Conjugate for immunisation and vaccination and method for improving immunogenicity

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

 

The invention relates to the field of immunology, in particular to the development of vaccines.

Vaccination in the world allowed to save humanity from smallpox and reduce the incidence of polio to individual cases. Despite these and other successes, the development of vaccines against many topical infections remains an unsolved problem. HIV, malaria, hepatitis C, many viral, bacterial and helminth infections remain common diseases against which so far have failed to develop effective vaccines.

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

In the first half of the twentieth century it was found that purified infectious antigens, in most cases, do not have sufficient immunogenicity, that is, their introduction in the mammalian organism does not cause immune response sufficient to protect against subsequent infection. Over the next decade was invented many ways to enhance the immunogenicity of antigens, among which the adsorption of antigens onto colloidal particles (for example, salts of aluminium the Oia or aluminium hydroxide), emulsification of the antigen in oil-water mixtures, as well as co-administration of antigen with various stimulators of immune responses (e.g., killed bacteria or components of bacteria, such as lipopolysaccharide, lipopeptide, peptidoglycan, and others).

One of the ways to increase the immunogenicity of antigens is the chemical compound with the immunogenic carrier. About 70 years ago, Karl Landsteiner showed that non-immunogenic antigens can be turned into immunogenic if they are chemically combine with protein substances. In particular, the introduction to the mammals of small molecules called haptens, does not lead to induction of the synthesis of antibodies that bind introduced the hapten. If the hapten is connected by covalent (chemical) bond with the protein carrier, such conjugates <a hapten-carrier> can induce the production of antibodies that recognize both the hapten and the carrier.

Since opening Clandestinely was offered a lot of different conjugated immunogens. Immunogenic properties of antigens and vaccines increased by covalent connection with lipopolysaccharide

As an analogue of the invention may be considered immunogenic conjugates of β-propionamido polysaccharide and N-propionamides is on oligosaccharide protein as well as the method of obtaining these conjugates. The conjugates used to produce vaccines against infectious diseases and cancer (RF patent 2249463). The disadvantages of the proposed conjugates is the complexity of manufacture and lack of effectiveness, due to the fact that protein is "inert" carrier slightly in the degree of activating the immune response.

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

Also, as an analogue of the invention may be considered acidic peptidoglycan (hereinafter CNG), with the properties of the activator of the immune system. CNG is a water-soluble acid peptidoglycan derived from plants with a molecular mass of 1200-40000 kDa. The sugar composition of the polysaccharide part of the following: gala is turanova acid 18±6%, glucose 9±3%, galactose 5.5V±2%, arabinose 3,8±1,3%, rhamnose 1,9±0,9%, mannose 0,7±0,25%. The peptide part is 13±3%, contains amino acids with primary amino groups (arginine 2,6%, lysine 1,8%), and amino acids with carboxyl groups (aspartic acid to 10.6%, glutamic acid 11,1%). CNG and its production method described in the patent of the Russian Federation 2195308.

CNG is a strong immunostimulant: activates NK-cells, dendritic cells, macrophages, monocytes, enhances the production of cytokines and interferon. CNG is an adjuvant. When the joint introduction of experimental animals a mixture of protein antigen and CNG increases the production of antibodies specifically binding the antigen used. According to the patent RF under the joint administration to mice of 20 µg CNG and 50 mg of egg albumin concentration of antibodies in the blood of mice was achieved titers of 1: 22000, which was significantly higher than the response to the introduction of 50 mg of egg albumin without CNG (1:13000). Adjuvant effect of CNG was comparable with the effect known adjuvant of lipopeptide Pam3Cys-Ser-Lys4. With the introduction of 20 µg of lipopeptide together with 50 mg of egg albumin in the blood of mice accumulated antibodies to egg albumin in titer of 1:20000.

The disadvantage of increasing the intensity of the immune reaction by mixing antigen and CNG is the need to introduce a sufficiently large doses of the Academy of Sciences of the Egan. So, in the experiment by immunization of mice protein antigen (egg albumin, bovine serum albumin) adding CNG can significantly reduce the dose of antigen, it is necessary to apply the usual dose of about 50 μg of antigen per mouse.

Despite the abundance of the proposed options, still not found a universal immunostimulating carrier that has the best combination of efficacy and safety. Often, effective immunostimulatory carriers are unsafe for humans and animals, cause more or less severe adverse (toxic) effects. On the contrary, secure media are not sufficiently effective Immunostimulants.

The present invention is directed to creating a safe drug with high efficiency as immunostimulating carrier and suitable for construction of immunogens and vaccines.

