KR20030059096A - Vaccine against influenza virus and method for producing thereof - Google Patents

Abstract

Vaccines against influenza viruses are provided, comprising compounds of heterochain polyamines with vaccine antigens of influenza virus. The compound may be prepared in the form of a conjugate having the following formula by carrying out the reaction of the vaccine antigens of the influenza virus with derivatives of heterochain polyamines by the azide method:

Wherein R represents the vaccine antigens of influenza virus;

n is the number of basic units;

q is the number of alkylation units;

z is the number of units oxidized.

The vaccine of the present invention can also be prepared in the form of a complex compound of vaccine antigens of influenza virus and derivatives of heterochain polyamines, which are prepared by forming a strong electrostatic bond by performing a complexing reaction. Can be.

Description

Vaccine against influenza virus and method for producing

Influenza acts on all groups of communities and is recognized as the most prevalent infectious viral-etiology disease. The rapid spread of disease and the high diversity of the influenza genome are responsible for serious pandemics. Therefore, vaccines against influenza virus are very important for the prevention of such diseases.

Live influenza virus vaccines are known in the art, including the preparation of whole virions (Peradze TV et al., "Anti-influenza Prophylactic Praparations", Moscow, Meditsina Publishers, 1986, pp. 79-81 (in Russian)). The harmlessness of live vaccines, the possibility of quickly producing the actual strains for them, the low cost and ease of administration make it possible to use such vaccines as vaccines for the prevention of influenza.

However, attenuated viruses of live vaccines can be mutated and reacquire virulence and other unexpected properties. Their inadequate antigenic activity, which requires non-standard reactiveity and at least double nasal administration of live vaccines, limits the use of live vaccines under large immunization environments.

These disadvantages can be overcome by using inactivated vaccines (Barry DW, Mayner RE, Staton E., et al., Comparative trial of influenza vaccines. Immunogenicity of whole virus and split product vaccines in man.-Amer. J.) Epidemiol., 1976, vol. 104, No. 1, pp. 34-46).

However, live vaccines and inactivated vaccines contain large amounts of ballast substances that may cause side effects and complications and are therefore unsafe for large scale prevention.

Main antigenic components isolated from virions: split and sub-unit vaccines are known, including hemagglutinin (H) and neuraminidase (N). . Split influenza vaccines induce high levels of immunity and do not contain major ballast substances. For example, a vaccine manufactured by Pasteur Merieux (France) is commercially available under the trade name "Vaxigrip" and is widely used. The "Vaxigrip" vaccine is characterized by high immunogenic activity and low reactivity, and is recommended for vaccination for infants from 6 months old to children and people at high risk.

Sub-unit vaccines are the least reactive, but also less effective, and therefore require at least two doses of sufficient dose at intervals of two to three weeks to obtain the required immune response, which is a large anti-influenza vaccine. It makes practically difficult to run speech programs. To enhance immunogenic activity, sub-unit vaccines are prepared with the addition of sorbents or complete virions, which also negatively affects the conformity to the safety and reactive properties of the formulation.

Of all known vaccines, the closest to the invention described herein is an anti-influenza vaccine prepared by reacting the vaccine carrier with the vaccine antigen of the influenza virus (RU 1580617, IPC A61K 39/145, publ. 1996 ). Antigen-polymer vaccines prepared by chemical binding provide phenotypic modification of the activation of immune cells and the gene regulation of the immune response. These vaccines are characterized by a weak response and are also harmless for vaccination in children and people at high risk.

However, in known vaccines, vaccine proteins bound to low molecular weight polymer carriers are not stable enough for use and storage of the formulation, and thus known vaccines, even if a special storage environment for them is provided. However, they do not always meet stringent shelf life requirements. Known vaccines also have relatively low immunogenic activity and require administration of sufficiently high formulation doses in order to obtain the required immune response.

Moreover, the polymer carriers used in known vaccines cannot form stable electrostatic complexes with vaccine antigens under physiological conditions, and thus the use of simple, safe and economical complexing techniques for preparing vaccines against influenza viruses. The possibility is excluded.

Known methods for making vaccines against influenza viruses by reacting the vaccine carrier with the vaccine antigen of the influenza virus provide a next generation vaccine: an effective immune response in the antigenic portion of the conjugate, which is independent of the genetic regulation of the immune response. Making it possible to obtain conjugated polymer sub-unit vaccines.

However, the performance of known methods involves the use of toxic succinimide esters, which not only complicates vaccine manufacturing techniques, imposes high requirements on working conditions and safety techniques, but also In carrying out the method, it is necessary to bring about ecologically undesirable environments and to take special means for carrying out precise purification. Inadequate technical effects of known methods in the manufacture of vaccines against influenza viruses involve significant material and labor resource requirements, but the yield of the target product does not exceed 20-30%. Known methods of making vaccines against influenza viruses do not provide effective and objective control at every stage of vaccine manufacture, which has a negative impact on the repeatability of the production parameters.

