RU2332231C2 - Method of obtaining acellular vaccine for pertussis immunoprophylaxis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves cultivation of Bordetella pertussis culture in casein carbon agar with further washing microbe cells off the nutrient medium surface. Then pH of 8.4±0.1 is established in the microbe suspension containing 70±10 colony-forming units of microbe cells. Then water solution of sodium desoxycholate is added to the suspension until the final concentration reaches 0.5%, the suspension is left at the temperature of (3±1)°C for 2 hours. The target product is separated from the biomass by centrifugation and undergoes diafiltration, ultrafiltration, neutralisation by adding 0.03% of formalin at pH 7.6±0.2 and temperature of (36±3)°C for 3 days. Neutralised target product is precipitated by hydrochloride acid at pH 3.5±0.3 in the presence of 0.30±0.05% of sodium hexametaphosphate. The obtained sediment is dissolved in 0.9% sodium chloride solution at pH 7.1±0.3, and sterilising filtration of ready product is performed.

EFFECT: anti-pertussis drug with reduced reactogenicity.

4 tbl, 1 ex

Description

The invention relates to medicine, and more specifically to the vaccine prophylaxis of human infectious diseases, and can be used in the production of highly immunogenic harmless drugs for the prevention of whooping cough.

A known method for producing acellular pertussis vaccines, providing for the release of purified Bordetella pertussis antigens with their subsequent combination in the finished product (K.M. Edwards, B.D. Meade, M.D. Decker et al. Comparison of 13 Acellular Pertussis Vaccines: Overview and Serologic Response. Pediatrics, 1995; 96: 548-557). The disadvantages of this method are the lack of adequate laboratory tests to quantify the protective activity of the obtained vaccines, the insufficient amount of epidemiological observations of their effectiveness, and the inability to date to establish which antigens should be present in the vaccines (Vaccines edited by Stanley A. Plotkin; Walter A. Orenstein - 3rd edition. 1999. USA).

Closest to the proposed invention, an analogue is a method for producing a corpuscular pertussis vaccine, a component of the DTP vaccine, comprising growing a Bordetella pertussis culture on KUA (casein-carbon agar) culture, washing microbial cells from the surface of the culture medium, inactivating the microbial suspension with formaldehyde (FS 42- 0504-6501-05 (vaccine pertussis-diphtheria-tetanus adsorbed liquid).

At the same time, DTP vaccine is the most reactogenic drug in the national vaccination calendar. In many ways, the complications of using this drug are associated with the pertussis component.

The technical result of the invention is to obtain a cell-free pertussis vaccine with reduced reactogenicity.

The invention consists in the following. The microbial suspension of Bordetella pertussis is treated with a solution of sodium deoxycholate, the target product is separated from the biomass by centrifugation, subjected to diafiltration, ultrafiltration, neutralization with formaldehyde and heat, purification with hydrochloric acid in the presence of sodium hexametaphosphate and sterilizing filtration.

Treatment of Bordetella pertussis cells with a solution of sodium deoxycholate allows you to select antigenic complex with protective activity. At the stage of diafiltration, the purification of the target product from low molecular weight ballast impurities is achieved. The addition of formaldehyde helps neutralize the potentially toxic components of the drug. Additional purification of the target product is achieved by treatment with hydrochloric acid in the presence of sodium hexametaphosphate. At the same time, the reactogenicity of the isolated, purified and neutralized antigenic complex is reduced in comparison with the whole-cell preparation.