The task of the invention is to increase the specific immunogenicity of the antigen, reducing the dose of antigen required for induction of the immune response, expanding Arsenal of immunostimulatory media. This task is solved by covalent crosslinking CNG and antigen.

The invention is carried out as follows. CNG can be obtained in various ways from vegetable raw materials. The following is the way p the receipt of CSG, which illustrates the invention, but does not restrict the scope of the claims.

To get CNG 5 kg of sprouts of the potato family, Solanaceae (S. tuberosum) are crushed, add 10 l of water and extracted at room temperature with stirring for 2 hours. The mixture is squeezed using a mechanical press. Aqueous extract cialiswhat and concentrate to a volume of 1 l by ultrafiltration on a 10 kDa filter. To the concentrate was added sodium chloride at the rate of 100 mg per 100 ml and 3 volumes of 96% ethanol. Sediment allocate by centrifugation at 4000 rpm for 30 minutes

The precipitate was washed with 300 ml of 96% ethanol, centrifuged and dried in air. Then re-pereosazhdeniya obtained precipitate salt agent is calcium chloride. For this purpose, 10 g of dry intermediate product is dissolved in 1 l of distilled water under stirring at room temperature for 1 hour. Nerastvorim sediment cast, and to the liquor add 150 ml of 5% solution of calcium chloride. The precipitation, which represents an acid peptidoglycan raw, separated by centrifugation. Then translate it into soluble form by adding a saturated solution of sodium chloride and incubated the mixture with stirring and heating at 50°C. until complete dissolution. The resulting solution was applied on a column of TSK HW-75F. Column ale the shape of water collect the first high molecular weight peak with a molecular weight of from 1200 up to 40,000 kDa. The solution is concentrated on a rotary evaporator and freeze-dried. The output of acid peptidoglycan is 170 mg Mass ratio between glucose and uronic acids is 1:4. Next, perform covalent connection CNG with immunogenum.

1. Covalent connection with CNG antigen containing sulfhydryl groups, is carried out using water-soluble heterobifunctional reagent containing maleimide and N-hydroxy-operations group. The conjugation reaction is carried out in two stages. In the first stage reaction is the activation of CNG by using heterobifunctional of this reagent sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). In this case N-hydroxy-operations group, Sulfo-SMCC reacts with amino groups CNG (arginine and lysine). After activation of the CNG remove unreacted Sulfo-SMCC and other low molecular weight products by dialysis or gel filtration with a threshold of 500 kDa. In the second stage reaction is carried out interaction maleimide group attached to the activated CNG with sulfhydryl group antigen. The reaction forms a covalent complex (conjugate) of the formula:

CNG-NH-R1-SH-R2,

where R1any water-soluble gets rolefunctioning reagent, containing maleimide and N-hydroxy-operations group, R2any antigen that contains a sulfhydryl group.

The absence of SH-groups in CNG allows to avoid the formation of complexes CNG-NH-R1-SH-CNG. After conjugation unbound antigen is removed by gel-filtration or ultrafiltration with a threshold of 500 kDa. The fact covalent compounds CNG and antigen proved by any method - spektrofotometrichesky, fluorimetrically, dyes, protein, antibodies that specifically bind to the antigen - emergence protein signal in the high molecular weight region (chromatography, electrophoresis, or other separation methods), typical CNG.

2. Covalent connection CNG with any antigen, containing amino groups, is carried out using heterobifunctional reagent that can react with carboxyl groups and amino groups. An example of such heterobifunctional reagent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

The reaction is carried out in two stages. In the first stage, carried out the reaction of the CNG with the EDC. While EDC reacts with a carboxyl group CSG: (galacturonic, aspartic and glutamic acid). Formed activated CNG modified O-utilizationa. This intermediate product is unstable. So the first reaction stage Provo is seeking in the presence of the stabilizer N-hydroxysultaine (Sulfo-NHS). In the second stage, carried out the reaction between CNG modified O-acilitation, and the antigen with a primary amino group. The reaction forms a covalent complex (conjugate) of the formula:

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 covalent compounds antigen and CNG proved by any method - fluorimetrically, spektrofotometrichesky, dyes, protein-specific antibodies - the emergence of the protein signal in the high molecular weight region (chromatography, electrophoresis, or other separation methods), typical CNG.

