RU2708561C1 - Method of obtaining a brucellarotic antigen for cell tests in vitro - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biotechnology. Disclosed is a method for producing a bruclease antigen. Method involves cultivation of Brucella abortus 19 BA on a dense nutrient medium followed by water-salt extraction with 2.5 % NaCl 1:1 solution for one day, destruction of microbial cells on a hydraulic press, centrifugation and lyophilization of the supernatant.

EFFECT: invention provides staging of antigen-stimulated cell responses in vitro for assessing the formation of postvaccinal immunity against the brucellosis agent.

1 cl, 1 tbl, 3 ex

Description

The invention relates to medicine and biotechnology, in particular to a method for producing a highly active antigen from a vaccine strain of Brucella (B.) abortus 19 VA and can be used to formulate antigen-stimulated cell reactions in vitro to evaluate the formation of post-vaccination immunity against the causative agent of brucellosis.

To study specific cell-mediated immunity against the causative agent of brucellosis, a skin-allergological reaction with brucellin was previously proposed (Bürne test). But due to the high risk of developing general and local adverse reactions, the development of antigen-specific in vitro methods is currently underway, which allows us to assess the formation and intensity of cellular immunity in in vitro tests. Researchers have demonstrated the high efficiency of applying methods of antigenic activation of leukocytes in vitro using flow cytometry technology to study the formation of post-vaccination immunity [1, 2, 3, 4].

In order to develop the most diagnostically informative methods for setting up antigen-stimulated cell tests in vitro, careful selection of a stimulating agent (antigen) is required, which has sufficient activating potential for T-lymphocytes and ensures the specificity of the reaction in vitro.

There is currently no commercially available brucellosis antigen for in vitro cell tests.

A known method of producing an antigen intended for the formulation of serological reactions (indirect hemagglutination reaction and complement fixation) to detect anti-brucellosis antibodies. The method includes culturing the causative agent of brucella in a liquid nutrient medium, followed by isolation of the target product. Cultivation is carried out at a temperature of 39-40 ° C for 18-24 hours, followed by a temperature increase to 45-50 ° C for 6-10 hours and a temperature increase to 60 ° C for 1-2 hours with further separation of the back mass and isolation Brucellosis antigen is obtained from the culture medium by ultrafiltration or chromatography, which is a substance in which sugars are determined by the Dubois method up to 40%, proteins by the Lowry method up to 30% and nucleotides by a spectrophotometric method up to 20% with a molecular weight of 10-20 kDa [5 ]. However, the use of this method allows to obtain the brucella antigen, intended only for the formulation of immuno-serological reactions, with which you can evaluate the level of antibodies in animals in response to the introduction of anti-brucellosis vaccines.

A known method of producing brucellosis antigen from strain B. abortus 19 VA for the manufacture of a single brucellosis antigen for RA, CSC and RDSK, including obtaining seed (B. abortus strain 19 VA) on a solid nutrient medium, its deep cultivation in a liquid nutrient medium containing Hottinger digest, glycerin, glucose, NaCl, yeast extract and water, concentration and purification of the grown Bakmass on an ultrafiltration unit using separation apparatuses using 0.5% phenolized physiological solution, and inactivation of the antigen in the bioreactor at a temperature of 80 ° C for an hour [6]. However, the method used to concentrate the bacterial mass on ultrafiltration columns is not efficient enough, energy- and labor-intensive, includes the collection and sterilization of the ultrafiltration unit, and requires continuous monitoring of the filtration process. In addition, when using an ultrafiltration column there is a risk of loss of back mass and its contamination by extraneous microflora due to rupture of the cassette fibers.

