RU2322259C1 - Carrier of antigens - Google Patents

Abstract

FIELD: biotechnology, immunology.

SUBSTANCE: invention proposes a carrier of antigens as a lipid complex consisting of glycoside, cholesterol and lipid. As glycoside a carrier comprises holotoxin A₁, and as lipid - monogalactosyldiacyl glycerides (MGDG) of sea macrophytes taken in the weight ratio 3 (holotoxin A₁) : 2 (cholesterol) : (2-6) (MGDG). Prepared glycoside-cholesterol-lipid carrier shows stretched threadlike-tubular structure. Use of the prepared carrier of antigens allows enhancing the immunogenic activity of vaccine preparations, and to decrease or remove completely hemolytic toxicity of holotoxin A₁, and to eliminate inflammatory, painful, toxic and hemolytic effects of vaccines.

EFFECT: improved and valuable medicinal properties of carrier and vaccines.

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Description

The invention relates to biotechnology and immunology, in particular to drugs that combine the delivery system of antigens and natural mediators, and can be used in the field of medicine and veterinary medicine.

The creation of a new generation of vaccines involves the use of isolated antigens that can provide a highly specific immune response to bacterial, viral pathogens and tumor cells. The advantages of highly purified subunit vaccines in comparison with traditional immunization agents are characterized by the economy and safety of production, a decrease in the risk of side effects, and the possibility of obtaining oral preparations. At the same time, the immunogenicity of most individual antigens due to a number of features, such as low molecular weight of the antigen, solubility, are directly dependent and are mediated by stimulation of the cellular component of the immune system, namely T-helper cells. Therefore, the development of effective subunit vaccines is accompanied by the search for reliable, safe and adequate adjuvants.

Of interest are lipid adjuvants used as carriers in vaccine formulations. Lipids are able to bind simultaneously adjuvants and amphiphilic antigens and present them to immunocompetent cells in a corpuscular form. Antigens in the composition of the carriers have high immunological activity, and the carriers themselves are biodegradable and lack of their own immunogenicity. Complexes based on the lipid matrix have great prospects for solving the problem of adequate and prolonged presentation of vaccine antigens to the immune system.

It is known in the art that lipid carriers can exist as emulsions. For example, water-oil solutions of reverse micelles and bilayer vesicles from plant galactolipid digalactosidiacylglycerol (DGDG) are described [US Pat. RU 2127124, publ. 03/10/1999]. However, oil adjuvants have several disadvantages:

- with subcutaneous and intramuscular administration, local reactions of an inflammatory-necrotic nature are caused, and increased viscosity makes their introduction difficult;

- the specified emulsion does not give a significant increase in the immunogenicity of protein and other antigens, since DGDG itself does not have immunoadjuvant properties;

- instability during storage of the vaccine in the finished form as a result of phase separation and non-standard quality.

It is also known from the prior art that liposomes are used as a carrier for presenting antigens, the main structure-forming component of which is lamellar phosphatidylcholine [Allison AG, Gregoriadis G. Liposomes as immunological adjuvants. Nature. 1974. Nov 15; 252 (5480): 252].

Antigens in liposomal preparations can be considered as immunoadjuvants. Unlike other adjuvants, the formation of granulomas does not occur at the injection site of liposomal preparations, and even the prevention of toxic manifestations of encapsulated drugs is observed.

Liposome vesicles can include both hydrophobic antigenic material in the lipid bilayer and hydrophilic in the internal phase. Complexes of liposomes with antigen are able to stimulate antibody formation, activate phagocytes and be absorbed by macrophages in various organs and tissues of the body, and ensure the delivery of drugs (antigens) to the cell. The restriction of their use is a consequence of the following disadvantages:

- chemical and physical instability of vesicles;

- a relatively moderate immune response;

- inability to stimulate a cellular immune response, providing antiviral and antitumor protection.

