RU2378015C2 - Conjugate for immunisation and vaccination and method for improving immunogenicity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and immunology and concerns a conjugate for immunisation and vaccination and method for improving immunogenicity. Substance of the invention involves said conjugate for immunisation and vaccination representing an immunogen covalently bonded with a high-molecular carrier that is acidic peptidoglycane of molecular weight 1200-40000 KDa, and having the mass ratio of glucose and uronic acids 1:2-4.

EFFECT: advantage of the invention consists in improved immunogenicity 10-100 times higher, that in turn allows reducing immunogen doses in 10-100 times to achieve the same effect.

6 cl, 6 ex, 3 tbl

Description

The invention relates to the field of immunology, namely to the development of vaccine preparations.

Vaccination around the world has allowed us to rid humanity of smallpox and reduce the incidence of polio to isolated cases. Despite these and other successes, the development of vaccines against many topical infections remains an unresolved problem. HIV infection, malaria, hepatitis C, many viral, bacterial and helminth infections remain widespread diseases against which effective vaccines have not yet been developed.

One of the main problems hindering the creation of new vaccines is the lack of a safe and effective immunostimulating carrier, the function of which is to provide an intense immune response to the infectious antigens included in the vaccine.

As early as the first half of the 20th century, it was found that purified infectious antigens, in most cases, do not have sufficient immunogenicity, that is, their introduction into the body of mammals does not cause an immune response sufficient to protect against subsequent infection. Over the next decades, many methods have been invented to increase the immunogenicity of antigens, including adsorption of antigens on colloidal microparticles (for example, aluminum salts or aluminum hydroxide), emulsification of antigens in water-oil mixtures, as well as co-administration of antigen with various stimulants of immune reactions (for example, killed bacteria or bacterial components such as lipopolysaccharide, lipopetid, peptidoglycan and others).

One way to increase the immunogenicity of antigens is to chemically combine them with an immunogenic carrier. About 70 years ago, Karl Landsteiner showed that non-immunogenic antigens can be converted into immunogenic antigens if chemically combined with protein substances. In particular, the introduction into the organism of mammals of small molecules called haptens does not lead to the induction of the synthesis of antibodies that bind the introduced hapten. If a hapten is linked by a covalent (chemical) bond to a protein carrier, then such hapten-carrier conjugates can induce the production of antibodies that recognize both the hapten and the carrier.

Since the discovery of K. Landsteiner, many different conjugated immunogens have been proposed. The immunogenic properties of antigens and vaccines were increased by covalently combining them with a lipopolysaccharide

As an analogue of the invention, immunogenic conjugates of a β-propionamide-linked polysaccharide and N-propionamide-linked oligosaccharide with a protein can be considered, as well as a method for producing these conjugates. Conjugates are used to produce vaccines against infectious diseases and cancer (RF patent 2249463). The disadvantages of the proposed conjugates are the complexity of manufacturing and lack of effectiveness, due to the fact that the protein is an “inert” carrier that slightly activates the immune response.

The closest analogue of the invention may be a method of enhancing the immunogenicity of antigen molecules having weak immunogenic properties, which turns them into antigens suitable for immunization. The method consists in preparing conjugates of antigen with weak immunogenic properties and a synthetic peptide carrier, which has the ability to significantly increase the immunogenicity of the specified antigen (RF application 95105991 or US patent 5736146). The disadvantages of this invention is the need to use potentially dangerous peptides as a carrier and the complexity of the design of the proposed conjugates.

Also, as an analogue of the invention, an acidic peptidoglycan (hereinafter referred to as “CNG”) having immunity activator properties can be considered. CNG is a water-soluble acidic peptidoglycan obtained from plants with a molecular weight of 1200-40000 kDa. The sugar composition of the polysaccharide part is as follows: galacturonic acid 18 ± 6%, glucose 9 ± 3%, galactose 5.5 ± 2%, arabinose 3.8 ± 1.3%, ramnose 1.9 ± 0.9%, mannose 0, 7 ± 0.25%. The peptide part is 13 ± 3%, it contains amino acids with primary amino groups (arginine 2.6%, lysine 1.8%), as well as amino acids with carboxyl groups (aspartic acid 10.6%, glutamic acid 11.1%). CNG and the method for its preparation are described in RF patent 2195308.