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

The advantages of this method are as follows:

- in 10-100 times will decrease the flow rate of the antigen in the immunization;

- significantly reduced the side effects of toxic antigens with reactogenicity (local and General the e a toxic reaction to an antigen), for example, microbial toxins and toxoids, viral hemagglutinin, neuraminidase and other substances the introduction of which not only leads to the development of the immune response, but also to the development of some pathological (side) effects.

The invention is illustrated by the following examples.

Example 1.

The conjugation of ovalbumin with CNG using Sulfo-SMCC.

As the antigen used natural protein ovalbumin. Conjugation of ovalbumin with CNG was performed using heterobifunctional reagent Sulfo-SMCC (commercial product of the firm Pierce, cat.N 22322). The reaction was carried out in accordance with the Protocol of the manufacturer, adapted to the peculiarities of CNG. Activation of CNG using Sulfo-SMCC.

27.7 mg CNG dissolved in 15 ml of distilled water under stirring on a magnetic stirrer for 30 min at room temperature. To the resulting solution was added 10 ml of 0.1 M phosphate buffer pH of 7.35 and sprinkled 3.1 mg Sulfo-SMCC. The mixture is left in a thermostat at 37°C for 1 hour, stirring occasionally. After incubation, the solution is cooled to room temperature and applied to a chromatographic column with gel TOYOPEARL HW-65F. The elution is conducted with distilled water, the detection of peaks - Refractometer. Collect the first high molecular weight peak with a molecular mass of more than 500 kDa. The solution of activated CNG evaporated on the rotor is ω evaporator to 13 ml. Preparation of ovalbumin to conjugation.

Commercial preparation of ovalbumin dissolved, treated with 2-mercaptoethanol for recovery of sulfhydryl groups, and then released from microaggregates by the method of gel permeation chromatography. to 17.6 mg of Ovalbumin (Calbiochem cat.N 32467) dissolved in 7 ml of a reducing buffer (0.1 M phosphate buffer pH 7.4, 2.5 mm ethylenediaminetetraacetic acid sodium salt EDTA, and 0.6% 2-mercaptoethanol). The resulting solution was incubated at room temperature for 2 hours under stirring on a magnetic stirrer and then filtered through a filter with pore diameter of 0.45 μm and is applied to the chromatographic column (950×30 mm) with gel TOYOPEARL HW-75F. Fractions elute purified water, detect by the method of refractometry. Collect the fraction of 40-50 kDa corresponding to the molecular mass of ovalbumin. By spectrophotometry to determine the maximum absorption of UV light (280 nm), in comparison with a calibration solutions of ovalbumin allows to determine the concentration of protein in the collected fractions, it was 0,31 mg/ml

The conjugation.

To 4 ml of activated CNG poured 3 ml solution of ovalbumin and 3 ml of conjugating buffer (150 mm sodium chloride, 20 mm phosphate buffer, pH to 7.2 with the addition of 1 mm EDTA). The reaction mixture was kept in refrigerator at 4°C for 15 hours, the Le which is to be applied on the column with gel TOYOPEARL HW -75 F. Fractions elute the purified water, the detector Refractometer. Collect the first peak with a molecular mass of more than 500 kDa. Assembled the first peak evaporated on a rotary evaporator to 10 ml and dried by freeze drying. Get to 2.3 mg conjugate CNG-ovalbumin.

The content of ovalbumin in the conjugate is determined by fluorescence (fluorimeter Hitachi 850, excitation 280 nm, the slit width of 3 nm, fluorescence at 320 nm, the slit width of 3 nm) during calibration with ovalbumin. The content of ovalbumin in the conjugate CNG-ovalbumin was about 1%.

Example 2.

The conjugation of recombinant protein of human immunodeficiency virus (rvic) and CNG using Sulfo-SMCC.

As the antigen used recombinant protein (commercial product of the firm ProSpec-Tany TechnoGene Ltd., Cat. No. HI V-110), comprising a polypeptide that contains immunodominant fragments of four proteins of HIV-1, in particular gag pl7, P24, gp120 and gp41. Protein rvic comes in the form of a solution of 1 mg/ml in buffer 20 mM PBS rn, 20 TM 0.5M NaCl containing 1 TM of digitial and 8 M urea.

Shift buffer was performed on a column of TOYOPEARL HW-40F (440×10 mm), pre-equilibrated with buffer containing 0.02 M phosphate, 0.02 M NaCl and 0.1 mm EDTA. Fractions were suirable buffer containing 0.02 M phosphate, 0.02 M NaCl and 0.1 mm EDTA. Detector - Refractometer. Collected fraction volume of 7 ml, corresponding to the peak of protein, rvic (molecular m the SSA ~100 kDa), which was then concentrated to 2 ml

The activation of CNG using Sulfo-SMCC and conjugation of rvic activated CNG conducted in accordance with the methods described in example 1. The output of the conjugate CNG-rvic 10.6 mg Content of rvic in the conjugate was 0.33%.

