RU2101355C1 - Method of selection of immunogenic strains and subcultures of live anthrax vaccines - Google Patents

Abstract

FIELD: medicinal microbiology, immunology. SUBSTANCE: method involves preliminary quantitative estimation of immunogenicity of the analyzed culture population by relative amount of toxin-producing colony-forming units in vitro on agar for immunological investigation. Low- and nonimmunogenic cultures were excluded from the further study of immunogenicity in vivo. Then method involves selection of immunogenic cultures, selection in population of lowimmunogenic and nonimmunogenic cultures of immunogenic clones, preparing cultures for the following confirmation of their immunogenicity and final selection in vivo. Invention ensures to increase effectiveness of selection by two-fold, decrease consumption of experimental animals by two-fold and enhance immunogenicity of cultures. EFFECT: improved method of selection. 2 tbl

Description

 The invention relates to medical microbiology and immunology, in particular, to the development, production and quality control of live anthrax vaccines.

The main factor determining the immunogenicity of the anthrax microbe is its exotoxin. All modern living anthrax vaccines are capsule-free toxin-producing strains. It was shown that a non-producing toxin devoid of the plasmid toxin formation pX01 variant of the vaccine strain 34F ₂ Sterne is non-immunogenic [1,2]
Closest to the invention is a method for the selection of immunogenic anthrax vaccine strains, which consists in a single immunization of guinea pigs with several doses of the studied cultures with infection after 21 days of immunized animals and the subsequent calculation of the index of immunity in comparison with the reference vaccine STI-1 [3]
The disadvantages of this method are the low efficiency due to the fact that all capsule-free cultures are subject to research, including non-toxin-producing cultures that obviously cannot be immunogenic, and also that negative cultures that are negative are excluded from further research as unpromising.

The experimental data of other authors on the absolute need for toxin production for immunogenicity were confirmed and it was found that different vaccine strains and their subcultures grown on agar for immunological studies containing antitoxic antibodies differ in the relative number of colony-forming toxin-producing units (Tox ⁺ KOE) in the cell population culture. Moreover, the degree of their immunogenicity, determined by the immunity index for guinea pigs, directly correlates with the amount of Tox ⁺ KOE.

 The aim of the invention is to increase the efficiency of the method of selection of immunogenic strains and subcultures of live anthrax vaccines and reduce the consumption of laboratory animals.

The goal is achieved in that the method of selection of immunogenic strains and subcultures involves a single immunization of guinea pigs with several doses of the studied cultures with infection after 21 days of anthrax by the causative agent of the anthrax followed by the calculation of the immunity index in comparison with the reference vaccine, and before immunization of animals in the studied cultures is determined exotoxin production in vitro and relative amount Tox ⁺ KOE neimmmunogennye selected at values nizkoimmunogennye 1-10% at 11-63% and immunogenic n and 64-100% followed by selection in populations of low non-immunogenic and immunogenic cultures of clones was obtained from these subcultures. Thus, the selection is made more purposefully than in the prototype method.

With respect to the prototype, the invention has the following distinctive features. Additional introduction of in vitro determination of toxin formation in cultures, selection by relative amount of Tox ⁺ KOE of non-immunogenic cultures with values of 1-10% low immunogenic at 11-63% and immunogenic 64-100% cultures, selection in populations of low and non-immunogenic cultures of immunogenic clones, obtaining subcultures of these clones.

The invention is as follows. Agar for immunological studies containing antitoxic antibodies (RYE) [4] is prepared by dissolving in 358 ml of distilled water the ingredients, which are taken in the ratio, g:
1-Arginine • HCl 0.125
1-Aspartic acid 0.184
1-Valine 0.137
1-Histidine • HCl 0.055
Glycine 0.065
1-monosodium glutamate 0.612
1-Isoleucine 0.170
1-Leucine 0.230
1-Lysine 0.230
1-Methionine 0.073
1-Proline 0.043
1-Serine 0.235
1-Threonine 0.120
1-tyrosine 0.144
1-Tryptophan 0.035
1-Phenylalanine 0.125
1-Cystine 0.025
Adenine 0.0021
Uracil 0.0014
Thiamine • HCl 0.001
CaCl ₂ • 2H ₂ O 0.012
MgSO ₄ • H ₂ O 0.0099
MnSO ₄ • H ₂ O 0,0009
K ₂ HPO ₄ 3,000
NaHCO ₃ 8,000
d (+) glucose 2,500
Yeast Extract 8,000
The pH of the medium is monitored and, if necessary, adjusted to 8.0. The resulting solution of the nutrient base of the medium is sterilized by filtration through a membrane filter with a pore diameter of 0.22-0.45 μm. A portion of agarose 15 is melted in 500 ml of distilled water and sterilized by autoclaving at 121 ^° C for 20 minutes. A 358 ml sterile nutrient base solution and lamb antiserum against spores of anthrax vaccine strain 34F ₂ in a 142 ml volume are added to molten and cooled to 50 ^° C agarose, mixed and poured into 10-12 ml Petri dishes.

