RU2355423C1 - Adjuvant - Google Patents

Abstract

FIELD: medicine; virology.

SUBSTANCE: claimed is application of birch bark extract as adjuvant. Invention can be used in veterinary and medicine.

EFFECT: birch bark extract increases immunogenity of target antigen in combined introduction.

3 cl, 4 tbl, 4 ex

Description

The present invention relates to the field of medicine and veterinary medicine, and more particularly to virology.

The threat of viral epidemics continues to worry mankind. In developing a strategy to combat viral pathogens, virologists are unanimous in their opinion that vaccines should take the leading place as the most effective medical means of preventing and reducing mortality.

Therefore, over the past few years, intensive development of vaccine preparations has been carried out all over the world, since vaccines (live and inactivated) have shown themselves to be harmless, safe for others immunogenic preparations, although, in some cases, their effectiveness leaves much to be desired.

In relation to inactivated vaccines, the greatest efficiency can be predicted if a virulent virus strain is used as the basis, but the production complexity sharply increases due to the danger of work and the need for strict control over the completeness of inactivation of the pathogen.

A distinctive feature of inactivated vaccines is the inclusion of adjuvants in their composition. An adjuvant is a substance added to a vaccine preparation to increase the body's production of antibodies. Most often, aluminum hydroxide is used for these purposes. Recent studies have shown that in addition to increasing the residence time of the antigen at the injection site and its adsorption, it increases the expression of class II HLA antigens, B 7-2 molecules (CD 86 and CD 83), the expression of costimulatory molecules, adhesion molecules and the production of interleukin-4 , which is accompanied by a change in morphology towards dendritic cells.

As a result of recent studies, other more active adjuvants have been developed. In animal experiments, the efficacy of immunization with GpG oligodeoxynucleotides isolated from bacterial DNA and synthetically created has been shown. The efficacy of mucosal ripposis vaccination is increased with the use of cholera vibrio toxins and enterotoxins of Escherichia coli, coraubazidan (a polysaccharide produced by extracellularly apatogenic fungus), chitosan (a polysaccharide obtained from the shell of crustaceans), baculovirus and a synthetic double-strand TLR4 synthetic agonist: TLR4: ) The prophylactic activity of inactivated vaccines with the new MF 59 adjuvant for immunization of the elderly, especially with chronic diseases, was higher than that of conventional sorbed drugs.

The adjuvants used in medical practice, in particular Freund's adjuvant, are expensive and contain residues of cellular structures that can cause the production of extraneous antibodies. Their high viscosity makes it difficult to work on small animals.

The present invention is the search for a new adjuvant, largely devoid of these disadvantages. Studies conducted by the authors of the present invention have revealed the possibility of using birch bark extract (white part of birch bark) with a predominant betulin content as such adjuvant. It is preferable to use the indicated preparation in the form of nanoparticles.

The object of the study was the experimental samples of inactivated viral antigens in combination with an adjuvant based on nanoparticles of “Dry birch bark extract” with a predominant content (65%) of betulin (hereinafter referred to as betulin). The specified extract is made by the applicant of this application - LLC “Birch world” and is used as a dietary supplement.

A comparative study of the immunogenic properties of the recombinant hepatitis B surface antigen and inactivated tick-borne encephalitis (CE) antigen in combination with adjuvants based on betulin nanoparticles was carried out.

The following drugs were used to immunize animals:

- Recombinant surface antigen of hepatitis B virus (semi-finished vaccine), the concentration of HBsAg - 50 μg / ml;

- Inactivated tick-borne encephalitis virus antigen (whole-virion semi-finished product of the vaccine), protein concentration E-10 μg / ml;

- Adjuvant 1 (AD-1): dry birch bark extract with 65% betulin content, (BES-65);

- Adjuvant 2 (AD-2): dried preparation of nanoparticles BES, (RY);

- Adjuvant 3 (AD-3): 0.5% aqueous solution of betulin nanoparticles (GIPC).

- Known adjuvant (aluminum hydroxide, Gal): 0.5% aqueous solution.

Adjuvants AD-2 and AD-3 were prepared as follows. Adjuvant AD-3 (0.5% aqueous solution of nanoparticles):

- dissolution of dry birch bark extract (BES-65) in an organic solvent at a concentration of 2.5-5 g / l;

- the addition (injection) to the specified solution of a large amount of water (up to 25 volumes of water relative to the volume of the solution);

- removal of solvent and the bulk of the water.

