RU2262949C1 - Antigenic preparations for preventing glanders in people and animals - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: the suggested preparation consists of formalin-inactivated suspension of B.mallei deep culture sorbed upon aluminum hydroxide. The content of components, 1 cu. cm of the preparation: inactivated cell B.mallei 0.1-10 billion, aluminum ions up to 2.5 mg, formaldehyde - not more than 0.005%. Vaccination should be performed once annually, stable immunity should be formed to 7-14 d.

EFFECT: increased immunogenicity.

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Description

The invention relates to medicines containing antigens, in particular to antigenic preparations, and can be used to prevent glanders in humans and animals.

There are known attempts to create a drug based on attenuated and recombinant strains of the glanders pathogen (see Shantyr N. Vaccination of glanders with frogs // Arch. Vet. Sciences. - 1902, No. 4. - P.756-781; Horomansky A. Bacillus glanders in the body of a pigeon // Arch.Vet.Sci. - 1911, No. 2. - P.149-154; Konev DF From works on the study of glanders // Proceedings of the meeting on bacteriology, on the development of measures against glanders ... - 1912. - Issue 1. - P.73-79; A. Dedyulin On the issue of immunization of horses against glanders // In the book: Collection of works in memory of I.M.Sadovsky. - St. Petersburg, 1912. - S.80- 86; Vyshelesky S.N., Maccabees V.I. Further research according to glanders // Transactions of the II Congress of Scientific and Practical Veterinarians of Ukraine. - Kiev, 1927; Krylov V.D. Weakening of the virulence of B.mallei // Practical Veterinary Medicine. - 1929, No. 5. - S.432-440; Bakulin M .K. et al. The use of genetic methods to develop vaccines against glanders and melioidosis // Materials of the Russian scientific conference: Immunology and specific prevention of especially dangerous infections.

Saratov, 1993; Patent WO Number 2004006857 "GLAND-ERS / MELIODOSIS VACCINES").

In common with the claimed drug is the use as a basis for the manufacture of a prophylactic agent of B. mallei strains with reduced virulence.

These drugs can not be used for the prevention of glanders due to their low immunogenicity and high frequency of development of the chronic form of this disease in immunized animals. This circumstance may be due to the instability of the biological properties of attenuated and recombinant strains, often observed in the process of their maintenance, and the ability of the Sap microbe to L-transformation under the influence of factors of immunobiological protection of the macroorganism.

A medicament is known intended to prevent glanders in susceptible B.mallei-sensitive mammals containing porin protein with a molecular weight of 37-40 kDa, which is isolated from the outer cell wall of the sap microbe (see Patent RU No. 2052993 "Method for the prevention of glanders").

In common with the claimed preparation is the use of the antigenic complex B.mallei as an immunogen, which induces specific immunity to the glanders pathogen when introduced into animals.

A significant disadvantage of this tool is its low immunogenicity, which necessitates the multiple immunization of golden hamsters to achieve the desired protective effect. The complex scheme for isolating the antigenic complex from the cell wall of the sap microbe allows one to obtain porin proteins only in preparative quantities. This tool cannot be used to prevent glanders in humans due to the lack of data on its reactogenicity and immunological safety in primates and volunteers. It is only known that membrane protein preparations with a molecular weight of 37-40 kDa do not have a toxic effect against golden hamsters in doses of 30 mg / kg of animal weight.

Closest to the claimed is the drug, known in domestic literature as the "Anamorph of Legra" (see Legroux. A new method of vaccination using anabacteria // La presse Medicale. - 1933, No. 6. - p. 118-120). It is a lysed in distilled water, inactivated by 0.3% formalin and heating at 60 ° C cell culture pathogen glanders. It is known that the drug in many cases protected guinea pigs and horses from glanders infection. The reaction to the introduction of "Anamorv Legra" was expressed in people with malaise within 24-48 hours and redness of the injection site.

A common essential feature with the claimed drug is the use of an inactivated bacterial culture of the pathogen glanders as an antigen.

The disadvantage of this drug is the instability of the properties of the culture of the pathogen glanders used for manufacturing, its low immunogenicity.

These shortcomings are due to the lack of control methods for both the finished form of the drug and the biological properties of the production strain (data on the strain are absent in the message), its maintenance scheme, which was probably the cause of the instability of the results and served as the main reason for the incompleteness of this work (see Legroux, A New Method of Vaccination with Anabacteria // La presse Medicale. - 1933, No. 6. - p. 118-120).