There is also a known method of manufacturing brucellosis antigen for "Rose Bengal Sample" (RBP). The method includes obtaining seed and industrial cultivation of strain B. abortus 19 VA in a liquid nutrient medium containing Hottinger digest, glycerin, glucose, NaCl, yeast autolysate and water. The cultivation is carried out in deep conditions for 19-21 hours with the regulation of the level of partial pressure of oxygen dissolved in the culture fluid throughout the entire cultivation process. The grown bacterial cells are separated and concentrated by ultrafiltration on fibers with a retention limit of 15-50 kD. Inactivate by heating. Stained with rose bengal, followed by resuspension in a buffer diluent and standardization of the target product [7]. However, the disadvantages listed above are also characteristic of this technical solution.

All antigens obtained by the above methods are complex substances of the surface layer of the brucella cell wall and are used to develop diagnostic drugs designed to detect specific antibodies.

The closest analogue is the method for producing brucellosis antigen proposed by E.N. Afanasyev (1983), which consists in the cultivation of different types of brucella strains on a solid nutrient medium, followed by inactivation of the back mass with a double volume of cold (minus 30 ° C) acetone at 4 ° C, 48 hours. Then it is washed twice with physiological saline by centrifugation at 4000g for 20 minutes. The biomass disinfected and tested for specific sterility is extracted with a 2.5% NaCl solution for 48 hours. It is then centrifuged at 4000 g for 60 minutes. After centrifugation, (I) the supernatant is separated from the precipitate. Microbial cells in the sediment destroy the X-press of the disintegrator in the chamber. In the next step, the biomass is subjected to ultrasonic disintegration and centrifugation at 20,000 g for 60 minutes. After centrifugation, the (II) supernatant is separated from the precipitate. The precipitate was extracted with 2.5% NaCl solution, followed by centrifugation at 20,000 g for 60 minutes. The precipitate formed is removed. All obtained supernatants are combined and dialyzed against distilled water for 48 hours. The resulting antigen is lyophilized. The amount of protein is 25-28 mg / ml [8].

All the above methods make it possible to obtain an antigen intended solely for the production of serological reactions (antibody search).

To test the basophil activation test in vitro, the researchers proposed the use of the brucellosis liquid allergen - “Brucellin” (produced by NPO Microgen) [9].

However, preliminary studies indicated a low specific activity of brucellin in relation to the activation of T-lymphocytes. Considering that cellular immunity is the leading one in immunological protection against brucellosis infection, the low activity of brucellin against T cells does not allow us to evaluate the specific reactivity of cell factors of specific resistance after immunization against brucellosis.

The objective of the invention is to obtain an antigenic complex from brucella, by an effective method of isolation of specific cell components from the bacterial masses of the causative agent of brucellosis for use in cell tests in vitro to assess the specific reactivity of cellular immunity after vaccination against brucellosis (assessment of the immunological effectiveness of vaccination).

The problem is solved by obtaining crops B. abortus 19 VA IV generations with the accumulation of biomass; disinfecting microbial mass with acetone chilled to minus 40 ° С; drying the microbial mass; isolation from the microbial mass of the active substance - brucellosis polysaccharide-protein complex (BPBC), followed by bottling, freezing, freeze-drying, sealing and labeling of the ampoules with the obtained product.

At the stages of preparation of seed crops, a check is made for the absence of foreign microorganisms and fungi, the concentration of microbial cells is determined, the specific sterility of the disinfected bacterial mass of brucella is monitored; at the stage of preparation of BSPK, protein concentration, pH, activity, specificity are determined.

Work with the culture of strain B. abortus 19 VA is carried out in compliance with SP 1.3.2322-08 “Safety of work with microorganisms of III-IV pathogenicity (danger) groups and pathogens of parasitic diseases”.

