RU2330681C1 - Polyvalent corpuscular antigen for production of medical and preventive biological products for brucellosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: antigen contains mixed cells of virulent strains №№ 21, 145 Br. melitensis and 544 Br. abortus in ratio 1:1:1 inactivated by heating at temperature 60°C during 1 h. Vaccines based on this antigen, provide more than 70% of animal protection at infection with virulent culture of brucellosis agent within 3 months after immunisation.

EFFECT: improved efficiency.

2 tbl, 3 ex

Description

The invention relates to the field of biotechnology and can be used for the manufacture of therapeutic and prophylactic biological products against brucellosis in humans.

Known vaccine for specific prophylaxis of brucellosis in humans, prepared using the vaccine strain Br. abortus 19 VA (Vershilova P.A., Feder M.L., Polyakova A.M. Vaccination of people against brucellosis with a live vaccine // Transactions of the USSR Academy of Medical Sciences. - Moscow. - 1952. - No. 2. - S.231-235). Over the years since the start of production of the indicated vaccine preparation, rich experience has been gained in its use. It is shown that the incidence among vaccinated people working with sick animals does not exceed 8% (Report on research work / research institute named after N.F. Gamalei. Head: P.A. Vershilova. - Topic No. 39, 1986). At the same time, the immunity caused by this vaccine significantly decreases over time. This determines its relatively low protective effect. So, according to the literature (Handbook on the immunization of infectious diseases in the Soviet Army and the Navy in peacetime. - M., 1991), the maximum effectiveness of the drug remains only for the first one and a half to two months after immunization. P.A. Vershilova points out (Vershilova P.A., Kurdina D.S. Study of the intensity of cross-immunity in brucellosis // Journal of Microbiol. - 1963. - No. 8. - P.34-36), which after 1, 4 and 12 months after vaccination, the incidence rate is 17.3%, 32.7% and 50.0%, respectively. In 1 year after immunization, positive serological and allergic reactions are detected only in 25-50% of vaccinated (Smirnov S.M., Tivugabylov M.K., Schmuter M.F. About skin immunization of people against brucellosis with the vaccine from strain Br. Abortus 19 / / Journal of microbiol. - 1961. - No. 1. - S.51-54). The pronounced sensitizing properties of the vaccine contribute to the appearance of both specific and non-specific sensitization of the body. In experiments on laboratory animals, it was found that after the second revaccination, dystrophic and degenerative changes in the organs develop, moreover, the vaccine instead of a stimulating effect on the immune system leads to its suppression associated with the depletion of the reticuloendothelial system (Grekova N.A. Influence of multiple revaccinations against brucellosis on the state of the animal organism // ZhMEI. - 1968. - No. 2. - S.131-140). The vaccine cannot be used with antibiotics if emergency prophylaxis is needed.

The closest to the claimed invention analogue is a composition based on purified antigen isolated from strains of the species Br. abortus (No. 19 or 19 VA or 99), or a species of Br. melitensis (No. 565 or 16M) in the S-form (Dranovskaya EA, Samoilenko II Method for producing antigen for the prevention of brucellosis. Auth. certificate. SU No. 957471). The antigen is obtained by treating the microbial biomass of Brucella with an organic solvent, followed by hydrolysis with acetic acid and lyophilization. The vaccine preparation consisting of the indicated antigen was intended both for the prophylaxis of brucellosis and for the treatment of sick people (Dranovskaya EA, Dranovskaya TN. The vaccine for the prevention and treatment of brucellosis. RF patent No. 2076493, application No. 94023720). The disadvantage of this chemical vaccine is its limited effectiveness, the duration of the created immunity did not exceed three months (Comparative study of the safety, reactogenicity and antigenic activity of brucellosis chemical and live vaccines in a controlled epidemiological experience / Sumarokov A.A., Karinskaya G.A., Dranovskaya E .E. et al. // ZhMEI. - 1984. - No. 2. - S.58-63). Along with this, the appearance of local reactions of moderate degree and pain in the injection area in vaccinated was noted (Studying the harmlessness and reactogenicity of the experimental-production series of brucellosis chemical vaccine with various methods of its application / Elshina G.A., Sumarokov A.A., Anisimova T.I. . et al. // ZhMEI. - 1991. - No. 9. - P.50-53). An attempt to improve the method for producing an antigen by exposing it to ionizing radiation also did not lead to a significant increase in the protective properties of the vaccine. In addition, it is known that exposure can negatively affect the subject of exposure, reducing its immunobiological activity.