The technical nature and the achieved result closest to the claimed technical solution is the immunostimulating complex (ISKOM). These are lipid-saponin particles, consisting of a mixture of Quil A plant triterpene glycosides, cholesterol and phospholipids [US Pat. RU 2120302, publ. October 20, 1998] (prototype). Such complexes have a very specific structure, confirmed by electron microscopy and are not lipid vesicles or liposomes. These are colloidal particles consisting of regular subunits with an open spherical structure that look like rings under a microscope. The basic structure of ISCOM is formed both in the presence of antigen and without it. The structural basis of ISCOM is the saponin-cholesterol complex, which contributes to the development of the immune response. ISCOM allows one to reduce the dose of antigens administered by an order of magnitude, induce significant antiviral immunity, and increase the level of the humoral immune response in adjuvant doses much lower than in conventional vaccines.

Serious disadvantages that prevent the widespread use of Quil A saponins as adjuvants and delivery systems in vaccines against human infectious diseases include the following:

- Quil A is a heterogeneous mixture of saponins, which is unacceptable for use in medicine, since a mixture of saponins with different chemical and biological properties complicates the standardization of ISCOM and can lead to unpredictable effects in vivo;

- high toxicity with intravenous administration of the drug due to high hemolytic activity;

- inflammatory and pain reactions in humans with intramuscular administration, therefore their use as a non-specific immunostimulant in humans is limited to oral administration;

- chemical instability in aqueous solutions due to hydrolysis of the ester bond in one of the side hydrocarbon chains;

- deacylated saponins lose their adjuvant properties, the ability to stimulate a first-class T-cell immune response and produce antigen-specific cytotoxic T-lymphocytes, which negatively affects the effectiveness of vaccines against cytopathic infections of a viral or bacterial nature, antitumor and antiparasitic vaccines.

Attempts to isolate from the Quil A fraction of more purified saponins with low cytotoxic properties lead to the fact that the obtained saponins either do not form structures in the form of ISCOM or do not possess the necessary immunostimulating activity.

The technical result of the proposed solution is to obtain a carrier of antigens having high immunological efficacy in the composition of subunit vaccines and characterized by the absence of toxicity, inflammatory and pain reactions when used.

The technical result is provided by obtaining a carrier of antigens in the form of a lipid complex consisting of glycoside, cholesterol and lipid; the complex contains holotoxin A ₁ as a glycoside, and monogalactosyl diacylglycerides (MGDG) as marine macrophytes as a lipid.

The technical result is optimally achieved by obtaining an antigen carrier in the form of a lipid complex in which the weight content of holotoxin A ₁ to cholesterol is constant and equal to 3: 2, and the weight content of monogalactosyldiacylglycerides (MGDG) from marine macrophytes can vary in the range (2-6).

The resulting lipid complex has an elongated filamentous-tubular structure with a particle length of 200 to 300 nm and a tube diameter of about 20 nm, which is different from the structure of the known immunostimulating complexes.

For the first time, an antigen carrier has been obtained that has high immunological and incorporating characteristics with respect to amphiphilic antigens, which provides an increase in the safety of vaccines and is characterized in that when it is used, a decrease in the toxicity of holotoxin A ₁ , which is part of the complex, is achieved.

The inventive antigen carrier is obtained by mixing holotoxin A ₁ , cholesterol and MGDG, taken in a weight ratio of 3: 2: (2-6) in the following order: mix solutions of cholesterol and MGDG in chloroform with a weight ratio of cholesterol and MGDG 2: (2-6 ), evaporated in vacuo to remove chloroform and add 3 parts by weight of a 0.4 percent aqueous solution of holotoxin A ₁ ; the mixture is solubilized, after which the total concentration of lipids (cholesterol and MGDG) is adjusted to 2 mg in 1 ml of suspension with phosphate-buffered saline at pH 7.2. The indicated concentration of lipids is optimal for electron microscopy studies. The resulting suspension is voiced using an ultrasonic disintegrator for 5 minutes and left for 2 hours until equilibrium is reached in the system (Example 1).