Example 3.

The conjugation of recombinant E protein (re) coronavirus and CNG using EDC.

As the antigen used re - recombinant envelope protein of a coronavirus associated with SARS (severe acute respiratory syndrom, SARS). Protein d is a polypeptide chain length of 76 amino acid residues. In this work, we used a commercial product of the firm ProSpec-Tany TechnoGene Ltd, Cat.No. SARS-252), which comes in the form of a solution of 1 mg protein d in 1 ml of buffer 50 mM Tris-HCl, 60 mM NaCl containing 50% glycerol.

Shift buffer were carried out as in example 2. Receive 2 ml of protein solution d in phosphate buffer. Conjugation and re CNG performed using heterobifunctional reagent EDC (we used a commercial product of the firm Pierce, Cat. No. 77149) in the presence of sulfo-N-hydroxysuccinimide (abbreviated, Sulfo-NHS, a commercial product of the firm Pierce, Cat. No. 24510). The reaction is carried out according to the Protocol of the firm Pierce, adapted to the peculiarities of CNG. For this 10,2 mg CNG dissolved in 5 ml of 0.1 M 2-(N-morpholino)econsultancy acid pH 4,16, stirring on a magnetic stirrer at room te is the temperature for 30 minutes To the resulting solution was activated CNG sprinkled 2.2 mg EDC and 3 mg Sulfo-NHS, stirred for 30 minutes Then add 2 ml of protein solution d, and the mixture was incubated at room temperature for 1.5 hours.

Conjugate CNG-re allocate to the chromatographic column with gel TOYOPEARL HW-75F, as described in example 1. Obtain 7 mg of the conjugate.

Content re-protein conjugate CNG-d was 1.6%.

Example 4.

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

The immunogenicity of the conjugate CNG-ovalbumin studied in laboratory mice males (CBA×C57B1/6)F1weight 20, the Conjugate was compared with the original antigen by ovalbumin and physical mixture of ovalbumin with CNG. The antigens were dissolved in physiological solution of 0.9% NaCl was injected into mice intraperitoneally in a volume of 0.2 ml of Repeated immunization with the same dose and in the same amount spent in 1 month.

Blood was obtained after 1 week after the second immunization. Blood from 10 mice of the same experimental group were pulirula was given the serum, which was poured into several portions of 100 μl and were frozen until testing.

Serum antibodies that specifically bind to ovalbumin was determined by ELISA according to standard methods. Commercial preparation of ovalbumin manufactured by Calbiochem was dissolved in carbonate buffet is e (0.05 M, pH of 9.5) at a concentration of 10 μg/ml, made of 100 μl into the wells of 96-Loup night tablet (Nunc, Maxisorb, Cat. No. 442404)were incubated overnight in the refrigerator. The wells were washed 3 times with 200 μl of phosphate buffer (0.15 M sodium chloride, 0.01 M phosphate, pH 7.4)containing 0.1% Tween-20 (Serva). "Clogging" conducted a 1% solution of casein in phosphate buffer containing 0.1% Tween-20 for 45 min (37°C), 150 μl per well. After 3 times washing with 250 μl of phosphate buffer with 0.1% Tween-20 in wells contributed dilution of the test serum of experimental mice. Serial dilution of the serum in phosphate buffer with 0.1% Tween-20 was prepared starting dilution 1:50, in increments of cultivation 3. After making holes serial dilution of the investigated sera the plates were incubated for 1 hour at 37°C. Washing was performed 3 times making 250 μl of phosphate buffer with 0.1% Tween-20.

For detection of mouse antibodies in the wells made of 100 ál conjugate goat antibodies specific for mouse IgG, horseradish peroxidase (firm "Sorbent", Moscow) in a dilution of 1:1000 in phosphate buffer containing 0.1% Tween-20, incubated for 1 hour at 37°C. After 3 times washing with 250 μl of phosphate buffer with 0.1% Tween-20 in wells contributed substrate for detecting horseradish peroxidase (0.6 mg/ml of orthophenylphenol hydrochloride in 0.1 M citrate buffer, pH 5, 3% hydrogen peroxide), incubated 10 min. Color reacts the Yu stopped, filling in wells 100 ál of 1 M sulfuric acid. The measurement of the intensity of the reaction was performed photometrically at 492 nm on a tablet photometer MRX company Dynex (USA).