A possible solution to the problem of specific prophylaxis and treatment of brucellosis is the development of inactivated vaccines based on killed virulent cultures of brucella, in which the whole variety of antigenic components of the microbial cell is preserved. At the same time, it is known that, in their pure form, dead cultures are rapidly eliminated from the body and, when administered once, are not able to form stable, intense immunity. However, this drawback can be eliminated with an adequate selection of the component composition, the use of adjuvants and carriers, the choice of an effective dose and immunization schedule. In addition, one of the advantages of killed vaccines is the possibility of their simultaneous use with antibiotics for emergency prevention and treatment of brucellosis, they are easier to dose, they are more standard and stable.

The objective of the invention is to obtain brucellosis antigen suitable for the preparation of highly immunogenic vaccines intended for the treatment of people with brucellosis with the aim of hyposensitization and immunomodulation, and for the prevention of brucellosis.

The problem is solved in that the brucellosis antigen polyvalent corpuscular in its composition contains a mixture of heat-inactivated cells of the causative agent of brucellosis of virulent strains No. 21, 145 Br. melitensis and 544 Br. abortus in a ratio of 1: 1: 1.

Brucella is grown on meat-peptone agar (MPA) or Albimi medium, modified by the introduction of a growth stimulator of hemophilic microbes. The accumulation of microbes is carried out according to two schemes. The first of these involves the sequential cultivation of bacteria in a dense nutrient medium, beveled in test tubes and mattresses. In the second case, brucella is additionally cultivated in a liquid nutrient medium consisting of fish-bone meal sulfuric acid hydrolyzate for 48 hours in 20-liter glass bottles or in cultivators under aeration conditions.

In agar cultures, from each mattress and in all deep cultures, the following are assessed: the presence of extraneous microflora in Gram stains and plating on “hungry” agar; the presence of dissociation in the sample with a solution of tripaflavin; optical concentration of microbes.

Based on the results of determining the concentration of microbes according to the standard turbidity, the suspension is diluted with physiological solution of sodium chloride to a concentration of 30 ... 40 · 10 ⁹ cells in 1 cm ³ . After that, the microbial suspension of cultures of each strain is thermally inactivated at a temperature of 60 ° C and a 5.0% solution of twice distilled phenol is added to its final concentration in suspension of 0.5%. Then the microbial suspension is placed in a thermostat and incubated for 48 hours at a temperature of (36-38) ° C.

The effectiveness of inactivation of cultures is evaluated by indicators of general and specific sterility, specific safety.

The antigen obtained by the above method, with the addition of a preservative - phenol solution to a final concentration of 0.25% in the finished form of the drug, is proposed to be used to prepare a therapeutic brucellosis liquid vaccine (vaccine No. 1), intended for the specific treatment of various forms of brucellosis in humans. To this end, uterine suspensions of cultures of strains No. 21, 145 Br. melitensis and strain No. 544 Br. abortus is mixed in a ratio of 1: 1: 1 and diluted with saline to 1 · 10 ⁹ MK in 1 ml containing twice distilled phenol to a final concentration of 0.25%. The finished vaccine suspension is poured into ampoules and sealed.

At the same time, it is possible to manufacture a vaccine of brucellosis adsorbed inactivated liquid for immunization of people (vaccine No. 2), consisting of the above inactivated microbial cells, adjuvant and preservative, with a concentration of 4.5-5.5 · 10 ⁹ microbial cells in 1 cm ³ in the following ratio of components, vol.%:

heat inactivated cells of virulent microbes of strains No. 21, 145 Br. melitensis and 544 Br. abortus in a ratio of 1: 1: 1 with a concentration of 30-40 · 10 ⁹ microbial cells in 1 cm ³ - 12.5-16.7;

5% phenol solution - 4.5-5.5;

aluminum hydroxide (up to the content of aluminum ions 0.07-0.12) - 16.3-28.0;

physiological solution of sodium chloride - up to 100.0.

The finished vaccine is packaged in bottles. Each series of vaccines is checked for general and specific sterility, toxicity to white mice, specific safety.

The strains used for the preparation of brucellosis antigen meet the requirements for the production of inactivated biological products, are deposited in the collection of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation, Kirov under the numbers: 21, 145, 544 and have the following passport characteristics and immunobiological properties.

Strain No. 21 Br. melitensis isolated from a sick person in the Dagestan Republic, strain No. 145 Br. melitensis is also isolated from a sick person in the Shymkent region, strain No. 544 Br. abortus is an international reference. These brucella strains belong to the 1st biotype of their species, are in a stable S-form, and possess typical cultural and biochemical properties.

Small brucella (0.3-0.6 × 0.6-2.5) microns, gram-negative, motionless coccobacteria.

Cultural and biochemical properties.