Ten-fold dilutions of the spores of the vaccine strains of the anthrax microbe in distilled water are prepared before dilution of 10 ^-7 . Sow three cups with Hottinger agar from dilutions 10 ^-5 , 10 ^-6 and 10 ^-7 of 0.1 ml of spore suspension for each. Crops are incubated for 18 hours at 37 ^° C, the number of colonies grown on plates is counted, and the average number of colonies for each dilution is determined. For sowing plates with RYE, a dilution is used that, when seeding from it, forms 25-50 isolated colonies on plates with Hottinger agar.

Suspend the spores of the studied strains of 0.1-0.2 ml per cup, seeded in 10-20 cups of RYE. To control the quality of the medium, the suspension of the reference preparation of the STI-1 vaccine prepared in the same way is seeded. Cups containing crops are placed in a microaerostat or CO ₂ incubator. 25% of the air is pumped out of the microaerostat by a vacuum pump, focusing on the readings of the manovacuum meter, and replaced with carbon dioxide. In a CO ₂ incubator, plates are cultured with 10% carbon dioxide. Crops are incubated at 37 ^o C for 48 h, then transfer the cups from the microaerostat or CO ₂ incubator to the refrigerator and incubated there for 3 days at a temperature of 4 ^o C.

 Toxin formation is detected by the method of [4]. To detect toxin formation, RYE cups are examined under natural light on a black background using a sheet of black paper. Around the toxin-producing colonies in the medium, concentric halos of immunoprecipitation are formed in the form of thin white rings. Non-toxin producing colonies lack such halos. Count the total number of colonies (at least 500), the number of colonies with halos of immunoprecipitation and their percentage.

 If the percentage of colonies with halos of immunoprecipitation is 1-10%, then the culture is assessed as non-immunogenic; when the content of the colonies is 11-63%, the culture is assessed as low immunogenic; 64-100% - corresponds to the immunogenic culture.

Among the colonies of cultures with insufficient immunogenicity, colonies are selected surrounded by halos of immunoprecipitation and transplanted into tubes with Hottinger broth. Crops were cultured at 37 ^o C for 18 hours and subcultured on mattresses with Gladstone-Fields agar. The latter are cultured at 32 ^o C for 7-10 days. On the seventh day, smears are made from mattresses, stained with Rebiger and microscopic, determining the degree of spore formation. If the degree of spore formation reaches 95-100%, spores are washed off with distilled water, spore suspensions are prepared, the percentage of toxin-producing CFU is determined in them, as described above, and subcultures containing 64-100% of toxin-producing CFU are selected. Suspensions of 10 ⁶ spores in 0.5 ml are prepared from spores of selected strains and subcultures and 30 guinea pigs weighing 320-350 g per culture are immunized subcutaneously with such doses. After 21 days, each of the animal groups was subcutaneously infected with an anthrax test strain 71/12 in doses of 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ spores, 6 animals per dose. As a control, the same strain 30 infected unvaccinated guinea pigs subcutaneously in a volume of 0.5 ml doses of 10, 10 ^2, 10 ^3, 10 ^4, 10 ⁵ and 10 ^6, 6 animals per dose. After 10 days, the number of surviving animals from each infectious dose is calculated and the LD ₅₀ for vaccinated and unvaccinated guinea pigs is determined according to Reed-Mench. The immunity index is calculated, which is equal to the ratio of LD _{50 of the} infecting strain for vaccinated strains to LD _{50 of} the same strain for unvaccinated animals. In parallel, the immunity index for the reference strain of the STI-1 vaccine is determined by the same technique. Those cultures whose immunity index is equal to or higher than the immunity index of the reference drug are selected as immunogenic.