Adjuvant AD-2 (dried nanoparticle preparation):

- a cryoprotectant (for example, sorbitol) is added to the resulting nanoparticle aqueous solution (see preparation of AD-3) and lyophilized.

Prior to immunization, AD-1 and AD-2 adjuvants were dissolved in phosphate buffer (pH 7.5), sonicated for 15 minutes, and then cooled.

In the experiments, drugs of the following composition were studied:

Based on Recombinant Hepatitis B Surface Antigen

Preparation 1, composition: HBsAg - 10 μg / ml, GAL - 500 μg / ml

Preparation 2, composition: HBsAg - 10 μg / ml, AD-1 - 500 μg / ml

Preparation 3, composition: HBsAg - 10 μg / ml, AD-2 - 500 μg / ml

Preparation 4, composition: HBsAg - 10 μg / ml, AD-3 - 500 μg / ml

Drug 5, composition: HBsAg - 10 μg / ml, AD-1 - 100 μg / ml

Preparation 6, composition: HBsAg - 10 μg / ml, AD-2 - 100 μg / ml

Drug 7, composition: HBsAg - 10 μg / ml, AD-3 - 100 μg / ml

Based on inactivated tick-borne encephalitis antigen (TBEV)

Drug 8, composition: TBEV protein E -1 μg / ml, GAL - 500 μg / ml

Preparation 9, composition: TBEV protein E - 1 μg / ml, AD-1 - 500 μg / ml

Preparation 10, composition: TBEV protein E - 1 μg / ml, AD-3 - 500 μg / ml

Preparation 11, composition: TBEV protein E - 1 μg / ml, AD-1 - 100 μg / ml

Preparation 12, composition: TBEV protein E - 1 μg / ml, AD-3 - 100 μg / ml

As reference drugs used:

Ref. 1: HBsAg - 10 mcg / ml

Ref. 2: TBEV protein E - 1 μg / ml

Ref. 3: AD-1 - 500 mcg / ml

Ref. 4: Saline

The titers of specific antibodies in the blood sera of immunized mice were determined by enzyme-linked immunosorbent assay (ELISA) and inhibition of hemagglutination (RTGA). For the quantitative determination of TBE virus, an experimental test system was used using monoclonal antibodies to surface glycoprotein E.

The protective activity of drugs with the TBE virus was determined by intraperitoneal administration of 300-400 lethal doses of the Absettarov reference strain one week after the last immunization. Within 14 days after infection, the specificity of dead animals was recorded.

Example 1

The immunogenic and protective activities of various viral preparations were evaluated by subcutaneous immunization of outbred white mice weighing 14-16 g. The drugs were administered once and twice with an interval of 14 days. As a control, white mice were injected subcutaneously once and twice with reference preparations.

21 days after the end of the immunization cycle, animals were taken from animals from each group and a combined pool of blood serum was prepared to determine the titer of AT in ELISA and rtga.

The results characterizing the immunological effectiveness of various drugs are presented in table 1.

Table 1 Drug No. Antigen Single dose titer Double administration titer IFA RTGA IFA RTGA one HBsAg 1: 320 1: 640 2 HBsAg 1: 640 1: 1280 3 HBsAg 1: 320 1: 2560 four HBsAg 1: 640 1: 1280 5 HBsAg 1:80 1: 320 6 HBsAg 1: 160 1: 160 7 HBsAg 1:80 1: 320 8 CE 1:80 1:40 1: 320 1:80 9 CE 1:80 1:40 1: 640 1:80 10 CE 1: 160 1:40 1: 1280 1: 160 eleven CE 1:40 1:20 1: 320 1:40 12 CE 1:20 <1:20 1: 160 1:80 Ref. 1 HBsAg 1:40 1: 160 Ref. 2 CE 1:20 1:10 1:40 1:40 Ref. 3 <1:20 <1:10 1:10 <1:10 Ref. 4 <1:10 <1:10 <1:10 <1:10

The immunogenicity of the preparations containing adjuvants did not differ much from each other, with the exception of ELISA titers when immunizing HBs Ag with AD-2.

As follows from the data obtained, under conditions of single and double immunization, experimental samples of antigens when administered with adjuvants based on nanoparticles of birch bark extract showed higher immunogenicity than the antigen itself and the antigen adsorbed onto aluminum hydroxide.

The highest immunogenic efficiency was shown by the HBs Ag preparation, administered with AD-2 adjuvant. This adjuvant increased the efficiency of immunization for both HBs Ag and inactivated CE. The difference was that for inactivated CE, more pronounced results were obtained after the second immunization.