The technical result of the invention is that the culture is sown on Albimi agar, poured into Petri dishes and grown at a temperature of (37 ± 1) ° C, (48 ± 1) h (2 generation). Then produce reseeding on the jambs of Albimi agar poured into test tubes, grown at a temperature of (37 ± 1) ° С, (48 ± 1) h (3 generation). The culture from mowed agar in the tubes was washed off with a 0.9% NaCl solution and inoculated on bacteriological mattresses with Albimi agar, grown (72 ± 1) h at a temperature of (37 ± 1) ° С (4th generation). To disinfect the brucellosis microbe, cold (minus 40 ° C) acetone is used. The biomass of brucellosis microbes disinfected and tested for specific sterility is poured into Petri dishes for 18-20 hours to evaporate acetone, obtaining a dry biomass into which a 2.5% NaCl solution 1: 1 is added for extraction, per day. It is then centrifuged at 12,000 g for 40 minutes. The precipitate obtained after centrifugation of biomass and dissolved in a 0.9: 1% NaCl solution of pH 7.2 in a 1: 1 ratio is placed in a chamber of a hydraulic X-press of cell disintegrator (X-25 Press LKB, Sweden), pre-cooled for 3 h at a temperature of minus 40 ° C. Microbial cells in the disintegrator are frozen at the same temperature for 12 hours. After this, the cells are destroyed in a hydraulic press, forcing the biomass four times through the calibrated hole of the disintegrator cartridge. Next, the biomass is thawed and centrifuged at 12,000 g for 40 minutes. In the supernatant receive BPBC.

The degree of destruction of microbial cells was evaluated by light microscopy (UV. 1000). The activity of BSPK was controlled in the immunodiffusion reaction (RID), which was 1: 128, the pH of the solution was 7.2 ± 0.2. The total protein concentration in the test samples was 536 μg / ml (the protein content was quantified by specific fluorescence (Qubit 2.0 fluorimeter, LifeTechnologies, USA). The qualitative composition of the BSPC hydrolyzate was also determined by ion exchange chromatography with refractometric detection. The following were typical for the antigen. monosaccharides - rhamnose and D-glucose The obtained antigen was lyophilized to increase its shelf life, which made it possible to transport it without observing cold single chain.

The specific activity of the obtained brucellosis antigenic complex was evaluated in cell tests in vitro. We used white laboratory mice (n = 50) immunized with a brucellosis vaccine based on the Brucella B. abortus 19 VA strain (NPO Mikrogen, Ministry of Health of Russia) at a dose of 3.4 × 10 ⁸ -4.6 × 10 ⁸ live microbial cells in 0.5 ml of saline. The control group (n = 50) consisted of laboratory mice, which were injected with sterile saline in a volume of 0.5 ml. Blood sampling from immunized and control biomodels was carried out before vaccination and on the 14th day after immunization. The studies were performed on a FACS Calibur flow cytometer (Becton Dickinson, USA), using monoclonal antibodies (MKAT) to surface antigens of mouse T-lymphocytes. The activation molecules CD25, CD69 expressed on T lymphocytes were determined. The lymphocyte population was identified by gating in FSC-SSC coordinates on a Dot Plot plot (BD Cell Quest ^Tm software). Events that entered the lymphocyte region were analyzed for expression of CD3 ⁺ CD69 ⁺ , CD3 ⁺ CD25 ⁺ receptors. Used MKAT (Invitrogen, USA) were labeled with FITC (fluorescein isothiocyanate) and PE (phycoerythrin).

To determine the background values (background) of activation markers, lymphocyte stimulation was carried out with sterile saline. The activation reaction was taken into account 24 hours after incubation with BSPK in vitro.

Studies have shown that the intensity of expression of markers of activation of T-lymphocytes in the control group when stimulated with saline and BSPK did not have a statistically significant difference in all periods of the study. This indicates that BSPK does not cause a non-specific lymphocyte response in vitro. Thus, the expression level of the marker CD25, when stimulated with a sterile isotonic solution, averaged 2.47 ± 0.46%, CD69-10.22 ± 2.23%.

Studies of the expression intensity of the “early” activation molecules after vaccination showed that on day 14 after immunization the background values did not have a statistically significant difference of 3.73 ± 0.40%, and after incubation with the antigenic complex, BPPC showed the level of IL-2 receptor expression ( CD25) increased by 2.8 times, averaging 8.65 ± 1.85%. Analysis of the specific activity of BSPK by the marker of early proliferation of lymphocytes - CD69 showed that on 14 days after immunization the antigenic complex contributed to an increase in CD69 exposure by more than 2 times compared to background values (23.07 ± 1.87%) - 46 , 81 ± 4.53%.