The possibility of obtaining an antigen carrier with a special tubular superstructure with a tube diameter of about 40 nm was first shown by us in the application for the invention "A method for producing lipid-based antigens from marine macrophytes and cucumarioside triterpene glycoside" when using cucumarioside A ₂ -2 as triterpene glycoside from Cucumaria cucumaria japonica (application for invention No. 2005131645/13 (035476) dated 12.10.2005).

The inventive carrier with different ratios of components has an ultramicroscopic size and an elongated filamentous-tubular structure with a particle length of 200 to 300 nm and a tube diameter of about 20 nm, which is confirmed by electron microscopy (examples 2,3, figure 2, pictures A and B) . Figure 2 shows electronic micrographs of a lipid carrier of antigens, including: holotoxin A ₁ : Chol: MGDG from Zostera marina in weight ratios of 3: 2: 2 (image A) and 3: 2: 6 (image B). A comparison of images A and B in figure 2 shows that a change in the weight content of MGDG from 2 to 6 during the formation of the antigen carrier complex leads to the formation of well-formed tubular structures.

It was also shown that the amount of MGDG from various marine macrophytes upon receipt of an antigen carrier by the claimed method does not affect its morphological characteristics if its weight ratio to holotoxin A ₁ and cholesterol varies in the range (2-6), i.e. the optimal ratio of holotoxin A ₁ , cholesterol and MGDG is 3: 2: (2-6) (examples 3-6, figure 2, pictures A-B).

It was found that complexes with a similar structure, but with formations of a smaller diameter, can be formed without the addition of MHD at different weight ratios of holotoxin A ₁ and cholesterol (examples 7, 8). This phenomenon is found for the first time - when used as a triterpene glycoside kukumariozida A ₂ -2 of sea cucumber Cucumaria japonica (application for invention № 2005131645/13 (035,476) on 12.09.2005), we have been registered complex formation between the A ₂ -2 kukumariozidom and cholesterol without the addition of a lipid matrix.

Photographs B and D of Fig. 2 show electron micrographs of sterol-glycosidic structures including holotoxin A ₁ : Chol in weight ratios of 4: 1 and 3: 2, respectively. The images show structures similar to the structure of complex formations in images A and B; their diameter is almost two times smaller than when adding MHD in the structure of complexes, and is 10-12 nm. An increase in the amount of cholesterol leads to the appearance of white spots in the image D (figure 2), indicating an excess of cholesterol.

However, the addition of a membrane-forming polar lipid MGDG enhances the binding of holotoxin A ₁ to sterol, causing a significant decrease in the hemolytic activity of the immunostimulating triterpene glycoside (Example 9), and is also a necessary condition for the spontaneous incorporation of amphiphilic in nature protein antigens of the outer membranes of microorganisms. The effective weight content of MHD in the holotoxin A ₁ system: Chol: MHD is in the range (2-6). Studies with a model surface protein of bacterial origin show that the optimal ratio of holotoxin A ₁ : Chol: MGDG for effective incorporation (more than 95%) of pseudotuberculous porin is 3: 2: 6 (example 10, figure 4). Figure 4 shows the distribution of pseudotuberculosis porin protein in fractions after deposition of samples of lipid-glycoside carriers with different contents of MHD by ultracentrifugation in phosphate-buffered saline.

The decrease in the content of MHD in the complex of the antigen carrier (less than two weight parts) leads to incomplete inclusion of the protein in the structure of the complex, and with its increase (more than 6 weight parts), there is no significant improvement in the immunological and morphological characteristics of the resulting antigen carrier.

Examples 2 and 3 (figure 2, pictures A, B) confirm that the introduction of MHD into a mixture of holotoxin A ₁ and cholesterol in the above ratios contributes to the formation of a stable lipid complex that has the properties required for an antigenic carrier and has a standard thread-like tubular structure.