CNG is a powerful immunostimulant: it activates NK cells, dendritic cells, macrophages, monocytes, enhances the production of cytokines and interferon. CNG is an adjuvant. When experimental animals are co-administered with a mixture of a protein antigen and CNG, the production of antibodies that specifically bind the antigen used is enhanced. According to RF2195308, when mice were injected together with mice 20 μg of CNG and 50 μg of egg albumin, the concentration of antibodies in the blood of mice reached 1: 22000 titers, which was significantly higher than the response to the introduction of 50 μg of egg albumin without CSG (1: 13000). The adjuvant effect of CNG was comparable to the effect of the known adjuvant Pam ₃ Cys-Ser-Lys ₄ lipopeptide. With the introduction of 20 μg of lipopeptide together with 50 μg of egg albumin in the blood of mice, antibodies to egg albumin accumulated in a titer of 1: 20,000.

The disadvantage of this method of increasing the intensity of the immune response by mixing antigen and CNG is the need to introduce sufficiently large doses of antigen. So, in an experiment when mice were immunized with protein antigens (egg albumin, bovine serum albumin), the addition of CNG does not significantly reduce the dose of antigen; one has to use the usual doses of about 50 μg of antigen per mouse.

Despite the abundance of the proposed options, a universal immunostimulating carrier has not yet been found that has the optimal combination of efficiency and safety. Most often, effective immunostimulating carriers are unsafe for humans and animals, causing more or less pronounced side (toxic) effects. In contrast, safe carriers are not effective immunostimulants.

The present invention aims to create a safe drug with high efficiency as an immunostimulating carrier and suitable for the construction of immunogens and vaccines.

The objective of the invention is to increase the specific immunogenicity of the antigen, reduce the dose of antigen necessary for the induction of an immune response, expand the arsenal of immunostimulating carriers. This problem is solved by covalent crosslinking of CNG and antigen.

The invention is as follows. CNG can be obtained in various ways from plant materials. The following is a method of producing CNG, which illustrates the invention, but does not limit the scope of claims.

To obtain CNG, 5 kg of potato seedlings of the Solanaceae family (S. tuberosum) are crushed, 10 l of water are added and extracted at room temperature with stirring for 2 hours. The mixture is squeezed using a mechanical press. The aqueous extract is dialyzed and concentrated to a volume of 1 l by ultrafiltration on a 10 kDa filter. Sodium chloride was added to the concentrate at the rate of 100 mg per 100 ml and 3 volumes of 96% ethyl alcohol. The precipitate was isolated by centrifugation at 4000 rpm for 30 minutes.

The precipitate is washed with 300 ml of 96% ethanol, centrifuged and dried in air. Then re-precipitation of the precipitate is carried out with a salt agent - calcium chloride. To do this, 10 g of dry intermediate is dissolved in 1 liter of distilled water with stirring at room temperature for 1 hour. The insoluble precipitate is discarded, and 150 ml of a 5% solution of calcium chloride are added to the mother liquor. The precipitate, which is a crude acid peptidoglycan, is separated by centrifugation. Then translate it into a soluble form by adding a saturated solution of sodium chloride and maintain the mixture with stirring and heating at 50 ° C until complete dissolution. The resulting solution was applied to a TSK HW-75F column. The column is eluted with water, the first high molecular weight peak with a molecular weight of from 1200 to 40,000 kDa is collected. The solution was concentrated on a rotary evaporator and freeze-dried. The yield of acidic peptidoglycan is 170 mg. The mass ratio between glucose and uronic acids is 1: 4. Next, covalent coupling of CNG with immunogens is carried out.

1. The covalent compound of CNG with an antigen containing sulfhydryl groups is carried out using a water-soluble heterobifunctional reagent containing maleimide and N-hydroxy-succinimide groups. The conjugation reaction is carried out in two stages. At the first stage of the reaction, CNG is activated by using the heterobifunctional reagent sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxalate (Sulfo-SMCC). In this case, the Sulfo-SMCC N-hydroxy-succinimide group reacts with the amino groups of CNG (according to arginine and lysine). After CNG activation, unreacted Sulfo-SMCC and other low molecular weight products are removed by dialysis or gel filtration with a threshold of 500 kDa. At the second stage of the reaction, the maleimide group attached to the activated CNG interacts with the sulfhydryl group of the antigen. As a result of the reaction, a covalent complex (conjugate) of the formula is formed:

KPG-NH-R ₁ -SH-R ₂ ,

where R ₁ is any water-soluble heterobifunctional reagent containing maleimide and N-hydroxy-succinimide groups, R ₂ is any antigen containing a sulfhydryl group.