Analysis of the results of the study showed that the BSPC obtained from B. abortus 19 VA strain according to a modified method has a pronounced specific activity in vitro. BSPK does not cause nonspecific reactions (activation) of T-lymphocytes in vitro, which makes it possible to use it as a test antigen in the formulation of the antigen-specific activation of T-lymphocytes in vitro to assess the formation of adaptive vaccine immunity against brucella.

The possibility of practical application of the invention is illustrated by examples of its specific implementation using a combination of the claimed features.

Example 1. The culture of B. abortus 19 VA is sown on Albimi agar, poured into cups and grown at a temperature of (37 ± 1) ° C, (48 ± 1) h (2 generation). Then produce reseeding on the jambs of Albimi agar poured into test tubes, grown at a temperature of (37 ± 1) ° С, (48 ± 1) h (3 generation). The culture from mowed agar in test tubes is washed off with a 0.9% NaCl solution and seeded on bacteriological mattresses, grown (72 ± 1) h at a temperature of (37 ± 1) ° С (4th generation). To inactivate the brucellosis microbe, cold (minus 40 ° C) acetone is used. The biomass of brucellosis microbes disinfected and tested for specific sterility is poured into Petri dishes for 18-20 hours for evaporation from acetone, obtaining dry biomass, into which a 2.5% NaCl solution 1: 1 is added for extraction, per day. Then centrifuged at 12000 g for 40 min in a centrifuge. The precipitate obtained after centrifugation of the biomass and dissolved in a 0.9: 1: 1 NaCl solution of pH 7.2 in a 1: 1 ratio is placed in the X-chamber of the cell disintegrator (X-25 Press LKB, Sweden), pre-cooled for 3 hours at a temperature of minus 40 ° C. Microbial cells in the disintegrator are frozen at the same temperature for 12 hours. After this, the cells are destroyed in a hydraulic press, forcing the biomass four times through the calibrated hole of the disintegrator cartridge. Next, the biomass is thawed and centrifuged at 12,000 g for 40 minutes. In the supernatant receive BPBC. The antigen activity in the RID was 1: 128, the pH of the solution was 7.2 ± 0.2. The total protein concentration in the test samples was 536 ± 2.0 μg / ml. Monosaccharides - ramnose and D-glucose were revealed.

The resulting antigen was lyophilized, controlling its physicochemical and biological parameters. During the year, the drug remained stable.

The results of a study of the specific activity of BSPK in terms of the expression of activation markers CD25, CD69 by T-lymphocytes indicate a high activation potential of the antigenic complex. On the 14th day after vaccination, the intensity of antigen-induced expression by T25 lymphocytes of CD25 left - 8.65 ± 1.85% (background - 3.73 ± 0.40%), CD69 - 46.81 ± 4.53% (background - 23.07 ± 1.87%). Thus, it was found that the Stavropol Anti-Plague Institute of Rospotrebnadzor BPPK developed at the Federal Public Health Institution based on the B. abortus 19 VA strain does not cause non-specific reactions (non-specific activation) of T-lymphocytes and has a pronounced specific activity in vitro. The results of specific antigen activity are presented in table 1.

Example 2. Similar to example 1, however, after the destruction of cells in a hydraulic press and centrifugation at 12000 g for 40 minutes, the biomass is extracted with 2.5% NaCl solution and further centrifuged at 20,000 g for 60 minutes. The precipitate formed is removed. The resulting antigen is lyophilized. The antigen activity in the RID was low and amounted to 1:16, the pH of the solution was 7.2 ± 0.2. The total protein concentration in the test samples was 470 ± 1.8 μg / ml. Monosaccharides - ramnose and D-glucose. The results of a study of the specific activity of the antigen in in vitro lymphocyte tests indicated on its tap a low stimulating potential for T cells - the values of the expression intensity of T-lymphocyte activation markers (CD25, CD69) upon antigen activation did not have a statistically significant difference with the background values (Tab. one).

