered with a cut-off threshold of 30 kDa protein and fractionated by gel filtration on Sephadex G-75 (Pharmacia, Sweden). Subsequent purification of the protein-containing fraction is carried out on DEAE-Sepharose in a NaCl gradient (from 0 to 50 M NaCl) to obtain fractions with a mol. weighing 30-50 kDa and a protein content of 4-5 mg / ml.

The belonging of the obtained fractions to protein-containing compounds was determined by immunoblotting. After electrophoretic separation of the studied fraction by molecular weight, hyperimmune serum of mice immunized with a preparation based on the indicated fraction (primary antibodies) was added to them, and then labeled antibodies to mouse immunoglobulins (secondary antibodies) were added. Precipitation bands when testing fractions containing immunologically active compounds were detected in the region of 35-40 kDa (Fig. 1).

The belonging of the studied fractions to secreted proteins of K. pneumoniae was determined by flow cytometry. The method is based on the processing of microbial cells with 0.5% paraform in 0.9% phosphate-buffered saline (FSB) and is recommended for fixing bacterial or mammalian cells when working on a flow cytometer. Before setting up the reaction, microbial cells were washed three times by centrifugation at 800 g in PBS without paraform. The washed cells were transferred in a volume of 200 μl to special tubes (50 · 10 ³ cells per tube) and 10, 50 or 100 μl of serum (primary antibodies) obtained after hyperimmunization of mice with the studied protein fractions were added. An appropriate amount of FSB buffer or serum from intact mice was added to control tubes (negative control). As a positive control, the blood serum of mice surviving after infection with a strain of K. pneumoniae K2 was used. Samples were incubated for 1 hour at 37 ° C. After that, 3 ml of an isotonic sodium chloride solution was added to each tube and the serum was washed three times by centrifugation at 800 g. The cells were then resuspended in 200 μl of FSB and secondary FITC-labeled rabbit antibodies against mouse immunoglobulins introduced (Caltag Labs, USA). The tubes were again incubated for 30 min at 37 ° C. The results were taken into account using a FacsCalibur flow cytometer (Becton Dickinson, USA). The level of fluorescence isolated in the gate of the microbial cell population in the FL-1H channel was measured. The geometric mean cell fluorescence intensity (Gm) was calculated using a generally accepted WinMdi 2.8 flow cytometer for data processing and expressed in arbitrary units (cu).

On figa shows the dot-plot of the microbial cells of K. pneumoniae. It is seen that the selected population of microbial cells is a homogeneous cell population, forming a clear "cloud". The fact of isolation of such a homogeneous population allows us to consider that the method used to isolate microbial cells does not cause their destruction or adhesion and, therefore, the cell population is suitable for analysis. On figb shows a histogram of the glow of the original population of microbial cells in the channel FL-1H. It is seen that the initial population of microbial cells had a low level of fluorescence in the presence of secondary FITC-labeled antibodies. The addition of hyperimmune serum containing antibodies to secreted protein-containing fractions with a molecular weight of 30-50 kDa in the presence of secondary FITC-labeled antibodies also did not increase the fluorescence of microbial cells (Fig. 2B). This indicates a lack of binding of these antibodies to the microbial cell preparation. The same results were obtained for the protein fraction with a molecular weight of 21 kDa. At the same time, the addition of blood serum from sick animals containing antibodies to microbial cell antigens was accompanied by increased fluorescence in the presence of secondary FITC-labeled antibodies (the average level of Gm fluorescence increased from 3 to 150 cu) (Fig. 2G).

The protein content is 4-5 mg / ml.

Determination of antigenic and immunogenic activity of the obtained protein-containing fractions. To immunize animals, protein-containing fractions of strain K2 are pre-sorbed on aluminum hydroxide. To assess the protective activity of the obtained fractions, immunization of mice is carried out 2 times with an interval of 2 weeks. Blood serum from immunized animals for the study of antigenic activity, preventive properties and other tests receive 14 days after the last immunization.