The optimum growth temperature is 37 ° C. The optimal pH of the culture medium is 6.8-7.2. When grown in solid nutrient medium, they give characteristic S-growth. On a dense nutrient medium form colorless, convex, round colonies with a smooth surface; in a liquid nutrient medium give uniform turbidity.

The strains are in a stable S-form. Microbial colonies (S-form) are not stained with a crystal violet solution or acquire a light yellow color. When a sample is placed with a 0.2% tripaflavin solution on glass, the bacterial cells remain uniformly suspended in the liquid.

Agglutinable properties.

The strains have type-specific agglutinability: Br. melitensis are agglutinated with M-serum, and A is not agglutinated with monoreceptor serum, Br. abortus are agglutinated with A - serum, and not agglutinated with M monoreceptor serum.

Antigenic properties.

With the introduction of rabbits 20 × 10 ^9- th suspension killed by heating brucella titer of agglutinins in the serum of rabbits 1: 800. The cultures of the strains are agglutinated to a titer of SOP of serum with diagnostic brucellosis rabbit agglutinating (with a titer of at least 1600).

Virulence of cultures of strains.

With subcutaneous administration of strains to guinea pigs, followed by bacteriological examination of spleen ID ₅₀ for strain 145 Br. melitensis ranged from 2 to 12 microbial cells for strain 21 Br. melitensis from 7 to 13, for strain 544 Br. abortus from 4 to 16 (p≥0.05).

The invention is illustrated by the following examples.

Example 1

Polyvalent corpuscular antigen is used to prepare vaccine No. 1, intended for the specific treatment of brucellosis in humans.

In preliminary experiments on guinea pigs weighing 250-300 g, infected subcutaneously with a virulent culture of strain 16M Br. melitensis at a dose of 10 ID ₅₀ , vaccine monotherapy was performed. Treatment was started 50 days after infection. The vaccine was administered subcutaneously in increasing doses of 40-80-200-400-2000-4000 thousand m.k., equivalent to a human dose, with an interval of 72 hours. The estimated amount of the drug was administered in a volume of 1.0 cm ³ in the inguinal region of the animal, opposite the site of infection.

During the experiment, specific parameters were monitored: serum agglutinins titers during RA, opsonophagocytic number and index, the presence and severity of an allergic reaction when setting a Burne test, plating microbes of the pathogen of brucellosis from organs of laboratory animals. The research results showed an intensive increase in antibody titer in animal serum after four injections of the drug, which was an order of magnitude higher than that in the group of untreated animals. In most guinea pigs after vaccination therapy, an increase in phagocyte activity was noted, the phagocytic index was 52-68.

At the same time, a 7-10-fold decrease in the intensity of the allergic reaction at the end of treatment was noted. A six-fold injection of the drug almost completely removes the high allergic rearrangement of the body, which is a leading factor in the pathogenesis of this disease, in comparison, in the group of untreated animals, the Burne test was pronounced and amounted to 0.8-1.4 cm. Conducting a course of monotherapy with a vaccine in 43 percent cases led to recovery. This result of the study was undoubtedly positive, because, not including antibacterial agents, it was possible to eliminate the pathogen from the body of almost half of sick animals.

Example 2

In preliminary experiments on guinea pigs weighing 250-300 g, infected subcutaneously with a virulent culture of strain 16M Br. melitensis at a dose of 10 ID ₅₀ , a comprehensive treatment with a therapeutic brucellosis vaccine was carried out using antibiotics highly effective against brucella - pefloxacin or doxycycline. Pefloxacin was administered in a daily dose of 0.024 g per os, doxycycline in a daily dose of 0.004 g per os daily for 20 days, therapeutic brucellosis vaccine in increasing doses (40-80-200-400-2000-4000 thousand m.k. ), equivalent to a human dose, with an interval of 72 hours.

During the experiment in guinea pigs, the indicators described in example No. 1 were monitored.

The results of the experiments showed that the implementation of both complex therapy and monotherapy with brucellosis vaccine leads to intensive stimulation of the humoral and cellular parts of the immune system. In comparison, with antibiotic therapy, serum agglutinin titers in most cases were inferior to the control and averaged 30 ME, which is due to the localization of the infection, and possibly the immunosuppressive effect of antibacterial drugs.

Conducting complex treatment with the vaccine again confirmed the high desensitizing effect of the drug, since vaccine therapy contributed to a multiple reduction in the skin allergic reaction. In comparison, the administration of pefloxacin or doxycycline alone reduced the intensity of the allergic reaction by only 30% compared with the control group. Inclusion of the vaccine in complex treatment together with pefloxacin or doxycycline made it possible to cure 83 and 87 percent of laboratory animals suffering from brucellosis, respectively, which was 1.5-2.0 times higher than this indicator compared with vaccine monotherapy and more than 20% antibiotic monotherapy .