The optimality of the weight ratio of holotoxin A ₁ and cholesterol (3: 2) is confirmed by electron microscopy. With this ratio, the formation of visible electron microscopic complexes is observed. The increase in the content of holotoxin A ₁ in the composition of the antigen carrier is not rational for two reasons:

- this does not lead to a change in the structure of the complex, but will lead to the appearance of its hemolytic activity;

- holotoxin A ₁ shows its immunomodulatory effect in ultralow concentrations.

Thus, it was experimentally established that the inventive lipid-cholesterol-glycosidic antigen carrier obtained at a weight ratio of holotoxin A ₁ : cholesterol: MGDG equal to 3: 2: (2-6) has optimal characteristics and does not have toxic effects with intraperitoneal ( in / b) and subcutaneous (s / c) administration to experimental animals (example 11). According to electron microscopy, the obtained antigen carrier has an elongated filamentous-tubular structure with a length of structural formations from 200 to 300 nm with a tube diameter of about 20 nm.

The closest structural analogues of the claimed carrier are lipid-saponin complexes of glycoalkaloids of plants of the nightshade family [Erik AJ Keukens, Truus de Vrije, Claudia van den Boom, Pieter de Waard, Henk H. Plasman, Felix Thiel, Vladimir Chupin, Wim MFJongen, Ben de Kruiff. Molekular basis of glicoalkaloid induced membrane disruption // Biochim Biophys Acta. 1995. Vol. 1240, P.216-228]. The proposed model seems to us fair for the claimed carrier. The structure of the claimed carrier is a three-layer tube (figure 1). The inner layer (2) of the structure consists of MHD, the molecules of which form a monolayer. In this case, the MGDG molecules are turned by their carbohydrate chains into the internal hydrated channel (1), while their fatty acid chains are turned in the opposite direction. Above the monolayer of glycolipids (2) is a layer of cholesterol-aglycon holotoxin A ₁ (3) complexes. Cholesterol-aglycone complexes also form a monolayer and are oriented in such a way that the carbohydrate chain of the holotoxin A ₁ molecule is exposed on the outer side of the carrier (4). It is known from the prior art that the formation of the cholesterol-holotoxin A ₁ complex is due to the interaction of the cholesterol and aglycon of this glycoside due to hydrophobic, van der Waals bonds and π-electrons of both molecules.

MGDG molecules form an internal monolayer of the complex due to the hydrophobic interaction of the fatty acid chains of neighboring glycolipid molecules. The formed monolayer in turn interacts with the aglycon-cholesterol monolayer, stabilizing the resulting structure.

A possible mechanism for the attachment of the porin antigen to the claimed complex is the interaction of its hydrophobic sites with the hydrophobic matrix of the carrier.

Golotoxin A _{1 is} obtained from available natural raw materials - commercial holothuria, Far Eastern trepang Apostichopus japonicus S. [Elyakov GB, Maltsev II, Kalinovsky AI, Stonik VA The structure of Golotoksin A ₁ (versoposide A), the main triterpene glycoside of the Pacific commercial holothurium Apostichopus japonicus // Bioorg. Chemistry, 1983. T. 9, No. 2. P.280-281].

The resulting feedstock includes the sum of triterpene glycosides, the main component of which is holotoxin A ₁ ; its structural formula is shown in figure 3 A.

Golotoxin A ₁ in low doses has adjuvant activity, providing a stimulating effect on the development of the immune response in different populations of immunocompetent cells upon oral and parenteral administration to animals. Under in vitro conditions, holotoxin A ₁ changes the functional activity of immunocompetent cells, increases the blastogenesis of spleen lymphocytes and the cytotoxic activity of blood macrophages. Macrophages activated by holotoxin A ₁ can play an important cooperating role in the immunostimulating activity of this glycoside [Popov AM, Atopkina LN, Samoshina NF, Uvarova NI Study of the immunomodulatory activity of tetracyclic triterpene glycosides of the dammaran and holostane series // Antibiotics and chemotherapy. 1994. T. 39, No. 7. S. 19-25].