The method of obtaining the drug is as follows. For the manufacture of the vaccine, 3-5 strains of the pertussis pathogen are used. Bordetella pertussis 1 phase. Microbes are grown on AMC in mattresses at (36 ± 1) ° C for 48 hours. After this time, the grown culture in each mattress is monitored for bacteriological purity and typical morphology of microbial cells by microscopy of Gram stained smears. From each mattress with a B.pertussis culture that meets the requirements for morphology and purity, the culture is washed off with a sterile 0.9% sodium chloride solution (pH 7.2). The bacterial suspension is poured into a sterile bottle. Standardize the suspension according to the optical turbidity standard, setting the concentration of 60-80 MOI of microbial cells in 1 ml of suspension. In a standardized suspension, a pH of 8.4 ± 0.1 is adjusted with a 5% sodium hydroxide solution. To a microbial suspension cooled to (3 ± 1) ° C, containing 60-80 MOE, add 10% sodium deoxycholate solution to a final concentration of 0.5% in the suspension, and maintain at a temperature of (3 ± 1) ° С for two hours with periodic stirring. After that, the target product is separated from the biomass by centrifugation. The resulting solution of the target product is subjected to clarifying filtration through membranes with a pore diameter of 1-3 μm. Then, the target product is purified on an ultrafiltration unit consisting of a peristaltic pump and membrane devices with a retention limit of substances by molecular weight of 15 kD. First, diafiltration is carried out against ten volumes of 0.9% sodium chloride solution. At the end of diafiltration, the solution is concentrated to the initial volume, 0.03% of the volume of 40% formaldehyde solution is added, the pH is adjusted to 7.6 ± 0.2, microfiltered through membranes with a pore diameter of 0.22 μm and kept at a temperature (36 ± 3) ° C for 3 days. Next, an additional purification of the target product is carried out by precipitation with hydrochloric acid at pH 3.5 ± 0.3 in the presence of 0.30 ± 0.05% sodium hexametaphosphate at a temperature of (4 ± 2) ° С. The precipitate formed is dissolved in a 0.9% sodium chloride solution at pH 7.1 ± 0.3 to a protein content of at least 0.4 mg / ml (according to the Lowry method), after which sterilizing filtration of the preparation is carried out.

Example.

Bordetella pertussis strains No. 39, 267, 305 (10 mattresses for each strain) were seeded on mattresses with AMC nutrient medium. Mattresses were incubated 48 hours at 36 ° C. After this time, the grown culture in each mattress was monitored visually and microscopically for bacteriological purity and typical morphology. 50 ml of a 0.9% sodium chloride solution were added to each mattress with a B. pertussis culture meeting the requirements for morphology and purity. The mattresses were left in a horizontal position with the seed surface up for 5 minutes, after which the culture was washed off by swaying.

The contents of the mattresses with the culture of each strain were combined in the appropriate bottles. The volume of microbial suspension of each strain was 450 ml, the content of microbial cells was 120 MOU. After that, 300 ml of 0.9% sodium chloride solution was added to each bottle. The content of microbial cells according to the control results was 75 MOU. Suspensions of all three strains were combined into a 5 liter common bottle. The volume of the suspension was 2.25 L, pH 7.0. The pH was adjusted with a 10% sodium hydroxide solution to 8.5.

112.5 ml of a sterile 10% sodium deoxycholate solution was added to the suspension and kept for 2 hours at a temperature of (4 ± 2) ° С with periodic shaking.

Next, the suspension was centrifuged at 3000 rpm for 1 hour. The resulting precipitate was discarded, and the supernatant with a volume of 2.3 L was freed from fine impurities by microfiltration on membranes with a pore diameter of 3 μm. After that, the target product was diafiltered on hollow fibers of VPU-15 grade against 25 l of 0.9% sodium chloride solution. After diafiltration, the target product was concentrated by ultrafiltration to 1.5 L, 0.45 ml of a 40% formaldehyde solution was added, the pH was adjusted with a 5% sodium hydroxide solution to 7.8, and filtered through plates with a pore diameter of 0.22 μm into a sterile bottle of 3 l The resulting solution was kept at a temperature of (36 ± 1) ° C for 72 hours.

Then, 15 ml of a 33% sodium hexametaphosphate solution was added to the preparation and the pH was adjusted with a 5% hydrochloric acid solution to a pH of 3.4. The precipitate formed was separated by centrifugation at 3000 rpm for 30 minutes and dissolved in 1.0 l of a 0.9% sodium chloride solution with alkalization with a 5% sodium hydroxide solution to pH 7.0. The resulting solution was subjected to sterilizing filtration through plates with a pore diameter of 0.22 μm. The protein content in the preparation was 0.4 mg / ml.

The drugs obtained using the proposed method were monitored according to Pharmacopoeia article 42-0504650105 “Vaccine pertussis-diphtheria-tetanus adsorbed liquid (DTP vaccine)”. In a study of the safety of drugs on outbred mice, it was shown that the drug is safe in doses of up to 2.0 mg (Table 1).

Table 1 Immunization Total weight Weight gain Group number Dose of the drug Number of animals before introduction, g on the 3rd day, g on the 7th day, g absolute., g rel.,% one 2 mg / ml cell-free preparation 10 149.2 176.5 202.8 53.6 85.6% 2 1 mg / ml cell-free preparation 10 149 173.4 203.4 54,4 86.9% 3 0.5 mg / ml cell-free preparation 10 149.8 175.8 200.3 50,5 80.6% four 0.25 mg / ml cell-free preparation 10 150.2 177.4 213.4 63,2 100.9% 5 Control (0.9% sodium chloride solution) 10 150.1 179.3 212.7 62,2 one hundred% 6 Corpuscular preparation 10 150.6 175.8 195.5 44.9 72.2%

As follows from table 1, the weight gain of the mice was maximum in the case of the introduction of the drug obtained by the proposed method. In this case, the minimum weight gain should be at least 60% of that in the case of the introduction of a 0.9% solution of sodium chloride.