Table 1 presents the results of testing the immunogenicity of conjugate CNG-ovalbumin (30 μg conjugate that is 0.3 μg of ovalbumin per mouse) in comparison with ovalbumin (0,3 μg and 30 μg per mouse) and physical mixture of CNG plus ovalbumin (30 µg CNG plus 0.3 µg and 30 µg of ovalbumin per mouse). Introduction mice conjugate CNG-ovalbumin caused the production of antibodies, more intense than the introduction of ovalbumin or a mixture of CNG with ovalbumin. When comparing the level of immune response to small doses of ovalbumin (0.3 ág per mouse), you can determine that the specific immunogenicity of ovalbumin in the composition of the conjugate in 100 or more times higher compared with the ovalbumin or a mixture of CNG plus ovalbumin.

Table 1.
Immune response of mice (CBA × C57B1/6)F1(in response to the introduction of conjugate CNG-ovalbumin in comparison with the introduction of ovalbumin or a mixture of CNG with ovalbumin.
ImmunogenThe antibody titers in the serum of mice immunized with different doses of antigen*
0.3 ág of ovalbumin30 μg of ovalbumin
Conjugate CNG-ovalbumin, 30 mcg55000Ni**
Blend: 30 µg CNG plus ovalbumin50030000
Ovalbumin10020000
Notes:
(*) presents the antibody titers after 1 week after the second injection of the indicated doses of immunogen (indicated doses for ovalbumin);
(**)Ni means not investigated.

Example 5.

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

The immunogenicity of the conjugate CNG-rvic studied in laboratory mice males (CBA × C57Bl/6)F1. The conjugate was compared with the original antigen, rwic and physical mixture of CNG plus rvic.

The immunization of mice and determination of antibody titers was performed as in example 4. As immunogen experimental mice were injected conjugate CNG-rvic (doses and 0.1 μg per mouse dose on CNG - 30 µg), or a mixture of CNG plus d (dose of 0.1 μg or 1 μg of rvic mouse plus 30 μg CNG), or rvic (dose of 0.1 μg, 1 μg, or 5 μg per mouse).

Table 2 presents the intensity of the production of antibodies specific is rvic, after 1 week after re-introduction of these immunogens. Obviously, the specific immunogenicity of the antigen rvic in the composition of the conjugate CNG-rvic was 50 times higher immunogenicity of rwic and 10 times higher immunogenicity of a mixture of CNG plus rvic.

Table 2.
Immune response of mice (CBA × C57B1/6)F1in response to the introduction of conjugate CNG-rvic in comparison with the introduction of rvic or a mixture of CNG with rvic.
ImmunogenThe antibody titers in the serum of mice immunized with different doses of antigen*
0.1 ág of rvic1 µg of rvic5 μg of rvic
Conjugate CNG-rvic (30 µg)22000Ni**Ni**
A mixture of 30 µg CNG plus rvic50020000Ni**
rvic100250020000
Notes:
(*) presents the antibody titers after 1 week after re-introduction pointed to by the x doses of immunogen (indicated doses for ovalbumin);
(**)Ni means not investigated.

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

The immunogenicity of the conjugate CNG-d was studied in laboratory mice males (CBA × C57B1/6)F1. The conjugate was compared with the original antigen d and physical mixture of CNG plus re.

The immunization of mice and determination of antibody titers was performed as in example 4. As immunogen experimental mice were injected conjugate CNG-d (dose re - 0.5 μg per mouse dose on CNG - 30 µg), or a mixture of CNG plus d (dose of 0.5 μg or 5 μg re on the mouse plus 30 μg CNG), or d (dose of 0.5 μg or 5 μg per mouse).

Table 3 presents the intensity of the production of antibodies specific to the PE, 1 week after re-introduction of these immunogens. From the presented data shows that the specific immunogenicity of the antigen d in the composition of the conjugate CNG-re was 8-10 times higher immunogenicity re and mixtures PE with CNG.

Table 3. Immune response of mice (CBA × C57B1/6)F1in response to the introduction of conjugate CNG-re in comparison with the introduction of a re or a mixture of CNG with PE.
ImmunogenThe antibody titers in the serum of mice immunized with different doses of antigen*
0.5 μg re5 µg re
Conjugate CNG-re, 30 mcg8000Ni**
A mixture of 30 µg CNG plus d50010000
d10010000
Notes:
(*) presents the antibody titers after 1 week after the second injection of the indicated doses of immunogen (indicated doses for ovalbumin);
(**)Ni means not investigated.