Holotoxin A ₁ refers to typical membranotropic substances. The membrane activity of holotoxin A ₁ is due to high cholesterol affinity. Even at low concentrations, it is able to form non-toxic glycoside-sterol or lipid-glycoside structures with a high degree of efficiency [A. Popov A comparative study of the cytotoxic and hemolytic effects of triterpenoids of ginseng and holothuria // Izv. RAS. Ser. biol. 2002. No. 2. S.155-164].

Thus, the use of holotoxin A ₁ in combination with cholesterol can increase the effectiveness of antiviral vaccines, namely: to increase the immunogenicity of antigenic substance due to the corpuscular presentation of antigens in the structure of the carrier having immune properties, and also to exclude the hemotoxicity of holotoxin A ₁ due to the strong binding of glycoside with cholesterol .

As the lipid component of the inventive antigen carrier, MHD was selected from seaweed, the structural formula of which is shown in figure 3 (B). This lipid combines in a complex structure-forming, immune properties and the ability to incorporate surface-membrane antigens of pathogenic microorganisms. MHD used as a lipid component from Ulva fenestrata (green algae department - Chlorophyta), Ahnfeltia tobuchiensis (red algae department - Rhodophyta), Laminaria japonica (brown algae department - Phaeophyta), Zostera marina (green sea grass - Magnoliophy department) method Sanina N., Goncharova S., Kostetsky E. Fatty acid composition of individual polar lipid classes from marine macrophytes // Phytochemistry. 2004. V. 65. No. 6. R. 721-730. The lipid complex preparations were prepared as described in the application. To obtain the final product, MGDG isolated from Zostera marina (Example 3), from Ulva fenestrate (Example 4), Ahnfeltia tobuchiensis (Example 5), Laminaria japonica (Example 6) were used.

Regardless of the source of isolation of MHD from marine macrophytes, the preparations obtained are an aqueous suspension of a lipid complex having a filament-tubular structure 200-300 nm long with a tube diameter of about 20 nm.

This lipid is the most unsaturated glycoglycerolipid and includes a wide range of polyene fatty acids. The prior art knows the effect of unsaturated fatty acids, in particular linolenic and arachidonic, on the state of the membranes of cells of the immune system, especially macrophages.

The effectiveness of using MGDG as a lipid in the formation of antigen carriers with a tubular structure was investigated and substantiated by us in the application for invention 2005131645/13 (035476) dated 12.10.2005. The application showed that MGDG exhibits independent immuno-adjuvant activity with i.v. and sc administration, comparable with the activity of Freund's adjuvant and even superior to it when immunized with a corpuscular T-dependent antigen - sheep erythrocytes (EB); has the ability to suppress the activity of delayed-type hypersensitivity effectors (HRT).

It should be noted that the new functions of holotoxin A ₁ and MGDG, which they exhibit in the claimed invention, do not follow with obviousness from the properties known to them.

On the basis of holotoxin A ₁ , cholesterol and MGDG, an antigen carrier was obtained for the first time, having a filamentous-tubular structure, which allows increasing the effectiveness of antiviral vaccines, namely: increasing the immunogenicity of antigenic substance due to the corpuscular representation of antigens in the structure of a lipid-glycosidic carrier having immune properties, as well as eliminate hemotoxicity due to the strong binding of glycoside with membrane-forming lipids - cholesterol and MGDG in the structure of the lipid-glycoside matrix (approx. p 9).

The authenticity of the drug is determined by the qualitative and quantitative composition of the structural components (holotoxin A ₁ , cholesterol and MGDG), which are the initial ones when receiving the carrier of antigens; its structure is confirmed by electron microscopy (Fig.2, pictures A, B). The drug is stable at a temperature (-4 ° C) of more than three months.