The absence of SH-groups in CNG allows avoiding the formation of CNG-NH-R ₁ -SH-KPG complexes. After conjugation, unbound antigen is removed by gel filtration or ultrafiltration with a threshold of 500 kDa. The fact of the covalent compound of CNG and antigen is proved by any method - spectrophotometrically, fluorimetrically, with protein dyes, antibodies specifically binding to the antigen - by the appearance of a protein signal in the high molecular weight region (chromatography, electrophoresis or other separation methods) characteristic of CNG.

2. The covalent compound of CNG with any antigen containing amino groups is carried out using a heterobifunctional reagent capable of reacting with carboxyl groups and amino groups. An example of such a heterobifunctional reagent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

The reaction is carried out in two stages. In the first stage, the CNG is reacted with EDC. In this case, EDC reacts with the carboxyl groups of CNG: (galacturonic, aspartic and glutamic acids). An activated CNG modified by O-acylisourea is formed. This intermediate is unstable. Therefore, the first stage of the reaction is carried out in the presence of a stabilizer N-hydroxysulfosuccinimide (Sulfo-NHS). In the second stage, a reaction is carried out between CNG, a modified O-acyl isourea, and an antigen having primary amino groups. As a result of the reaction, a covalent complex (conjugate) of the formula is formed:

CNG-CO-NH-R,

where R is any antigen containing a primary amino group.

Unbound antigen is removed by gel filtration or ultrafiltration with a threshold of 500 kDa. The fact of a covalent compound of antigen and CNG is proved by any method - fluorimetrically, spectrophotometrically, with dyes on a protein, specific antibodies - by the appearance of a protein signal in the high molecular area (chromatography, electrophoresis or other separation methods) characteristic of CNG.

3. The use of conjugates obtained in accordance with claim 1 and claim 2 for the immunization of experimental animals induces an intense immune response specific for the introduced antigen. Moreover, the maximum intensity of the immune response is achieved with the introduction of 10-100 times lower doses of antigen compared with doses of the same antigen required for immunization with a mixture of antigen + CNG.

The advantages of this method are as follows:

- 10-100 times lower antigen consumption during immunization;

- the side effect of toxic antigens with reactogenicity (local and general toxic reactions to the antigen), for example, microbial toxins and toxoids, viral hemagglutinins, neuraminidases and other substances, the introduction of which leads not only to the development of an immune response, but also to the development of which will be significantly reduced pathological (side) effects.

The invention is illustrated by the following examples.

Example 1

Conjugation of ovalbumin with CNG using Sulfo-SMCC.

The natural protein ovalbumin was used as an antigen. Conjugation of ovalbumin with CNG was performed using the Sulfo-SMCC heterobifunctional reagent (commercial product from Pierce, cat. N 22322). The reaction was carried out in accordance with the protocol of the manufacturer, adapting to the characteristics of CNG. CNG activation using Sulfo-SMCC.

27.7 mg of CNG are dissolved in 15 ml of distilled water with stirring on a magnetic stirrer for 30 minutes at room temperature. To the resulting solution was added 10 ml of 0.1 M phosphate buffer pH 7.35 and 3.1 mg of Sulfo-SMCC was sprinkled on. The mixture is left in a thermostat at 37 ° C for 1 hour, stirring occasionally. At the end of the incubation, the solution was cooled to room temperature and applied to a TOYOPEARL HW-65F gel chromatography column. Elution is carried out with distilled water, peak detection with a refractometer. The first high molecular weight peak with a molecular weight of more than 500 kDa is collected. The activated CNG solution was evaporated on a rotary evaporator to 13 ml. Preparation of ovalbumin for conjugation.

The commercial ovalbumin preparation is pre-dissolved, treated with 2-mercaptoethanol to restore sulfhydryl groups, then freed from microaggregates by gel permeation chromatography. 17.6 mg of Ovalbumin (Calbiochem cat.N 32467) was dissolved in 7 ml of reduction buffer (0.1 M phosphate buffer, pH 7.4, 2.5 mM ethylenediaminetetraacetic acid, sodium salt - EDTA, 0.6% 2-mercaptoethanol). The resulting solution was kept at room temperature for 2 hours with stirring on a magnetic stirrer, then filtered through a filter with a pore diameter of 0.45 μm and applied to a chromatographic column (950 × 30 mm) with TOYOPEARL HW-75F gel. Fractions elute with purified water and are detected by refractometry. A 40-50 kDa fraction corresponding to the molecular weight of ovalbumin is collected. The spectrophotometry method determines the maximum absorption of UV light (280 nm), which, in comparison with calibration solutions of ovalbumin, allows you to determine the protein concentration in the collected fraction, it was 0.31 mg / ml.