FIELD OF THE INVENTION The present invention relates to the medical field and can be used for immunity, in particular for the prevention of influenza and acute respiratory diseases.

The present invention relates to the provision of a vaccine against influenza virus, which is safe for all age groups, and to increasing the prophylactic effect of the vaccine even with a very low vaccination dose.

Another object of the present invention is to enhance the technical effectiveness of the vaccine manufacturing process, to broaden the technical potential by using new methods of forming compounds of polymer carriers and vaccine antigens, and to improve safety technology and ecological manufacturing conditions. To reduce the requirements for the product and to improve the quality of the target product.

The object is achieved by a vaccine against influenza virus comprising a compound of a polymer carrier and the vaccine antigens of influenza virus, a derivative of a heterochain polyamine having the formula (1) is used as the polymer carrier :

Wherein n is the number of basic units;

q is the number of alkylated units;

z is the number of oxidized units;

A is CH or N.

According to the present invention, it is advantageous to use derivatives of heterochain polyamines having a molecular weight of 50000 to 150000 Daltons (Da).

According to the invention, it is advantageous to choose an antigen / carrier ratio of 1: 5 to 30.

According to the invention, it is advantageous to choose n = 350 to 1000, q = (0.2 to 0.4) n, z = (0.4 to 0.8) n, x = 2 or 4, y = 1 or 2.

The object is achieved by a compound comprising the vaccine antigens of influenza virus covalently linked to derivatives of heterochain polyamines in the form of conjugates of the general formula (2):

In which R represents the vaccine antigens of influenza virus.

The object is also achieved by a compound comprising a complex of vaccine antigens of influenza virus bound by electrostatic bonding to derivatives of heterochain polyamines.

As derivatives of the heterochain polyamines, it is preferable to use a copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide of the following formula (3):

According to the present invention, as the derivatives of the heterochain polyamines, a copolymer of 1,4-ethylene piperidine N-oxide and (N-carboxyethyl) -1,4-ethylene piperidinium bromide It is advantageous to:

It is preferable to use surface proteins of influenza virus as the vaccine antigen of influenza virus.

According to the invention, it is advantageous to use hemagglutinin and / or neuraminidase as surface proteins of influenza virus.

According to the present invention, it is advantageous to use internal proteins, matrix proteins (M-proteins) and nucleic acid proteins (NP-proteins) of influenza viruses as vaccine antigens of influenza viruses.

According to the invention, it is advantageous to use a combination of surface and internal proteins of the influenza virus as the vaccine antigen.

The above object is also achieved by a method for preparing a vaccine against influenza virus, which comprises reacting a vaccine carrier with a vaccine antigen of an influenza virus, and derivatives of heterochain polyamines are used as polymer carriers, and their influenza virus The reaction with the vaccine antigen is carried out by the azide method.

According to the invention, it is advantageous to use derivatives of heterochain polyamines having a molecular weight of 50000 to 150000 Daltons.

According to the present invention, the reaction between the derivatives of the heterochain polyamines and the vaccine antigens of the influenza virus is advantageously made with an antigen / carrier ratio of 1: 5 to 30.

According to the present invention, it is preferable to pre-activate the derivatives of the heterochain polyamines.

The activation of the derivatives of the heterochain polyamines is preferably carried out by introducing hydrazide groups.

The above object is also achieved by a method for preparing a vaccine against influenza virus, which comprises reacting a vaccine carrier with a vaccine antigen of an influenza virus, and derivatives of heterochain polyamines are used as polymer carriers, and their influenza virus The reaction with the vaccine antigens is carried out with the help of the complexation reaction.

According to the present invention, it is advantageous that the complexation reaction of the derivatives of heterochain polyamines with the vaccine antigens of influenza virus is performed at an antigen / carrier ratio of 1: 5 to 30.

This task is achieved by a complexing reaction carried out between the derivatives of the oppositely charged heterochain polyamines and the macromolecules of the vaccine antigens of the influenza vice.

According to the present invention, as derivatives of heterochain polyamines, copolymers of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide are used. It is advantageous to use hemagglutinin and neuraminidase, the surface proteins of the influenza virus, and carry out the complexing reaction at a temperature of 2 to 4 ° C. under phosphate buffer conditions of pH 5.8.

By using derivatives of heterochain polyamines which themselves have immunotropic activity as polymer carriers, the present invention provides high immunogenicity and stability of influenza virus antigens, formation of immune memory, substantial reduction of antigen inoculation amount (Approximately three times compared to the world standard for influenza vaccines) and to increase the organism's resistance to other infections.