The objective of the invention is to develop a highly immunogenic, low reactogenic and immunologically safe antigenic drug for the prevention of glanders in humans and animals.

The problem is solved due to the fact that the preparation contains a formalin inactivated suspension of B.mallei deep culture adsorbed on aluminum hydroxide in the following ratio of components of 1 cm: inactivated B.mallei cells - from 0.1 to 10 billion, aluminum ions - up to 2, 5 mg, formaldehyde - not more than 0.005%.

The dose for immunizing people is contained in 0.5 ml of the proposed antigenic drug. Immunization is carried out once a year, subcutaneously.

Comparing the values of the indices of indirect tests and the levels of protection against subcutaneous and aerogenic infection with virulent cultures of the sap microbe, determined in experimental animals, with the dynamics of the indicators of the cellular and humoral parts of the immune system in humans, it can be extrapolated that a tense immunity after a single use of the claimed drug provides protection against development infections of at least 70% vaccinated. Immunity is formed by 7-14 days and lasts for 6 months, and after 12 months, positive seroconversion persists in about 25-35% of vaccinees.

Along with this, the protective effect of porin protein (prototype), studied only on golden hamsters, was achieved only using a complex scheme of repeated administration of the drug. Data on the quantitative expression of the protective effect of “Anamorous Legra” (analogue) due to incomplete work in the literature are not available.

Between the set of essential features of the claimed object and the achieved technical result there is a causal relationship, namely:

1. The development of full-fledged immunity against glanders is achieved in the claimed solution using an inactivated antigen as an immunogen, which is a suspension of formalin-treated deep culture of strain 11, which has stable biological properties and is not inferior in immunogenicity to highly inactivated cultures of the highly virulent strain C-5 B. mallei.

2. Low reactogenicity, immunological safety of the antigenic drug are ensured by the use of 11 B.mallei strain 11 as an avirulent production strain, which has specific safety and security for personnel working with it, as well as the use of a protein-free synthetic nutrient medium for its cultivation.

3. The long periods of formation of specific immunity even after a single administration of the inventive antigenic drug are achieved by using an adjuvant, aluminum hydroxide, as part of the preparation.

The invention allows:

1. To increase the safety of people and animals in epidemic foci and in case of deterioration of the sanitary-epidemiological situation on glanders, as well as personnel directly working with B.mallei.

2. To carry out immunization on the background of antibiotic therapy glanders.

Antigenic preparation is a homogeneous suspension of a yellowish color without impurities and flakes. When stored at the bottom, a precipitate forms, which easily breaks when shaken.

The possibility of carrying out the claimed invention is shown by the following examples.

1. Obtaining antigenic drug

For the manufacture of the drug using strain 11 .mallei from the Federal collection of standardized microorganisms NIIM MO RF, registration number - KSM-4-2.

Standardization of the antigenic preparation begins with the control of the specified strain, which is characterized by the following features and properties.

Morphological and tinctorial properties: gram-negative immobile polymorphic bacilli.

Cultural properties: when grown in solid nutrient medium, after 46-50 hours it forms translucent S-colonies.

Biochemical properties: ferments glucose, galactose, glycerin with the formation of acid without gas, ferments milk.

Serological properties: agglutinates in the reaction (RA) with a standard enterprise sample (SOP) of the diagnostic multivalent serum diagnostic serum (see Patent RU No. 2188035) to its maximum titer.

Pathogenic properties: virulence, LD ₅₀ live microbial cells for golden hamsters, subcutaneous - more than 1 · 10 ⁹ ; virulence, ID ₅₀ live microbial cells for guinea pigs, subcutaneous - more than 1 · 10 ⁹ ; toxicity, LD ₅₀ live microbial cells for guinea pigs, intraperitoneally 36 hours after infection - (5-7) · 10 ⁹ .

Strain 11 B.mallei is cultivated on a solid nutrient medium, and then in a fermenter in a liquid nutrient medium at 36-38 ° C for 72-96 hours.

The grown microbial culture is clarified on a separator and the microbial suspension is inactivated with formalin for 20-30 days at a temperature of 36-38 ° C.