1. Conjugate for immunization and vaccination containing the immunogen covalently coupled with a macromolecular carrier, characterized in that the high molecular media use acid peptidoglycan molecular weight 1200-40000 kDa, having a mass ratio between glucose and uronic acids are equal, 1:2-4.

2. The conjugate according to claim 1, characterized in that the peptide portion of the molecule acid peptidoglycan is (13±3) wt.%.

3. The conjugate according to claim 2, characterized in that the sugar composition of the polysaccharide part of the acid peptidoglycan contains galacturonic acid 18±6%, glucose 9±3%, galactose 5.5V±2%, arabinose and 3.8±1.3%) and rhamnose 1,9±0,9%, mannose 0,7±0.25%, and peptide frequent which contains arginine 2,6%, and lysine to 1.8%, and aspartic acid 10.6% and glutamic acid 11,1%.

4. The conjugate according to any one of claims 1 to 3, characterized in that the immunogen is a recombinant protein of 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 (re) coronavirus associated with SARS.

6. The way to increase immunogenicity by covalent compounds immunogen with acid peptidoglycan molecular weight 1200-40000 kDa, having a mass ratio between glucose and uronic acids is 1:2-4.



 

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FIELD: medicine.

SUBSTANCE: invention is related to new compounds of common formula IC1: , where A represents cyano; B represents hydrogen; R1, R2, R3 and R4 independently represent hydrogen; alkyl; halogen or nitro; R5 and R6 independently represent hydrogen; alkyl; cycloalkyl; cycloalkylalkyl; heteroaryl; heteroarylalkyl; alkenyl; carboxyalkyl; cyanoalkyl; diphenylalkyl; aryl, arylalkoxyaryl, arylalkyl, arylalkylaryl, arylcarbonylaryl or aryloxyaryl, or R5 and R6, together with atom of nitrogen, to which they are connected, create heterocyclic ring system; or to salts of such compound; at the same time "heteroaryl" used separately or in combination, is related to mono-, bi- or tricyclic aromatic ring system, which contains up to 14 atoms included in ring, in which at least one ring contains at least one heteroatom, independently chosen from nitrogen, oxygen or sulfur, besides specified heteroaryl group may be unsubstituted or substituted with one to three substituents, independently selected from alkyl and alkoxy; "diphenylalkyl" is related to alkyl group, where each of two atoms of hydrogen is substituted with unsubstituted phenyl group; "aryl" is related to carbocyclic group, selected from group, which consists of phenyl, biphenyl, 1,2,3,4-tetrahydronaphthyl, naphthyl, antryl, phenantryl, fluorenyl, indanyl, 2,3-dihydrobenzo[1,4]dioxynyl and benzo[1,3]dioxolyl group, besides specified aryl group may be unnecessarily substituted with functional groups in number from one to three, which are separately and independently selected from alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, cyano, halogen, halogenlkoxy, halogenalkyl and nitro groups, where in certain specific cases, if aryl group represents condensed system from several rings, in which not all the rings are aromatic, one of carbon atoms of which is not included into aromatic ring may be in oxidised condition, and according fragment of ring -CH2- will be substituted by fragment-C(O); "arylalkoxy", used separately or in combination, is related to aryl group, which is connected to initial molecular fragment via alkoxygroup, where aryl group is unsubstituted; "arylalkyl", used separately or in combination, is related to aryl group, which is connected to initial molecular fragment via alkyl group, where aryl group may be unsubstituted or substituted with 1-3 substituents, independently selected from group, which consists of halogen; "aryloxy", used separately or in combination, is related to aryl group, which is connected to initial molecular fragment via oxygen bridge, where aryl group may be unsubstituted or substituted with 1-3 substituents, independently selected from group, which consists of halogen; "arylcarbonyl", used separately or in combination, is related to aryl group, which is connected to initial molecular fragment via carbonyl group, where aryl group is unsubstituted; "heterocyclic ring system", used separately or in combination, is related to monocyclic, bicyclic or polycyclic ring system, which contains up to 15 atoms included into ring, at least one of which represents heteroatom, independently selected from nitrogen, oxygen or sulfur, besides specified ring system may be saturated, partially unsaturated, unsaturated or aromatic, where specified heterocyclic fragment may be unnecessarily substituted with one or more substituents, every of which separately and independently is selected from group made of halogen and halogenalkyl, excluding the following compounds: {3-[(E)-2-cyano-2-(4-fluorophenylcarbamoyl)vinyl]indole-1-yl}acetic acid; [3-((E)-2-cyano-2-m-tolylcarbamoylvinyl)indole-1-yl]acetic acid; (3-[(E)-2-(3-bromophenylcarbamoyl)-2-cyanovinyl]indole-1-yl}acetic acid; [3-((E)-2-cyano-2-phenylcarbamoylvinyl)indole-1-yl]acetic acid; [3-((E)-2-benzylcarbamoyl-2-cyanovinyl)indole-1-yl]acetic acid; [3-((E)-2-cyano-2-o-tolylcarbamoylvinyl)indole-1-yl]acetic acid; [3-((E)-2-cyano-2-t-tolylcarbamoylvinyl)indole-1-yl]acetic acid; (3-[(E)-2-(4-bromophenylcarbamoyl)-2-cyanovinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(4-ethylphenylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(4-methoxyphenylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2~cyano-2-(4- ethoxyphenylcarbamoyl)vinyl]indole-1-yl}acetic acid; [3-((E)-2-cyano-2-isopropylcarbamoylvinyl)indole-1-yl]acetic acid; {3-[(E)-2-cyano-2-(3-etoxyphenylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-3-[[2-(1H-indole-3-yl)ethyl]amino]-3-oxo-1-propenyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(4-chlorophenylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-3-(4-methyl-piperidine-1-yl)-3-oxopropenyl]indole-1-yl}acetic acid; {3-[(E)-2-(3-chloro-4-methylphenylcarbamoyl)-2-cyanovinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(3-phenylpropylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(2,3-dichlorophenylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-(5-chloro-2-methylphenylcarbamoyl)-2-cyanovinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(4-methoxybenzylcarbamoyl)vinyl]indole-1-yl}acetic acid; {3-[(E)-2-cyano-2-(2-fluorophenylcarbamoyl)vinyl]indole-1-yl}acetic acid; and {3-[(E)-2-cyano-3-oxo-3-(4-phenyl-piperazine-1-yl)propenyl]indole-1-yl}acetic acid. Invention is also related to pharmaceutical composition, and also to application of compounds of clause 1.