The data obtained allow us to conclude that the vaccine, actively stimulating humoral and cellular immunity, significantly reduces the significance of the leading cause of late complications in brucellosis infection - sensitization, and thereby contributes to the removal of the pathogen from the macroorganism, i.e. virtually completely removes the main symptom complex of brucellosis infection.

Example 3

Polyvalent corpuscular antigen is used for the preparation of vaccine No. 2, intended for the prevention of brucellosis infection.

The immunogenic properties of experimental vaccine samples were evaluated on guinea pigs weighing 300-400 g. The drug was administered subcutaneously in the inguinal region at 1 cm ³ . After 30 and 90 days, the immunized guinea pigs were infected with a virulent culture of strain 16M Br. melitensis. At the same time, a group of non-immunized guinea pigs was infected (control).

After 30 days, blood was collected from the animals for RA, an allergic test was performed, and euthanized with ether. During bacteriological examination, seeding on nutrient media (MPB and MPA) was made from inguinal, submandibular, paraaortic, cervical lymph nodes, liver, spleen, and bone marrow. Crops were incubated at a temperature of (36-38) ° C and visually controlled after 4-6 days and after 14 days. The research results are presented in tables 1, 2.

Analysis of the data presented in table 1 shows that when the vaccine is administered in doses of 1.25 to 10.0 billion, 30 days after immunization, the guinea pigs have the same level of protection against infection of cultures of the highly virulent 16M Br strain. melitensis, equal to 73-77%. At the same time, the fact of the presence of a close level of agglutinins in all groups of immunized animals is noteworthy.

At the same time, similar studies 3 months after immunization of guinea pigs showed that the level of protection with the introduction of 2.5 billion cells decreases to 55% (table 2), while with the introduction of 5.0 billion cells it remains at the level 78%, and at a dose of 10.0 billion, it rises to 88%.

During the experiments, it was found that 30 days after immunization in infected guinea pigs, the agglutinin titer averaged 244 ME for all groups, which is statistically significantly lower than for non-diseased animals - 450 ME (P ≥ 0.05). In contrast to this fact, 120 days after vaccination, the agglutinin titer of 600 ME in diseased animals exceeded that of healthy guinea pigs remaining healthy - 67 ME.

Thus, the use of the proposed brucellosis antigen, consisting of inactivated virulent strains No. 21, 145 Br. melitensis and 544 Br. abortus in the ratio of 1: 1: 1, an adjuvant and preservative for the preparation of an inactivated adsorbed vaccine at a dose of 5 billion cu.m., can achieve more than 70% protection of animals from infection with a virulent culture of the pathogen brucellosis 3 months after immunization. The data obtained allow us to conclude that it is possible to further use this drug as a preventive vaccine.

Table 1
The results of determining the effectiveness of the vaccine for guinea pigs at 30 days after immunization Dose of the vaccine, billion mk cm ³ The number of animals taken in the experiment The number of sick animals Protection percentage Group average agglutinin titer in RA at 30 days after infection, ME, x (x _{min ...} x _max ) 1.25 13 3 77 460 (320 ... 1280) 2,5 eleven four 73 460 (320 ... 640) 5,0 13 3 77 430 (320 ... 1280) The control fifteen fifteen 0 80 (40 ... 120) Note - animals are infected subcutaneously with a culture of strain 16M Br. melitensis at a dose of 105 live microbes.

table 2
The results of determining the effectiveness of the vaccine for guinea pigs on 90 days after immunization Dose of the vaccine, billion mk cm ³ The number of animals taken in the experiment The number of sick animals Protection percentage Group average agglutinin titer in RA at 30 days after infection, ME, x (x _{min ...} x _max ) 2,5 eleven 5 55 60 (20 ... 160) 5,0 9 2 78 80 (20 ... 160) 10.0 8 one 88 80 (20 ... 160) The control fifteen fifteen 0 40 (20 ... 80) Notes: 1. Animals are infected subcutaneously with a culture of strain 16M Br. melitensis at a dose of 120 live microbes.
2. In all diseased animals, a positive allergic reaction to the intradermal administration of brucellin was determined.

Claims (1)

Polyvalent corpuscular antigen for the manufacture of therapeutic and prophylactic preparations against brucellosis, containing a mixture of virulent strains No. 21, 145 Br, inactivated by heating at a temperature of 60 ° C for 1 h. melitensis and 544 Br. abortus in a ratio of 1: 1: 1.