Inactivated suspension is a microbial semi-finished product that is used to prepare an antigenic preparation. In a microbial semi-finished product, it is determined: the completeness of inactivation and the presence of extraneous microflora by microscopy of Gram-stained smears and seeding on nutrient media; the concentration of microbial cells by direct counting in hemocytometric chambers; the content of antigens in the reaction of diffusion precipitation with a standard sample of an enterprise serum diagnostic sapny agglutinating rabbit dry; pH formaldehyde concentration. Microbial semi-finished product must meet the following quality indicators:

do not contain contaminating agents;

be specifically sterile, not contain live cells of the production strain;

contain (0.1-10) · 10 ⁹ microbial cells in 1.0 cm ³ ;

give a positive agglutination reaction (not less than 3+) with SOP of serum of diagnostic sap in a dilution of 1:25 in a titer of at least 1:32;

formaldehyde content should be from 0.2 to 0.4%;

have a pH of 6.3 to 7.0.

The formaldehyde is neutralized by adding a 30% sodium metabisulfite solution, the pH is adjusted with a 0.5N sodium hydroxide solution.

Alumina hydrate is added to the microbial suspension to a final concentration of aluminum ions of up to 2.5 mg in 1.0 cm ³ . In the antigen preparation control:

appearance;

authenticity in the reaction of diffusion precipitation with a standard sample of an enterprise serum diagnostic sapa agglutinating rabbit dry;

pH according to the method described in the Global Fund, XI, ed., issue 1, p.113;

sealing according to the method described in MUK 4.1 / 4.2.588-96, p.63;

nominal volume according to the methodology, Global Fund XI ed., issue 2, p. 141;

mechanical inclusions in accordance with MUK 4.1 / 4.2.588-96, p.126;

total nitrogen according to FS 42-3874-99, p. 7;

general and specific sterility according to the method described in MUK 4.1 / 4.2.588-96, p.25;

toxicity in accordance with the methodology described in the Global Fund XI ed., issue 2, p. 182;

specific safety by staging a bioassay on golden hamsters;

specific activity in guinea pigs during their subcutaneous infection;

aluminum ion content according to FS 42-3874-99, p.7, analysis conditions - according to MUK 4.1 / 4.2.588-96, p. 110;

formaldehyde content according to FS 42-3874-99, p.7.

Antigenic drug should meet the following quality indicators:

look like a homogeneous suspension of a yellowish color without impurities and flakes, during storage of which a transparent supernatant and sediment are formed, which easily breaks when shaken;

to form in the RDP one line of precipitation with the SOP of the serum of diagnostic polyvalent sap in a dilution of 1: 4 in a titer of at least 1: 2;

have a pH of from 6.8 to 7.4;

be airtight;

have a nominal volume of at least 3.0 ml;

the total nitrogen content should not exceed 0.3 mg in 1.0 cm ³ ;

must not contain contaminating agents and live microbes B.mallei;

should not form necrosis, abscesses, weight loss or death of white mice and guinea pigs during the observation period;

must protect at least 70% of guinea pigs when infected with B. mallei C-5 culture at a dose of 10 ID ₅₀ from disease or their death during the observation period;

the concentration of aluminum ions should be up to 2.5 mg in 1.0 cm ³ ;

the residual formaldehyde content should not exceed 0.005%.

All operations are carried out in compliance with aseptic rules of the requirements of the Sanitary Rules SP 1.3.1285-03.

2. The specific activity of the claimed antigenic drug and analogues

Comparison of the specific activity of the claimed antigenic drug and analog drugs was carried out in experiments on golden hamsters. Animals were vaccinated once, subcutaneously, with 0.2 ml of the developed preparation and preparations prepared on the basis of strains C-5 and 5584 B. mallei from the Federal Collection of Standardized Microorganisms of the NIIM MO RF (registration numbers - KSM-4-1 and KSM-4- 3, respectively) by the method of Legra (prototype). For the porin proteins (analogue), various administration schemes were used, which are presented in Table 1. 15 days after the last immunization, the animals were infected subcutaneously with a two-day-old agar culture of the highly virulent strain C-5 glanders pathogen used to control the immunobiological properties of the antigenic preparation. The resistance index (IR) was calculated by the ratio of the LD ₅₀ value for immunized individuals to the LD ₅₀ value of the control (without drug administration) group of animals. The results are shown in table 1.

Analysis of the data shown in table 1 shows that the effectiveness of the proposed antigenic drug is more than 1.5-2 times greater than the protective effect of the preparations prepared according to Legroux (prototype), as well as the sap microbe isolated from the cell wall (analogue), even with repeated introduction of the latter.