The proposed lipid-glycosidic complex with subcutaneous and intraperitoneal administration to mice does not cause inflammatory, pain, toxic, as well as high cytolytic effects (example 11).

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

Example 1. Preparation of a lipid-glycoside carrier preparation consisting of holotoxin A ₁ , cholesterol and MGDG.

The preparation of solutions of the starting components is carried out as follows: 5 mg of MGDG is dissolved in 1 ml of chloroform and 5 mg of cholesterol is dissolved in 1 ml of chloroform, 4 mg of holotoxin A _{1 is} dissolved in 1 ml of distilled water. The necessary amount of cholesterol and MGDG dissolved in chloroform is selected with a microdoser, evaporated in vacuo and the dry residue is solubilized with a 0.4% holotoxin A ₁ solution. After thorough mixing, salt phosphate buffer, pH 7.2, is added to the mixture to a final concentration of lipids in the mixture of 2 mg in 1 ml. The resulting suspension is voiced by the ultrasonic disintegrator SONOPULS Ultraschall - Homogenizatoren HD 2070 (Germany) at a frequency of 20 kHz, power 7 W in mode 7 (0.7 s operation, 0.3 s break) for 5 minutes, and then incubated for 2 hours until complete formation of complexes.

Example 2. Preparation of a lipid-glycoside carrier preparation consisting of three parts by weight of holotoxin A ₁ , two parts by weight of cholesterol and two parts by weight of MGDG from Zostera marina according to the procedure described in Example 1. 200 μl of cholesterol solution and 200 μl of MGDG solution are taken with a microdoser, evaporated to dryness under vacuum and the dry residue is solubilized with a 0.4% holotoxin A ₁ solution, the volume of which is 375 μl. After thorough mixing, 625 μl of phosphate buffered saline, pH 7.2, are added to the mixture. The final lipid concentration is 2 mg per 1 ml. The suspension is voiced for 5 minutes on an ultrasonic disintegrator. The resulting preparation is an aqueous suspension comprising particles of a carrier having an ultramicroscopic tubular lipid structure with a tube length of 100-300 nm. Figure 2 (snapshot A) shows an electron micrograph of the obtained lipid-glycosidic carrier (magnification X 50,000).

Example 3. The preparation of the lipid carrier was obtained according to the procedure described in example 1 at a weight ratio of the starting components of 3: 2: 6 using MGDG from Zostera marina. Figure 2 (image B) shows an electron micrograph of a lipid carrier obtained using MGDG from Zostera marina (magnification X 50,000).

Example 4. The preparation of the lipid carrier was obtained according to the procedure described in example 3 using MGDG from Ulva fenestrate. The use of MGDG isolated from Ulva fenestrate did not alter the morphology of the carrier.

Example 5. A lipid carrier preparation was prepared according to the procedure described in Example 3 using MGDG from Ahnfeltia tobuchiensis. The use of MGDG isolated from Ahnfeltia tobuchiensis did not alter the morphology of the carrier.

Example 6. A lipid carrier preparation was prepared according to the procedure described in Example 3 using MGDG from Laminaria japonica. The use of MGDG isolated from Laminaria japonica did not alter the morphology of the carrier.

Example 7. The preparation of a glycoside-sterol complex was obtained according to the procedure described in example 1 based on holotoxin A ₁ and cholesterol at a weight ratio of 4: 1 without adding MGDG. According to electron microscopy, holotoxin A ₁ in the presence of cholesterol and in the absence of MGDG forms threadlike structures 100-200 nm long. Figure 2 (snapshot B) shows an electron micrograph of a glycoside-sterol complex obtained under the conditions of example 4 (magnification X 50,000).