EFFECT: production of biologically active compounds, which have activity of antagonist coupled with G-protein of chemoattractant receptor of molecules homologue released by Th2-cells.

11 cl, 156 ex, 8 tbl

FIELD: medicine.

SUBSTANCE: invention relates to a method of producing 2-amino-2-[2-[4-(3-benzyloxy-phenylthio)-2-chlorophenyl]ethyl]-1,3-propanediol hydrochloride or its hydrate, involving the following steps: reacting 4-(3-benzyloxyphenylthio)-2-chlorobenzaldehyde and diethylphosphonoacetate ethyl in a solvent in the presence of a base, obtaining ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate; reducing the formed ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate with subsequent mesylation, iodizing, and nitration, obtaining 1-benzyloxy-3-[3-chloro-4-(3-nitropropyl-phenylthio]benzol; hydroxymethylation of the formed 1-benzyloxy-3-[3-chloro-4-(3-nitropropyl-phenylthio] benzol with formaldehyde, obtaining 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol; as well as reduction of the formed 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol, obtaining the end product. The invention also relates to intermediate products: ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate, 1-benzyloxy-3-[3-chloro-4-(3-nitropropylphenylthio]benzol, 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol or to their hydrates.

EFFECT: industrial production of said compound with good output and high purity.

4 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to a method of producing 2-amino-2-[2-[4-(3-benzyloxy-phenylthio)-2-chlorophenyl]ethyl]-1,3-propanediol hydrochloride or its hydrate, involving the following steps: reacting 4-(3-benzyloxyphenylthio)-2-chlorobenzaldehyde and diethylphosphonoacetate ethyl in a solvent in the presence of a base, obtaining ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate; reducing the formed ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate with subsequent mesylation, iodizing, and nitration, obtaining 1-benzyloxy-3-[3-chloro-4-(3-nitropropyl-phenylthio]benzol; hydroxymethylation of the formed 1-benzyloxy-3-[3-chloro-4-(3-nitropropyl-phenylthio] benzol with formaldehyde, obtaining 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol; as well as reduction of the formed 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol, obtaining the end product. The invention also relates to intermediate products: ethyl 3-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]acrylate, 1-benzyloxy-3-[3-chloro-4-(3-nitropropylphenylthio]benzol, 2-[2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl]-2-nitro-1,3-propanediol or to their hydrates.