To study the protective activity of protein-containing fractions sorbed on aluminum hydroxide, mice were immunized with two 10-fold decreasing doses of the preparations and infected with K. pneumoniae K2 strain at a dose of 500 microbial cells, which corresponded to 178 LD ₅₀ (Tables 1, 2). Contagious dose control was performed on intact animals. The protein-containing fraction with a molecular weight of 21 kDa was found to be low immunogenic. The protein-containing fraction with a molecular weight of 34-35 kDa was characterized by high protective activity, which had a dose-dependent nature. Protection against infection during immunization with an immunizing dose (10 μl) was 70%.

Protein-containing fractions with a molecular weight of 30-50 kDa with a protein content of 4-5 mg / ml, obtained from strain K16 and non-typable 204, had intraspecific cross-protective activity upon infection with strain K2 (Table 2, groups 3.4). In turn, the preparation from strain K2 protected mice from infection with strains K16 and 204 (Table 3).

The protein-containing fraction of K. pneumoniae K2 with a molecular weight of 30-50 kDa in the homologous system had protective activity without adjuvant (Table 4). The multicomponent bacterial vaccine Immunovac-VP-4, consisting of the antigens of the microbial cells Klebsiella pneumoniae, Proteus vulgaris, Escherichia coli and Staphylococcus aureus, containing as its main component LPS, associated with the outer membrane protein of gram-negative microorganisms, was used as the reference drug. peptidoglycan, teichoic acids and labile protein antigens of S. aureus. The drugs were administered according to a single scheme.

The protective activity of the 30–50 kDa protein-containing fraction was higher than Immunovac-VP-4 (it contains the antigens of the microbial cell of the heterologous strain K. pneumoniae 204, which has interspecific cross-protective activity).

Using the solid-phase ELISA, it was found that when immunizing mice with a fraction of 21 kDa, an increase in the level of antibodies does not occur or they have low affinity (Table 5). When immunizing mice with a fraction of 30-50 kDa, an increase in the level of antibodies was revealed, depending on the size of the immunizing dose. Cross antigenic activity between the studied fractions was not detected, which indicates a good degree of purification of the studied drugs.

Hyperimmune serum obtained after 4-fold vaccination of mice with a protein containing fraction of 30-50 kDa in a dose of 50 μl, administered to mice 2 hours, 1 day and 2 days after infection, protected them from lethal Klebsiella infection caused by K.pneumoniae K2. The serum obtained by immunizing mice with a fraction of 21 kDa was ineffective (Table 6).

Example 2. The protective activity of secreted protein-containing fractions of pneumococcus.

Protein-containing fractions were obtained as in example 1.

When immunizing mice with a fraction of 10-30 kDa, a weak protective effect was revealed (IE 1.99), which practically did not differ from the control (Table 7). A significant effect was obtained by immunizing mice with a fraction> 30 kDa (IE 8.9).

Next, fractionation of the supernatant obtained after cultivation of S. pneumoniae type 3 into a fraction of 30-50 kDa and more than 50 kDa was carried out (Table 8). The fraction with a molecular weight of 30-50 kDa, IE = 50.1 was characterized by the highest protective activity. A fraction with a molecular weight of more than 50 kDa was less immunogenic, IE = 7.9.

An advantage of the invention is the creation of a universal method for producing protective antigens based on secreted protein-containing fractions of gram-negative and gram-positive bacteria, upon infection of which toxin formation is not the leading pathogenetic mechanism in the development of the disease.