The duration and intensity of the created immunity was evaluated in experiments on guinea pigs immunized with the claimed drug and analogous drugs according to the schemes shown in table 2. Control infection of animals with a culture of highly virulent strain C-5 at a dose of 10 ID _{50 was} carried out subcutaneously after 15, 30, 60 and 90 days after the introduction of the immunogen.

The experimental results are shown in table 2, indicate that the claimed antigenic drug is able to create in experimental animals a certain level of resistance to infection with the test strain, which stably remains for 3 months. At the same time, vaccination of guinea pigs with preparations prepared according to Legra, as well as triple administration of porin proteins in animals creates a less intense immunity to infection by the sap microbe, which tends to decrease already in the first months after subcutaneous administration.

3. Harmlessness and reactogenicity

The safety and reactogenicity of the claimed antigenic drug and analogue drugs were studied in the experience of "acute" toxicity in various types of experimental animals.

All preparations were administered once: white mice intraperitoneally, 0.2 ml each, golden hamsters and guinea pigs subcutaneously, 1.0 ml each, rabbits intravenously, 5.0 ml every 20 seconds, primates subcutaneously, 0 5 ml. The doses of protein isolated from the outer wall of the Sap microbe for these animal species were 1, 5, and 25 mg, respectively. Assessment of toxicity was carried out according to the results of observations of the behavior, body temperature and weight of animals within 7 days after administration of the drug.

Studies have shown that the introduction of the claimed drug, porin proteins and a prototype based on strain 5584 B. mallei does not cause any changes in experimental animals, which, after immunization, ate food well and did not seem to respond to immunization.

The use of an inactivated preparation based on strain C-5 B. mallei led to weight loss, an increase in body temperature, the development of abscesses at the injection site in some individuals, as well as the death of up to 30% of small laboratory animals. In 25% of hamadrilian baboons and 60% of rabbits, immunization was accompanied by a transient temperature reaction. The data obtained testified to the toxicity of the anamorvus Legra, prepared on the basis of strain C-5.

Thus, comparative studies have established that the inventive antigenic drug, along with the drug-analogue and prototype, prepared on the basis of strain 5584, is low reactogenic.

In addition, in accordance with the requirements of the guidelines that determine the procedure for preclinical trials of new MIBPs, immunological safety, “chronic” toxicity, local effects, pyrogenicity, the effect on hematological parameters, central nervous system, and detoxifying liver function of an antigenic drug were evaluated.

The results of his laboratory experimental (preclinical) study testified to the benign course of the immune process in various types of experimental animals and, therefore, the safety and low reactogenicity of this drug.

4. Vaccination schedule in humans

The vaccination scheme of the claimed antigenic preparation was determined according to the principle of selecting the minimum dose and the frequency of its administration, which ensure that vaccines develop a high level of specific protection and do not cause an increased number of post-vaccination reactions. For this purpose, three drug administration regimens were tested: once in 0.5 ml, once in 1.0 ml, twice in 0.5 ml (with an interval of 30 days). In accordance with the "Program ... approved by the chairman of the committee of the IIBP of the Ministry of Health of Russia, 21 volunteers were administered the antigenic drug according to the indicated schemes. The results of the study are shown in table 3.

The data shown in table 3 indicate that the pre-selected vaccination dose of the inventive antigenic drug, which provides protection for at least 70% of model animals when infected with B. mallei, is sufficient for the formation of specific immunity in humans. The increase in the vaccination dose or the frequency of administration of the drug in two times was not accompanied by a significant change in the indices of indirect tests. In addition, a single injection of the inventive antigenic drug in the number of general and local reactions was less reactogenic in comparison with other schemes of its use.

The results of the State trials of an antigenic drug for the prevention of glanders in humans and animals at a dose and according to the scheme selected at the stage of limited clinical trials confirmed its immunological effectiveness, immunological safety and low reactogenicity.

Thus, as a result of studies on volunteers, the size of the vaccination dose of the claimed antigenic drug for humans was selected: once, subcutaneously, 0.5 ml each.

Based on the results, experts of the GISK them. L.A. Tarasovich, a conclusion was drawn on the advisability of registering the claimed drug and the approval of all regulatory and technical documentation for this drug ("Production Regulations ...", "Pharmacopoeia Article of the Enterprise", "Instructions for Use").

For the production of antigenic drug in the NIIM MO RF equipped with a hardware-technological line.

The drug is used as follows.