Conjugation.

3 ml of ovalbumin solution and 3 ml of conjugation buffer (150 mM sodium chloride, 20 mM phosphate buffer, pH 7.2 with the addition of 1 mM EDTA) are added to 4 ml of activated CNG. The reaction mixture was kept in a refrigerator at 4 ° C for 15 hours, after which it was applied to a TOYOPEARL HW-75 F gel column. Fractions were eluted with purified water; the detector was a refractometer. The first peak with a molecular weight of more than 500 kDa is collected. The collected first peak is evaporated on a rotary evaporator to 10 ml and dried on freeze drying. Obtain 2.3 mg of the conjugate CNG-ovalbumin.

The ovalbumin content in the conjugate is determined by fluorescence (Hitachi 850 fluorimeter, excitation 280 nm, 3 nm gap, 320 nm fluorescence, 3 nm gap) when calibrated with ovalbumin. The ovalbumin content in the CNG-ovalbumin conjugate was about 1%.

Example 2

Conjugation of recombinant human immunodeficiency virus protein (rHIV) and CNG using Sulfo-SMCC.

A recombinant protein (commercial product of ProSpec-Tany TechnoGene Ltd., Cat. No. HI V-110), which is a polypeptide that contains immunodominant fragments of four HIV-1 proteins, in particular gag pl7, p24, gp120, was used as an antigen and gp41. The rHIV protein is supplied as a solution of 1 mg / ml in a 20 mM PBS pH7.8 buffer, 20 tM 0.5M NaCl, containing 1 tM digiotreigol and 8 M urea.

The buffer was changed on a TOYOPEARL HW-40F column (440 × 10 mm), previously equilibrated with a buffer containing 0.02 M sodium phosphate, 0.02 M NaCl and 0.1 mM EDTA. Fractions were eluted with a buffer containing 0.02 M sodium phosphate, 0.02 M NaCl and 0.1 mM EDTA. The detector is a refractometer. A 7 ml fraction was collected corresponding to the peak of the rHIV protein (molecular weight ~ 100 kDa), which was then concentrated to 2 ml.

Activation of CNG using Sulfo-SMCC and conjugation of rHIV with activated CNG was carried out in accordance with the methods described in Example 1. The yield of the CNG-rHIV conjugate was 10.6 mg. The rHIV content in the conjugate was 0.33%.

Example 3

Conjugation of recombinant E-protein (pE) coronavirus and CNG using EDC.

As an antigen, pE, a recombinant envelope protein of the coronavirus associated with SARS (SARS), was used. Protein pE is a polypeptide chain with a length of 76 amino acid residues. We used a commercial product from ProSpec-Tany TechnoGene Ltd, Cat.No. SARS-252), which is supplied as a solution of 1 mg pE protein in 1 ml of 50 mM Tris-HCl and 60 mM NaCl buffer containing 50% glycerol.

The buffer change is carried out, as in example 2. Receive 2 ml of a solution of protein pE in phosphate buffer. The conjugation of pE and CNG is carried out using an EDC heterobifunctional reagent (commercial product from Pierce, Cat. No. 77149 was used) in the presence of sulfo-N-hydroxysuccinimide (for short, Sulfo-NHS, commercial product from Pierce, Cat. No. 24510) . The reaction is carried out according to the protocol of the company Pierce, adapting to the characteristics of CNG. For this, 10.2 mg of CNG is dissolved in 5 ml of 0.1 M 2- (N-morpholino) ethanesulfonic acid, pH 4.16, stirring on a magnetic stirrer at room temperature for 30 minutes. 2.2 mg of EDC and 3 mg of Sulfo-NHS are added to the resulting solution of activated CNG, mixed for 30 minutes. Then add 2 ml of a solution of protein pE, and the mixture is kept at room temperature for 1.5 hours.

The CNG-pE conjugate was isolated on a TOYOPEARL HW-75F gel chromatography column as described in Example 1. 7 mg of conjugate was obtained.

The content of pE protein in the CNG-pE conjugate was 1.6%.

Example 4

Evaluation of the immunogenicity of the CNG-ovalbumin conjugate obtained in Example 1.