Example 1. Spore suspensions of the reference preparation of STI-1 vaccine, four variants of the vaccine strain 228/8 and five capsuleless variants of strain 228 were prepared, which were selected as possible new vaccines, 25-50 colonies being formed on the plates from which were sown on Hottinger agar. RYE cups were prepared in accordance with the words and each of the options was sown for 10-20 cups, the crops were placed in an anaerostat, from which 25% of the air was pumped out and replaced with carbon dioxide. Crops were incubated at 37 ^° C for 48 h, then the plates were removed from the anaerostat and transferred to the refrigerator, kept at 4 ^° C for 3 days, after which the plates were examined under natural light against a dark background using a sheet of black paper. The total number of colonies, the number of colonies surrounded by halos of immunoprecipitation were calculated and their percentage for each of the options was determined. Suspensions of 10 ⁶ spores in 0.5 ml were prepared for all variants and 30 guinea pigs weighing 320-350 g per culture were immunized subcutaneously with such doses. After 21 days, each of the animal groups was subcutaneously infected with an anthrax test strain 71/12 in doses of 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ spores, 6 animals per dose. As a control, 30 unvaccinated guinea pigs were subcutaneously infected with the same strain in a volume of 0.5 ml in doses of 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 6 animals per dose. After 10 days, the number of surviving animals from each infectious dose was calculated and the LD _{50 was} determined for vaccinated and unvaccinated guinea pigs according to Reed-Mench. The immunity index was calculated, which is equal to the ratio of LD _{50 of the} infecting strain for vaccinated strains to LD _{50 of} the same strain for unvaccinated animals. In parallel, the immunity index for the reference strain of the STI-1 vaccine was determined by the same method. The results are shown in table 1.

 Example 2. The selection of immunogenic strains and their subcultures among 18 test strains and vaccine series in accordance with the above descriptions of the proposed method and the prototype method. Comparative determination results are given in table.2.

From table 1 it is seen that the selected ranges of the percentage of colonies with immunoprecipitation correspond to the assessment of the immunogenicity of the strains obtained in vivo. The interval of 1-10% corresponds to non-immunogenic strains, since their immunity index is 1-2, which does not exceed the spread within the same order and may reflect the spread of the LD ₅₀ values of the infecting strain with several determinations.

 An intermediate interval of 11-63% corresponds to low immunogenic strains, the immunity index of which is more than 10, but less than the immunity index of the reference drug. These strains provide protection against infection only part of the animals taken in the experiment. In the range of 64-100% of colonies with immunoprecipitation, all those strains are placed whose immunity index is equal to or higher than the immunity index of the reference strain STI-1 and which, by definition, are immunogenic.

 Thus, the percentage range of immunoprecipitated colonies formed on SOPEC directly correlates with the immunity index of vaccine strains and can be used as a criterion for the selection of immunogenic strains and their subcultures in vitro.

 The data given in table 2 show that using the proposed method, it is possible to select 17 immunogenic strains and subcultures of anthrax vaccines, while in the prototype method selection is limited to nine strains, and the selection of immunogenic subcultures of non-immunogenic strains is impossible. This consumes the same number of laboratory animals for 540 guinea pigs. The strain Sterne v2 is a culture of a non-immunogenic clone selected by us in the population of the STERNE immunogenic strain, the population of which does not contain toxin-producing CFU and is taken as a negative control. If the task is reduced only to the selection of immunogenic strains, then in this case, to select the same 9 strains by the proposed method, instead of 540, only 270 guinea pigs will be required. Thus, in any case, the proposed method is twice as effective as the prototype method. The inventive method is especially effective in checking the immunogenicity of strains for which the immunity index was previously determined, for example, in vaccine series after the expiration of the shelf life, since in this case we can limit ourselves to the first step of determining in vitro experiment, and then only 180-360 cups of RYE will be required for this instead of 540 guinea pigs. In addition, if the immunogenicity during storage decreased, it can be increased by selecting an immunogenic subculture in vitro, which is unattainable in other ways.

 Therefore, the proposed method for the selection of immunogenic strains and subcultures of anthrax vaccines has higher efficiency than the existing method, and allows you to use it for a new purpose to increase the immunogenicity of vaccines.

Sources offormation
1. Uchida I. Hashimoto, K. Terakado N. Virulence and immunogenicity in experimental animals of Bacillus anthracis strains harboring or lacking 110 Mda and 60 Mda plasmids // J. Gen. Microbiol.-1986.-Vol. 132. P.557-559.

 2. Welkos S.L. Friedlander A.M. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice // Microbiol. Pathogenesis. 1988. Vol. 5. P.127-139.

 3. The main criteria for the selection of anthrax vaccine strains for the manufacture of live anthrax vaccines for immunization of people. M.1982.

 4. Ivins V.E. Welkos S.L. Cloning and expression of Bacillus anthracis protective antigen gene in Bacillus subtilis // lnfect.and Immun. 1986. Vol. 54.

Claims (1)

The method of selecting immunogenic strains and subcultures of live anthrax vaccines, which consists in a single immunization of guinea pigs with several doses of the studied cultures with infection after 21 days of immunized animals and the subsequent calculation of the index of immunity in comparison with the reference vaccine, characterized in that before the immunization of animals in the studied cultures is determined exotoxin production in vitro and relative number of CFU collected Toh ⁺ nonimmunogenic when a value of 1 to 10% nizkoimmunogennye at 11 and 63% immunogenic etc. 64 100% followed by selection in populations of low and non-immunogenic cultures immunogenic clones was obtained from these subcultures.

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