Vaccination is carried out subcutaneously by a syringe method by introducing one inoculative human dose of an antigenic preparation ((0.05-5) · 10 ⁹ microbial cells) in a volume of 0.5 cm ³ into the region of the lower corner of the left shoulder blade. Revaccination (a single administration of an antigenic preparation in a volume of 0.5 cm ³ - (0.05-5) · 10 ⁹ microbial cells) is carried out 1 year after the previous immunization.

Contraindications:

pregnancy;

severe anaphylactic reactions (shock, angioedema of the larynx, etc.);

allergic reactions to the components of the antigenic drug or to the previous vaccination with this drug - vaccinations are carried out no earlier than 6 months after the reaction, according to the conclusion of the allergist;

acute infectious and chronic nonspecific diseases during the period of exacerbation - vaccinations are carried out no earlier than 1 month after recovery (remission);

immunodeficiency states.

Release form: on 3 ml in an ampoule. The package contains 10 ampoules.

Storage and transportation conditions, shelf life: the drug is stored and transported in accordance with the Sanitary Rules (SP) 3.3.2.1228-03. Storage - at a temperature of 2 to 10 ° C. Transportation - at a temperature of (8 ± 2) ° C, allowed - at a temperature of (10-25) ° C for no more than 10 days. The drug, subjected to freezing, is not subject to use. Shelf life is 1 year. Shelf life of the opened ampoule is 24 hours at a temperature of 0 to 8 ° C.

Table 1
A comparative assessment of the specific activity of the claimed antigenic drug and analogue drugs when immunizing golden hamsters The drug used for immunization Method of preparation (isolation) Dose for immunization The frequency of immunization Resistance index The inventive antigenic drug According to example 1 0.2 ml Once 12.5 Twice 18.6 Three times 19.8 Porin proteins Based on strain C-5 B.mallei, according to Novikova O.D., 1986 3 mg Once 1.8 Twice 3.0 Three times 5.8 Anamorv Legra Based on strain C.-5 B.mallei, according to Legroux, 1933 0, 2 ml Once 7.6 Twice 13,4 Three times 14,4 Based on strain 5584 B. mallei, according to Legroux, 1933 0.2 ml Once 4.6 Twice 7.8 Three times 8.6 The control - The drug was not administered 1 Notes
1 Porin proteins were administered at intervals of 21 days;
2 Doses of porin proteins were standardized for protein; Anamorva Legra - by the number of inactivated cells in 1 ml of the drug table 2
The results of assessing the tension and duration of immunity in guinea pigs during immunization with the claimed antigenic drug and analogues The drug used for immunization Scheme of drug administration Infection after immunization after ... days Number of animals E% total experience dead or infected The claimed Once fifteen 10 2 80 antigenic 0.2 ml each thirty 10 3 70 a drug 60 10 5 fifty 90 nine 4 56 Porinovye Three times fifteen 10 5 fifty squirrels 3 mg s thirty 10 6 40 interval 60 10 7 thirty 21 days 90 nine 8 eleven Anamorv Legra Once fifteen 10 4 60 based 0.2 ml each thirty 10 6 40 strain C-5 60 nine 6 33 90 7 5 29th Anamorv Legra Once fifteen 10 6 40 based 0.2 ml each thirty 10 7 thirty strain 5584 60 nine 7 22 90 7 6 14 The control A drug fifteen 10 10 0 did not enter thirty 10 10 0 60 10 10 0 90 nine nine 0

Table 3
The results of determining the indicators of direct and indirect methods in people immunized according to various schemes Scheme of drug administration

The value of the indicator of the indirect method ...,

IS RBTL, c.u. The content of specific antibodies in RPGA Lg x (x is the inverse of the titer) % seroconversions Once, 0.5 ml each 1.72 ± 0.12 0.94 ± 0.33 79 Once, 1.0 ml each 1.59 ± 0.12 0.76 ± 0.39 57 Twice, 0.5 ml 1.51 ± 0.08 0.89 ± 0.49 71

Claims (1)

A preparation for the prevention of glanders in humans and animals, containing an inactivated antigen of the sap microbe, characterized in that as an inactivated antigen it contains a formalin-treated suspension of B.mallei deep culture adsorbed on aluminum hydroxide in the following ratio of components in 1 cm ^{3 of the} preparation: inactivated B.mallei cells - from 0.1 to 10 billion; aluminum ions - up to 2.5 mg; formaldehyde - not more than 0.005%.