The present invention also provides for the simplification and acceleration of the processes for making vaccines against influenza virus, the reduction of energy density and the improvement of the ecological safety of manufacture, thereby improving the quality of the target product and enhancing its yield.

The present invention also makes it possible to obtain a vaccine against influenza virus, which stably reproduces physiological properties exhibiting high stability against physicochemical parameters and external factors.

Other objects and advantages of the present invention will become apparent from the detailed description of the vaccine against influenza virus and the method for producing the same and the specific embodiments of the method and vaccine described below.

The vaccine against influenza virus according to the present invention is a conjugate having a strong covalent bond between a vaccine antigen of an influenza virus and water-soluble derivatives of heterochain polyamines or a complex having a stable electrostatic bond between these components. Formulation.

Vaccines against influenza viruses are polymeric compounds having a molecular weight of 1 to 3 mln daltons and a molecular diameter of 50 to 80 nm and have an isoelectric point of 7.25.

Typically, influenza vaccines have surface antigens of influenza virus: virion's surface proteins (supercapsid sub-units) and an isoelectric point of 6.9 and 7.1, respectively, of hemagglutinin (HA) and a molecular weight of 77000 daltons; A neuraminidase having a molecular weight of 220000 Daltons is used.

These antigens cause the formation of an antibody immune response because HA antibodies have virus-neutralizing activity and protect life against infection by cognate viruses.

Internal antigens (nucleocapsid sub-units) of influenza virus used for vaccine preparation-nucleic acid protein (NP-protein) with molecular weight 170000 daltons and isoelectric point 6.5 and substrate protein (or M with molecular weight 64000 daltons and isoelectric point 6.8) Protein)-also plays an important role in the infection process. In humans infected with influenza or inoculated with complete virion vaccines, it has been found that antibodies against both surface HA and NA antigens and internal M- and NP-proteins are produced.

In order to isolate vaccine antigens from the virus of the corresponding strain, techniques using nonionic surfactants are used. Influenza viruses are grown in the allantoic cavity of eggs developed for 10 days, and are purified and concentrated by sucrose density step gradient technology.

Heterochain polyamine derivatives are water soluble, non-toxic, fully biochemical compounds with significant immunomodulatory properties.

Heterocyclic polyamine derivatives are obtained by synthesizing polyethylene piperidines by the "live" cationic polymerization method and oxidizing and alkylating the synthesized heterochain polyamines. The materials thus obtained have appropriate physiological and pharmacological activities.

Modification of the vaccine antigens by the polymer soluble immunoregulatory carrier makes it possible to enhance the immunogenic activity of the sub-units. Due to the formation of stable covalent or electrostatic bonds (depending on the method of preparing the compound) between the heterologous polyamine derivatives described above and the vaccine antigens of the influenza virus, the stability of these antigens and their immunogenicity Enhancement is possible, thus lowering the dose of antigen. Formation of immune memory against influenza virus antigens becomes more effective. At the same time, the protective properties of the organism against other infections are increased, resulting in an increase in the overall prophylactic activity of the vaccine. In addition, the use of non-toxic water soluble carriers contributes to increasing the safety of the vaccine, making it suitable for vaccination from 6 months infants to children.

The method for preparing a vaccine against influenza virus based on conjugates of heterochain polyamine derivatives with the prophylactic antigens of influenza virus is carried out in the following manner.

1,4-ethylenepiperazine N-oxide and (N-carboxyethyl) -1,4-ethylpiperazinium bromide for the sake of clarity in describing the vaccine preparation method for influenza virus according to the invention. Commercially produced "polyoxidonium", a copolymer of (CPL), is selected as the starting derivative of heterochain polyamines.

Preliminarily, heterochain polyamine derivatives CPL in the activated form are prepared. To this end, the starting CPL is modified by hydrazine hydrate.

After activation, the CPL hydrazide is mixed with the protective antigens of influenza virus at a ratio of 5 to 30: 1 and reacted by the azide method, and the result is a covalent combination of the vaccine antigens of the influenza virus and a polymer synthetic carrier. A stable covalent amide bond is formed between the carboxyl group of the polymer carrier and the primary amino group of the lysine residue of the protein molecules.

Such synthesis is performed under strictly controlled conditions, making it possible to obtain agents with a predetermined structure and protein / polymer ratio. Using the azide method with thorough observation of the reaction conditions makes it possible to completely avoid intermolecular and intramolecular crosslinking, preserve active centers, and obtain high yields and quality of target products.

A method for producing a vaccine against influenza virus, based on a complex compound of a high molecular polymer carrier from a water-soluble derivative group of heterochain polyamines and protective antigens of influenza virus, is carried out in the following manner.

CPL is dissolved in phosphate buffer at pH 5.8. A solution of vaccine antigens of influenza virus in the same buffer is added to the CPL solution.