The immunogenicity of the CNG-ovalbumin conjugate was studied in laboratory mice of males (CBA × C57B1 / 6) F ₁ weighing 20 g. The conjugate was compared with the initial antigen ovalbumin and a physical mixture of ovalbumin with CNG. Antigens were dissolved in 0.9% NaCl in physiological saline, and 0.2 ml were injected intraperitoneally into mice. Re-immunization with the same dose and in the same volume was carried out after 1 month.

Blood was obtained 1 week after re-immunization. Blood from 10 mice of one experimental group was pooled, serum was obtained, which was poured into several portions of 100 μl and frozen before testing.

The serum content of antibodies specifically binding to ovalbumin was determined by the enzyme immunoassay according to a standard method. A commercial ovalbumin preparation manufactured by Calbiochem was dissolved in carbonate buffer (0.05 M, pH 9.5) at a concentration of 10 μg / ml, and 100 μl was added to the wells of a 96-well night plate (Nunc, Maxisorb, Cat. No. 442404) incubated overnight in the refrigerator. Wells were washed 3 times with 200 μl of phosphate buffer (0.15 M sodium chloride, 0.01 M sodium phosphate, pH 7.4) containing 0.1% Tween-20 (Serva). Clogging was carried out with a 1% casein solution in phosphate buffer containing 0.1% Tween-20 for 45 min (at 37 ° C), 150 μl per well. After 3 times washing with 250 μl of phosphate buffer with 0.1% Tween-20, dilutions of the test serum of experimental mice were added to the wells of the plate. Serial dilutions of serum in phosphate buffer with 0.1% Tween-20 were prepared starting from an initial dilution of 1:50 with a dilution step of 3. After serial dilutions of the test sera were added to the wells, the plates were incubated for 1 hour at 37 ° C. Washing was performed by 3-fold application of 250 μl of phosphate buffer with 0.1% Tween-20.

To detect murine antibodies, 100 μl of goat antibody-specific goat antibody conjugate with horseradish peroxidase (Sorbent, Moscow) was diluted 1: 1000 in a phosphate buffer containing 0.1% Tween-20 and incubated 1 hour at 37 ° C. After washing 3 times with 250 μl of phosphate buffer with 0.1% Tween-20, a substrate for detecting peroxidase was added to the wells of a plate (0.6 mg / ml orthophenylenediamine hydrochloride in 0.1 M citrate buffer, pH 5, 3% hydrogen peroxide) incubated for 10 minutes The color reaction was stopped by pouring 100 μl of 1 M sulfuric acid into the wells of the plate. The reaction intensity was measured photometrically at 492 nm on an MRX plate photometer from Dynex (USA).

Table 1 presents the results of testing the immunogenicity of the CNG-ovalbumin conjugate (30 μg conjugate, which is 0.3 μg of ovalbumin per mouse) compared with ovalbumin (0.3 μg and 30 μg per mouse) and a physical mixture of CNG plus ovalbumin ( 30 μg CNG plus 0.3 μg and 30 μg ovalbumin per mouse). Administration to mice of the CNG-ovalbumin conjugate caused antibody production more intense than the administration of ovalbumin or a mixture of CNG with ovalbumin. When comparing the level of the immune response to small doses of ovalbumin (0.3 μg per mouse), it can be determined that the specific immunogenicity of ovalbumin in the conjugate is 100 or more times higher compared to ovalbumin itself or a mixture of CNG plus ovalbumin.

Table 1.
The immune response of mice (CBA × C57B1 / 6) F ₁ (in response to the administration of the CNG-ovalbumin conjugate compared to the administration of ovalbumin or a mixture of CNG with ovalbumin. Immunogen Antibody titers in the serum of mice immunized with different doses of antigen * 0.3 mcg ovalbumin 30 mcg ovalbumin CNG-ovalbumin conjugate, 30 mcg 55,000 n.i. ** Mixture: 30 mcg CNG plus ovalbumin 500 30000 Ovalbumin one hundred 20000 Notes:
(*) antibody titers are presented 1 week after the repeated administration of the indicated doses of immunogens (the doses for ovalbumin are indicated);
(**) n.i. means - not investigated.

Example 5

Evaluation of the immunogenicity of the CNG-rHIV conjugate obtained in Example 2.

The immunogenicity of the CNG-rHIV conjugate was studied in laboratory male mice (CBA × C57Bl / 6) F ₁ . The conjugate was compared with the original rHIV antigen and the physical mixture of CNG plus rHIV.