The drug had a lower histamine-sensitizing activity in comparison with the corpuscular analogue. The results are shown in Table 2.

table 2 A drug Breeding The number of animals in the experience taken survivors CCA 5 (industry standard) 20 MY - 1 ml 5 0 4 MY - 1 ml 5 one 0.8 MY - 1 ml 5 3 Cell-free preparation (the invention) 2 mg - 1 ml 5 5 0.4 mg - 1 ml 5 5 0.08 mg - 1 ml 5 5 Corpuscular preparation (prototype) 20 MY - 1 ml 5 2 4 MY - 1 ml 5 four 0.8 MY - 1 ml 5 5

Lymphocytosis-stimulating activity of corpuscular and acellular preparations in the same doses was moderate - 300-450 lymphocytes in 100 squares of the Goryaev chamber; against 250 lymphocytes with the introduction of 0.9% sodium chloride solution.

The regulated Kendrick test was used to evaluate the protective properties of pertussis drugs. When setting up the test, first-generation mouse hybrids (CBAxC57BL / 6J) were used. A comparative assessment of different series of cell-free drug and corpuscular vaccine shows their comparable immunogenicity (table 3).

Table 3 A drug Infective dose of neurotoxic strain B. pertussis 18323, cells Taken mice to experience Mice survived Survival rate Cell-free preparation p.1 100,000 eighteen 13 72.2% Cell-free preparation s.2 100,000 twenty fifteen 75.0% Cell-free preparation p.3 100,000 16 16 one hundred% Corpuscular preparation p.1 100,000 19 fourteen 73.7% Corpuscular preparation p.2 100,000 eighteen fourteen 77.8% Corpuscular preparation p.3 100,000 eighteen fifteen 83.3%

Thus, when using a cell-free drug, the percentage of survival of immune mice ranged from 72.2 to 100%, while when using a corpuscular preparation, from 73.7 to 83.3%. The regulated indicator of protective activity is 70%.

When evaluating humoral immunity in experiments on outbred guinea pigs, it was shown that the antibody content in the sera of animals twice immunized with acellular and corpuscular preparations did not differ significantly (Table 4).

Table 4 A drug Geometric mean titer by agglutination reaction * First vaccination Second vaccination Cell free 160 (73.4-347.1) 1470 (659.2-3279.4) Corpuscular 149 (39.5-564.6) 1575 (887.3-2798.8) * - the reciprocal of the dilution of serum

The study of local immunomorphological reactions to the administration of the patented drug and the corpuscular vaccine (prototype) was carried out on tetrahybrid mice (CBA / C57BL / 6J). As a result of the studies, it was found that the immune inflammation when using a cell-free drug proceeds as a delayed-type hypersensitivity, while when using a corpuscular preparation, the immunocomplex mechanism prevails, which can lead to the development of such serious adverse reactions and complications as vasculitis, arthritis, neuropathies.

Thus, the proposed method for producing acellular pertussis vaccine allows to obtain drugs with high immunogenic activity and significantly reduced reactogenicity compared to the corpuscular vaccine.

Claims (1)

A method of obtaining a cell-free pertussis vaccine by growing a Bordetella pertussis culture on casein-coal agar followed by washing off the microbial cells from the surface of the nutrient medium, characterized in that the pH is adjusted to 8.4 ± 0.1 in a microbial suspension containing 70 ± 10 MY of microbial cells then an aqueous solution of sodium deoxycholate is added to the suspension to a final concentration of 0.5% and kept at a temperature of (3 ± 1) ° C for 2 hours, then the target product is separated from the biomass by centrifugation, diafiltered, ultrafiltered radios are neutralized by adding 0.03% formalin at pH 7.6 ± 0.2 and temperature (36 ± 3) ° C for 3 days, the neutralized target product is precipitated with hydrochloric acid at pH 3.5 ± 0.3 in the presence of 0 , 30 ± 0.05% sodium hexametaphosphate, the resulting precipitate is dissolved in a 0.9% sodium chloride solution at pH 7.1 ± 0.3 and sterilized filtration of the finished preparation is carried out.