Example 2

For the preparation of the experimental vaccine, the adjuvant AD-2 SBES = 2% was mixed with the Newcastle disease virus antigen in a 1: 1 ratio. The mixture was thoroughly shaken and injected into the pectoral muscle in a volume of 0.5 ml. Another group of chickens was inoculated with an inactivated vaccine against Newcastle disease, made at FSI ARRIAH based on the ISA-70 adjuvant (30: 70 ratio Ag: Hell). The third group of chickens remained unvaccinated.

Blood sera were taken on the 14th, 21st and 31st days after vaccination and were examined in the RTHA of rooster erythrocytes by the Newcastle disease virus.

The test results are presented in table 2.

table 2 Drug name The number of goals in the group Dose of the drug Group average antibody titer in log2 Before vaccination 14 days after vaccination 21 days after vaccination 31 days after vaccination Sample No. 2 SBEC = 2% with Newcastle disease virus antigen 1: 1 10 0.5 ml 2 7.8 8.6 7.9 Newcastle disease inactivated emulsion vaccine based on ISA-70 10 0.5 ml 2 7.7 8.7 8.8 Unvaccinated chickens 10 0 2 2 four four

Example 3

A prototype vaccine with AD-2 adjuvant was prepared by mixing the drug with the inactivated avian reovirus antigen in a 1: 1 ratio and thoroughly mixing them. At the same time, using the same antigen, a sample was prepared based on the ISA 70 adjuvant in an antigen-adjuvant ratio of 30:70, which was used to compare the immune response.

Chicks of 45 days of age were vaccinated with a test sample with a nanoparticle preparation and a control sample based on ISA 70 at a dose of 0.5 ml in the chest area. A group of chickens was left unvaccinated.

Blood serum was collected 15 and 30 days after vaccination and was tested in ELISA using the appropriate kit for determining the level of antibodies.

The research results are presented in tables 3 and 4.

Table 3 Drug name The number of goals in the group Dose of the drug Group average titer Before vaccination. 15 days after vaccination 30 days after vaccination Sample 1.
Bird reovirus antigen with the preparation of nanoparticles No. 2 SBES = 2% in a ratio of 1: 1 10 0.5 ml 1125 2473 3104 Sample 2.
ISA-70 adjuvant bird avian reovirus antigen (30:70) 9 0.5 ml 1034 3233 4026 Unvaccinated chickens 10 0 1164 1235 1267

Example 4

A prototype vaccine with AD-2 adjuvant was prepared by mixing it with the inactivated antigen of metapneumovirus birds in a ratio of 1: 1 and thoroughly mixing. As in experiment 2, a control sample was prepared based on the ISA 70 adjuvant.

Chickens of 45 days of age were vaccinated with experimental samples with a preparation of nanoparticles and control samples based on ISA 70 at a dose of 0.5 ml in the chest area. A group of chickens was left unvaccinated.

Blood serum was collected 15 and 30 days after vaccination and was tested in ELISA using the appropriate kit for determining the level of antibodies.

The research results are presented in table 4.

Table 4 Drug name The number of goals in the group Dose of the drug Average% block per group Before vaccination 15 days after vaccination 30 days after vaccination Sample 3.
Antigen of metapneumovirus of birds with the preparation of nanoparticles No. 2 SBES = 2% in a ratio of 1: 1 9 0.5 ml 22.5 (negative) 46.2 (positive) 43.1 (positive) Sample 4.
Bird metapneumovirus antigen with adjuvant ISA-70 (30:70) 7 0.5 ml 24.3 (negative) 63.9 (positive) 66.5 (positive) Unvaccinated chickens 10 0 27.6 (negative) 25.4 (negative) 27.8 (negative)

The results of the studies show that the use of the drug of birch bark extract nanoparticles together with antigens of Newcastle disease virus, avian reovirus and meta pneumovirus birds in a ratio of 1: 1 and intramuscular administration at a dose of 0.5 ml per chicken has a pronounced adjuvant effect, which is manifested in an increase in the titer of antibodies against the corresponding antigen and not inferior in importance to the action of the adjuvant ISA70 for Newcastle disease virus and avian reovirus.

Claims (3)

1. The use of birch bark extract as adjuvant. 2. The use according to claim 1, characterized in that the birch bark extract is used in the form of nanoparticles. 3. The use according to claim 2 in the form of an aqueous solution of nanoparticles.