Table 1 The protective activity of drugs from the strain Klebsiella pneumoniae K2 when infected with a strain of K. pneumoniae K2 at a dose of 178 LD ₅₀ Group number A drug Immunizing dose, μl Infecting Dose, mic. cells Survived ED ₅₀ μl Abs. % ± m one Protein-containing fraction of K. pneumoniae K2 with a molecular weight of 21 kDa one 500 1/10 10.0 ± 9.5 > 10.0 10 500 0/10 0 2 The protein-containing fraction of K. pneumoniae K2 with a molecular weight of 34-35 kDa one 500 4/10 40.0 ± 15.5 * 2,5 10 500 7/10 70.0 ± 14.5 * 3 Aluminum hydroxide one 500 0/10 0 > 10.0 10 500 0/10 0 four Unvaccinated (control) - 500 0/10 0 Note: ^{1) the} significance of the difference between the percentage of surviving animals in the experimental groups and in the control, * p <0.01

table 2 Direct and cross-protective activity of drugs from the strain Klebsiella pneumoniae K2, K16 and 204 when infected with a strain of K. pneumoniae K2 at a dose of 15.8 LD ₅₀ Group Protein-containing fraction of K. pneumoniae Molecular weight, kDa Immunizing dose, μl The infectious dose, micr. cells Survived ED ₅₀ μl Abs. % ± m one K2 21 one 500 0/10 0 > 100 10 500 0/10 0 one hundred 500 3/10 30.0 ± 14.5 2 K2 34-35 one 500 1/10 10.0 ± 9.5 3.0 10 500 10/10 100.0 ± 6.0 ** one hundred 500 9/10 90 ± 9.5 ** 3 K16 34-35 one 500 3/10 30.0 ± 14.5 60.0 10 500 3/10 30.0 ± 14.5 one hundred 500 1/10 10.0 ± 9.5 four 204 34-35 one 500 1/10 10.0 ± 9.5 25.0 10 500 5/10 50.0 ± 15.8 * one hundred 500 5/10 50.0 ± 15.8 * 5 Unvaccinated (control) - 500 1/10 10.0 ± 9.5 Note: ^{1) the} significance of the difference between the percentage of surviving animals in the experimental groups and in the control, * p <0.05, ** p <0.01

Table 3 Direct cross-protective activity and preparations from the strain Klebsiella pneumoniae K2, K 16 and 204 K.pneumoniae when infecting strain K at a dose of 16 2,3 LD ₅₀ Group Protein-containing fraction of K. pneumoniae Molecular weight, kDa Immunizing dose, μl Infecting dose, × June ¹⁰ md. cells / ml Survived ED ₅₀ μl Abs. % ± m one K2 34-35 one 20,0 4/10 40.0 ± 15.5 8.0 10 20,0 6/10 60.0 ± 15.5 * one hundred 20,0 6/10 60.0 ± 15.5 * 2 K16 34-35 one 20,0 3/10 30.0 ± 14.5 8.0 10 20,0 6/10 60.0 ± 15.5 * one hundred 20,0 7/10 70.0 ± 145 ** 3 204 34-35 one 20,0 3/8 37.5 ± 15.3 2,5 10 20,0 6/8 75.0 ± 13.7 ** one hundred 20,0 8/8 100.0 ** four Unvaccinated (control) - 20,0 1/10 10.0 ± 9.5 Note: ^{1) the} significance of the difference between the percentage of surviving animals in the experimental groups and in the control, * p <0.05, ** p <0.01

Table 4 The protective activity of the secreted K.pneumoniae K2 protein fraction with a molecular weight of 34-35 kDa not adsorbed on aluminum hydroxide at a dose of 2 LD ₅₀ No. A drug Multiplicity of drug administration Immunizing dose Infecting Dose, mic. cells Survived / Total % ± m Reliability of the difference between experience and control, p one Protein-containing fraction of K. pneumoniae K2 with a molecular weight of 34-35 kDa, not sorbed on aluminum hydroxide 2 40.0 μl 25.0 9/9 100.0 * <0.001 2 Multicomponent vaccine Immunovac-VP-4 ²⁾ 2 200 mcg 25.0 6/10 60.0 ± 15.5 0.05 Unvaccinated (control)  -  - 25.0 2/10 20 ± 12.6 Note. ¹⁾ The formulations were administered 2-fold, alternating between intraperitoneal and subcutaneous administration at intervals of 14 days. Infection was carried out with a strain of K. pneumoniae K2 at a dose of 25 microbial cells on day 12. * The significance of the difference between the groups of vaccinated mice (groups 1 and 2), p <0.05