EFFECT: industrial production of said compound with good output and high purity.

4 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to manufacturing of vaccines for intranasal introductions. There is offered composition to be delivered through mucous membrane, containing two or more following agents: a capsular oligosaccharide antigen which induces immune response on Haemophilus influenzae; and (b) a capsular oligosaccharide antigen which induces immune response on Neisseria meningitidis; with specified antigens conjugated with a carrier protein.

EFFECT: combination allows for single introduction to immunisation against two separate agents of the systemic disease, namely bacterial meningitis.

20 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and aims at treatment of cancer in an individual. It involves introduction to the specified individual of amount of purified heat-shock protein (HSP) preparation. The purified HSP preparation contains purified HSP-peptide complexes containing HSP covalently or non-covalently attached to peptide and where HSP-peptide complexes express antigenicity of said cancer. In addition, an immunogenic reagent is introduced to an individual, where the immunogenic reagent specifically reacts with antigen chosen from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20, or is being anti-CTLA-4 or anti 41BB antibody.

EFFECT: combined introduction of HSP-peptide complexes and immunogenic reagent allows intensifying immune response in antibody therapy of cancer.

36 cl, 4 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.

EFFECT: improved pharmacokinetic properties of erythropoietin.

23 cl, 14 ex, 6 tbl, 11 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.

EFFECT: enlarging the application range of medicine.

9 dwg

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns compositions and medicinal forms on the basis of Gastrinum, application and reception methods. The essence of the invention includes the bond of Gastrinum representing conjugates of fragments of amino-acid sequence of Gastrinum, possessing functional ability to contact Gastrinum/SSK receptor, with various carriers, including application amino-acid spacers, and application of bifunctional sewing agents, and also a method of treatment by the compositions including bond of Gastrinum, sick of diabetes.

EFFECT: advantage of the invention consists in action prolongation.

7 cl, 8 ex, 3 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns vaccines, particularly vaccines for meningococcal infections and diseases. Invention claims immunogenic composition for transport via mucosa, including capsular saccharides of at least two of the following serological groups: A, C and W135 and Y N. Meningitidis, as well as trialkylated chitosan.

EFFECT: enhanced production of immune response to meningococci in mucosa, possible balance shift of Th1/Th2 type responses.

16 cl, 24 dwg

FIELD: chemistry.

SUBSTANCE: invention pertains to modified polysaccharide in particular to modified polysaccharide Neisseria meningitidis of serogroup A, which preserves immunogenicity, but has improved stability. The modified polysaccharide is obtained from reaction of capsular polysaccharide, or its fragment - oligosaccharide, with CDI type bifunctional reagent, accompanied by reaction with an amino-compound, such as dimethylamine. Description is also given of modified polysaccharide conjugates and vaccines, which are obtained from such conjugates.

EFFECT: obtaining modified saccharide.

70 cl, 17 dwg

FIELD: medicine; veterinary science.

SUBSTANCE: method of higher meat production of broilers provides single injection for day birds of liposomal forms of preparation containing chimeric protein with water insoluble enzyme-inactive chloramphenicol acetyltransferase without 10 S-terminal aminoacids, aminoacid spacer (Sp)n, where n=1, 2, 4, 8 and somatostatin-14 with aminoacid sequence AGCFWKTFTSC, with median size of liposomes 250±50 nm. And preparation is introduced in combination with Marek's disease factor vaccine.

EFFECT: invention allows for higher effective meat production of broilers using single injection of preparation during the whole fattening period.

2 cl, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention concerns aldehyde derivatives and conjugates of di-, oligo- or polysaccharide, of the general formula (I), methods of obtaining them, and pharmaceutical composition based on them and capable of staying in blood flow for prolonged time. , where R is -CH(CHO)CH2OH, -CH2CHO, -CH(CH2NHR1)CH2OH, -CH(CH2NHNHR1)CH2OH, -CH(CH=NNHR1)CH2OH, -CH2CH2NHR1, -CH2CH=N-NHR1, -CH2CH2NHNHR1; R1 is polypeptide or albumen; GlyO is a sialic acid bond; R3 is H; R4 is OH; n is 2 or more.

EFFECT: obtaining pharmaceutical composition based on aldehyde derivatives of sialic acid capable of staying in blood flow for prolonged time.

20 cl, 7 tbl, 22 dwg, 10 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.

EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.

31 cl, 6 dwg

FIELD: immunology.

SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.

EFFECT: higher efficiency.

1 dwg, 2 ex, 8 tbl

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