Immunization of laboratory mice and determination of antibody titers was carried out as in Example 4. As an immunogen, the mice were injected with the CNG-rHIV conjugate (doses of 0.1 μg per mouse, doses of CNG 30 μg), or a mixture of CNG plus pE (dose - 0.1 μg or 1 μg rVICH per mouse plus 30 μg CNG), or rVICH (doses - 0.1 μg, 1 μg, or 5 μg per mouse).

Table 2 presents the intensity of production of antibodies specific for rHIV, 1 week after repeated administration of these immunogens. It is obvious that the specific immunogenicity of the rHIV antigen in the composition of the CNG-rHIV conjugate was 50 times higher than the immunogenicity of rHIV and 10 times higher than the immunogenicity of the mixture of CNG plus rHIV.

Table 2.
The immune response of mice (CBA × C57B1 / 6) F ₁ in response to the administration of the CNG-rHIV conjugate compared to the administration of rHIV or a mixture of CNG and rHIV. Immunogen Antibody titers in the serum of mice immunized with different doses of antigen * 0.1 mcg rHIV 1 mcg rHIV 5 mcg rHIV CNG-rHIV conjugate (30 mcg) 22000 n.i. ** n.i. ** Mixture of 30 mcg CNG plus rHIV 500 20000 n.i. ** HIV one hundred 2500 20000 Notes:
(*) antibody titers are presented 1 week after the repeated administration of the indicated doses of immunogens (the doses for ovalbumin are indicated);
(**) n.i. means - not investigated.

Example 6. Evaluation of the immunogenicity of the CNG-rE conjugate obtained in Example 3.

The immunogenicity of the CNG-pE conjugate was studied in laboratory male mice (CBA × C57B1 / 6) F ₁ . The conjugate was compared with the original pE antigen and a physical mixture of CNG plus pE.

Immunization of laboratory mice and determination of antibody titers was carried out as in Example 4. As an immunogen, experimental mice were injected with the CNG-pE conjugate (doses of pE - 0.5 μg per mouse, doses of CNG - 30 μg), or a mixture of CNG plus pE ( doses - 0.5 μg or 5 μg pE per mouse plus 30 μg CNG), or pE (doses - 0.5 μg or 5 μg per mouse).

Table 3 presents the intensity of production of antibodies specific for pE 1 week after repeated administration of these immunogens. From the presented data it can be seen that the specific immunogenicity of the pE antigen in the composition of the CNG-pE conjugate was 8-10 times higher than the immunogenicity of pE and a mixture of pE with CNG.

Table 3. The immune response of mice (CBA × C57B1 / 6) F ₁ in response to the introduction of the conjugate of CNG-pE compared with the introduction of pE or a mixture of CNG with pE. Immunogen Antibody titers in the serum of mice immunized with different doses of antigen * 0.5 μg pE 5 μg pE CNG-rE conjugate, 30 mcg 8000 n.i. ** A mixture of 30 μg CNG plus pE 500 10,000 re one hundred 10,000 Notes:
(*) antibody titers are presented 1 week after the repeated administration of the indicated doses of immunogens (the doses for ovalbumin are indicated);
(**) n.i. means - not investigated.

Claims (6)

1. The conjugate for immunization and vaccination, containing an immunogen covalently linked to a high molecular weight carrier, characterized in that the acidic peptidoglycan with a molecular weight of 1200-40000 kDa, having a mass ratio between glucose and uronic acids equal to 1: 2-, is used as a high molecular weight carrier. four. 2. The conjugate according to claim 1, characterized in that the peptide portion of the acidic peptidoglycan molecule is (13 ± 3) wt.%. 3. The conjugate according to claim 2, characterized in that the sugar composition of the polysaccharide part of the acidic peptidoglycan contains galacturonic acid 18 ± 6%, glucose 9 ± 3%, galactose 5.5 ± 2%, arabinose 3.8 ± 1.3%, rhamnose 1.9 ± 0.9%, mannose 0.7 ± 0.25%, and the peptide portion contains arginine 2.6%, and lysine 1.8%, as well as aspartic acid 10.6% and glutamic acid 11, one%. 4. The conjugate according to any one of claims 1 to 3, characterized in that the immunogen is a recombinant protein of the human immunodeficiency virus. 5. The conjugate according to any one of claims 1 to 3, characterized in that the immunogen is a recombinant E-protein (pE) of coronavirus associated with SARS. 6. A method of increasing immunogenicity by covalently combining an immunogen with an acid peptidoglycan with a molecular weight of 1200-40000 kDa having a mass ratio between glucose and uronic acids of 1: 2-4.