Example 3. Similar to example 1, however, after the destruction of cells in a hydraulic press and centrifugation at 12000 g for 40 minutes, the biomass is subjected to ultrasonic disintegration and centrifuged at 20,000 g for 60 minutes After centrifugation, the (II) supernatant is separated from the precipitate. Sediment - extracted with 2.5% NaCl solution with further centrifugation at 20,000 g for 60 minutes. The precipitate formed is removed. And all supernatants are combined and dialyzed against distilled water for 48 hours. The resulting antigen is lyophilized. The antigen activity in the RID was 1:64, the pH of the solution was 7.2 ± 0.2. The total protein concentration in the test samples was 986 ± 2.5 μg / ml. Monosaccharides - ramnose and D-glucose. The resulting antigen is lyophilized. Analysis of the results of studying the intensity of the activating effect of the antigen on T-lymphocytes indicated the absence of induced activation of the studied population of lymphocytes, which indicates a low stimulating potential of this antigen (Table 1).

* - the difference in values is statistically significant at p≤0,05

Thus, the proposed method for producing antigen from brucella by isolating specific antigenic components from the bacterial mass of the causative agent of brucellosis according to example 1 is effective for antigen-stimulated cellular reactions in vitro. The use of the developed antigen helps to increase the sensitivity of cellular reactions in vitro. The obtained experimental antigen has a real prospect of application in the development of algorithms for laboratory diagnosis of brucellosis and the assessment of the actual vaccination of risk groups after vaccination against brucellosis.

Used Books

1. Darmov I.V., Elagin G.D., Bogacheva N.V., Kryuchkov A.V., Tikhvinskaya O.V. Modern laboratory methods for evaluating the effectiveness of immunization against dangerous and especially dangerous infections // Clinical laboratory diagnostics. - 2011. - No. 6. - S. 39-42

2. Kulichenko A.N., Abzaeva N.V., Gostischeva S.E., Rakitina E.L., Ponomarenko D.G., Kostyuchenko M.V. The use of antigen-specific cell tests in vitro to assess the formation of post-vaccination anti-plague immunity // Infection and immunity. - 2017. - T. 7. №2. - S. 203-208.

3. Firstova V.V., Bakhteeva I.V., Titareva G.M., Zyrina E.V., Ivanov S.A., Kiseleva N.V., Kopylov P.Kh., Anisimov A.P., Dyatlov I.A. Determination of expression of a marker of early activation of CD69 on lymphocytes of immune mice after stimulation with plague microbial antigens // Problems of especially dangerous infections 2010. - No. 103. - S. 56-59.

4. Ponomarenko D. G., Logvinenko O. V., Sargsyan N. S., Rakitina E. L., Pigeon O. G., Kulichenko A. N. / A new approach to allergodiagnosis of brucellosis // Infection and immunity. 2013. V. 3. No. 1. S. 89-92.

5. RF patent No. 2627897. Published: 08/14/2017 Bull. Number 23.

6. RF patent 2085212. Published: 07/27/1997.

7. RF patent No. 2163141. Published 02/20/2001. Bull. No. 5.

8. Comparative characteristics of the antigenic structure of bacteria of the genus Brucella / E.N. Afanasyev // dis. Cand. honey. sciences. Stavropol. 1983

9. FS Allergen liquid brucellosis (Brucellin). Instead of GF X, Art. 114, FS 42 - 3552-98.

Claims (1)

The method of producing brucellosis antigen for in vitro cell tests, which consists in the fact that obtaining the antigen involves culturing the causative agent of Brucella abortus 19 VA in a solid nutrient medium (4 generations), water-salt extraction with 2.5% NaCl 1: 1 during the day, destruction of microbial cells in a hydraulic press, centrifugation and lyophilization of the supernatant, achieving full recovery of the polysaccharide-protein antigenic complex of brucella with high activity and increased shelf life.