Example 8. The preparation of a glycoside-sterol complex was obtained according to the procedure described in example 1 based on holotoxin A ₁ and cholesterol at a weight ratio of 3: 2 without adding MGDG. According to electron microscopy, holotoxin A ₁ forms threadlike structures in the absence of MGDG and in excess of cholesterol. Figure 2 (snapshot D) shows an electron micrograph of a glycoside-sterol complex obtained under the conditions of Example 5 (magnification X 50,000).

Example 9. Lipid carriers shown in examples 2 and 3 were tested for hemolytic activity in vitro using erythrocytes from outbred mice as a test system. Heparinized blood obtained by decapitation of the mouse was washed in an isotonic sodium chloride solution by 2-fold centrifugation at 3000 rpm for 15 min at room temperature. A 0.5% cell suspension was obtained from the erythrocyte sediment. The studied preparations were prepared in phosphate-buffered saline, pH 7.4, and successively dispensed in 10 μl per well of the plate by the method of two serial dilutions followed by the addition of a suspension of red blood cells of 90 μl per well. 10 μl of phosphate-buffered saline was added to control wells. Incubation was carried out for 1 and 2 hours in a thermostat at 37 ° C. The hemolytic activity of the studied drugs was evaluated by visual inspection of the wells. The minimum effective dose of a substance that causes 100% hemolysis (ED ₁₀₀ ) was determined by the appearance of a clear solution of “varnish” blood.

The hemolytic effect of holotoxin F ₁ , included in the complex of the claimed antigen carrier, in relation to white mouse erythrocytes within two hours of observation in the composition of lipid carriers decreased by 9.8 times compared with the action of the same amount of individual triterpene glycoside.

Example 10. Obtained according to the method described in example 1, preparations of lipid carriers were mixed with 0.6 mg of pseudotuberculosis porin protein. The weight ratios of the structure-forming components of holotoxin A ₁ : Chol: MGDG were 3: 2: 2, 3: 2: 6 or 3: 2: 10. At the same time, the content of holotoxin in all the studied preparations was constant and amounted to 0.6 mg. After ultrasonic disintegration, protein-containing carrier samples were deposited on an ultracentrifuge at a speed of 45,000 × g for 30 min. 12 fractions were taken from centrifuge tubes, from top to bottom, and the protein content was determined in each according to the Lowry method.

As a result of the experiment, it was shown (Fig. 4) that more than 95% of the protein after mixing and ultrasound treatment is included in the composition of lipid carriers containing holotoxin A ₁ , cholesterol and MGDG in a ratio of 3: 2: (6-10). A decrease in the amount of MGDG by 3-5 times leads to a partial loss of the incorporating properties of the carrier.

Example 11. Obtained by the claimed method (example 2, 3) the lipid carrier of antigens was tested on a group of outbred mice (10 in the control and experimental group) for its safety. During the observation, the appearance of inflammatory or pain reactions, signs of toxicity, or death was controlled in animals.

A single intraperitoneal injection of a white lipid mouse into a preparation of a lipid carrier at the rate of 100 and 200 μg of holotoxin A ₁ per mouse does not cause animal death and toxic effects within three days of observation. The tested doses of holotoxin A ₁ correspond to 5 and 10 μg / kg of body weight. The optimal immunotropic dose of holotoxin A ₁ is 0.05-50 μg per 1 kg of weight.

A single subcutaneous injection of the lipid complexes shown in examples 2 and 3, based on 100 and 200 μg of holotoxin A ₁ per mouse, does not cause inflammatory and pain effects in animals within three days of observation.

Claims (1)

The carrier of antigens in the form of a lipid complex composed of a glycoside, cholesterol and lipid, characterized in that the glycoside it contains holotoxin A _1, and as a lipid-monogalaktozildiatsilglitseridy (MGDG) from marine macrophytes, taken in a weight ratio of 3 (holotoxin A ₁ ): 2 (cholesterol) :( 2-6) (MGDG) and is an elongated filamentous-tubular structure.

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