Table 5 Antigenic activity of secreted protein-containing antigens of K. pneumoniae K2 with a molecular weight of 34-35 kDa in ELISA The molecular weight of the protein fraction for sorption on tablets and immunization of mice, kDa The immunizing dose of the investigated protein fraction, μl / mouse The level of antibodies in the blood serum of mice immunized with fractions with a molecular weight ..., kDa OP ₄₉₂ Δ OP 34-35 one 0.320 ± 0.022 0,066 10 0.368 ± 0.029 0.114 one hundred 0.601 ± 0.152 *** 0.347 Unvaccinated 0.254 ± 0.016 Note. * OD ₄₉₂ - optical density at a wavelength of 492 nm; ** Δ OD - difference in serum OD between vaccinated and unvaccinated mice * * * Reliability of the difference in the level of antibodies with the sera of vaccinated and unvaccinated mice p <0.001

Table 6 Therapeutic activity of anti-Klebsiella serum in experimental infection caused by K. pneumoniae K2 No. Serum of mice immunized with a protein containing molecular weight fraction The frequency of administration of serum Infectious dose, microbial cells Survived / Total % ± m one 21 kDa 2 500 0/10 0 2 34-35 kDa 3 500 7/10 70.0 ± 14.5 * 3 Unvaccinated - 500 0/10 0 * Reliability of the difference p ₂ and _1.3 <0.05

Table 7 The protective activity of the preparations from the strain of serotype 3 pneumococcal infection when S.pneumoniae type 3 at a dose of 10,1 LD ₅₀ Group number Protein-containing fraction, S. pneumoniae type 3 immunizing dose 100 μl Infecting Dose, mic. cells Survived / Total LD ₅₀ , mic. cells IE one 10-30 kDa, 6 hours of cultivation 7 × 10 9/10 1380 1.99 7 × 10 ² 7/10 7 × 10 ³ 2/10 2 > 30 kDa, 6 hours of cultivation 7 × 10 10/10 6165 8.9 7 × 10 ² 10/10 7 × 10 ³ 4/9 3 Unvaccinated (control) 7 × 10 9/10 692 - 7 × 10 ² 3/10 7 × 10 ³ 3/10 Note: IE - performance index - ratio of LD ₅₀ in the experiment to LD ₅₀ in the control;

Table 8 The protective activity of drugs from the pneumococcal strain of serotype 3 upon infection with S. pneumoniae type 3 at a dose of 12.6 LD ₅₀ . Group number Protein-containing fraction, S. pneumoniae type 3, immunizing dose 100 μl Infecting Dose, mic. cells Survived / Total LD ₅₀ , mic. cells IE one 30-50 kDa 10 ³ 8/10 39810.7 50.1 10 ⁴ 6/10 10 ⁵ 7/10 2 > 50 kDa 10 ³ 4/10 6309.6 7.9 10 ⁴ 5/10 10 ⁵ 4/10 3 Unvaccinated (control) 10 ³ 4/10 794.3  - 10 ⁴ 0/10 10 ⁵ 0/10 Note: IE - performance index - ratio of LD ₅₀ in the experiment to LD ₅₀ in the control;

Claims (2)

1. A method of obtaining a protective protein-containing fraction of bacteria, including cultivating bacteria on liquid nutrient media, separating the culture medium from bacteria, characterized in that the culture medium containing secreted protein-containing fractions is purified and concentrated by ultrafiltration with a protein cut-off threshold of 30 kDa and column chromatography using gel filtration on Sephadex G-75 and subsequent purification on DEAE-Sepharose in a NaCl gradient to obtain fractions with a molecular weight of 30-50 kDa and Protein holding 4-5 mg / ml. 2. The method according to claim 1, characterized in that to obtain protective antigens based on secreted protein-containing fractions, non-toxigenic strains of gram-negative or gram-positive microorganisms are used.

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