The strength of the complex varies depending on the selection of derivatives and protective antigens of certain heterochain polyamines subjected to the reaction. In order for strong electrostatic bonds to be formed, an optimal proportion of the components in the solution must be selected. The complexing reaction is carried out by slowly adding the protein solution to the polymer solution at an antigen / carrier ratio of 1: 5 to 30. By the indicated pH value, macromolecules of the polymer carrier CPL and influenza virus surface antigens are reversely charged and as a result of this reaction they form stable electrostatic bonds.

The formulation is separated by chromatographic methods.

Analysis of the composition of the obtained compounds and their specific properties was performed using methods such as SDS-electrophoresis, electron microscopy, fluorescence spectroscopy and circular dichroism methods in polyacrylamide gel (PAAG). The methods made it possible to qualitatively analyze the degree of binding of the protein to the carrier, determine the location of the binding, and support the conformational stability of the protein in the conjugate composition.

The immunogenic and protective properties of the compounds obtained were evaluated by examining the response of the immune system and the response of the organism as a whole of the experimental animals and also testing the vaccine against volunteers.

The investigations performed show that vaccines against influenza viruses prepared in the form of conjugates or complex preparations in accordance with the invention described herein provide the following advantages:

Immunogenic activity is 10-fold higher than sub-unit vaccines,

Optimal active pH is 7.25, which is similar to the pH value of physiological body fluids, providing high bioavailability,

Absolutely safe to use formulations, since only the vaccine vaccines of highly purified influenza virus are used and the vaccination amount in terms of protein is 3 to 5 times lower than conventional vaccines,

-The vaccine is effective for single immunization throughout the epidemiological season,

The stability of the vaccine antigens in the conjugate or complex formulation composition is at least 100 times higher than the stability of natural proteins,

Since polymer carriers based on polymer water-soluble derivatives of heterochain polyamines have very suitable immunomodulatory activity, a marked improvement in the immunogenicity and prophylactic activity of the vaccine can be obtained.

Investigations into volunteers were conducted during a seasonal increase in morbidity due to influenza and acute respiratory disease (ARD) in the case of single vaccination. From the analysis of morbidity due to influenza and ARD among adults, the influenza morbidity in the external control group was 92.3 per 1000 observers, representing the population vaccinated with the vaccine of the present invention (26.7 per 1000 inoculated subjects) and the internal control ( It is evident that this is significantly higher than 36 per 1000 observers). Serological interpretation of the etiology diagnosis of influenza was performed.

The properties established from epidemiological experimental data make it possible to determine the most important properties as a vaccine by using the appropriate formulas:

The efficiency index is 3.4,

The prophylactic efficiency factor is 77%,

In the presence of 50% immune interlayer in the population, the anti-epidemic protection factor is 68%.

In certain embodiments of the invention described below, Examples 1-12 describe several specific variables in carrying out methods for making vaccines against influenza viruses, and Examples 1-15 illustrate Examples 1-12. An experimental evaluation of the effectiveness of the formulations obtained in is provided. The efficiency evaluation results of the formulations are reflected in Tables 1-4.

Example 1 Preparation of a Vaccine Based CPL Conjugate with Surface Proteins HA and NA of Influenza Viruses Using the Azide Conjugation Method

Step 1: Carrier-CPL hydrazide synthesis in activated form.

500 mg CPL (n = 700, q = 0.2, z = 0.8, MW = 80000, MWD = 1.33) were dissolved in 25 ml of methyl alcohol. 0.2 ml (2 × 10 ⁻³ M) hydrazine hydrate was added at a temperature of 20 ° C. The reaction was carried out for 24 hours. After methanol was distilled off on a rotary evaporator, the residue was dissolved in water and ether extraction was performed. The final product was separated by ultrafiltration using a “Millipore” membrane and subsequent freeze drying.

The content of hydrazide groups was determined by standard methods of titrating primary amino groups with 2,4,6-trinitrobenzenesulfonic acid. The content of hydrazide groups was 15%.

Step 2: Preparation of the conjugate by condensation reaction of CPL hydrazide with HA and NA.

100 mg of CPL hydrazide was dissolved in 4 ml of 1N HCl. The solution was cooled to 0-2 ° C. Next, 1.15 ml of 3% sodium nitrite solution was added while stirring and cooling. After 15 minutes, the pH of the solution was adjusted to 8.5 by adding 2N NaOH.

To the CPL hydrazide solution, a 20 mg solution of HA and NA protein mixture (95: 5 ratio) in 10 ml of 0.05M buffer at pH 8.5 was added. The reaction pH was maintained at 8.5 by adding 2N NaOH. The reaction was carried out with stirring for 12 hours and cooling to 0-2 ° C. To isolate and purify the conjugate, the reaction mixture was added to a column (2.6 x 90) filled with Sephadex G-100, and the eluent was 0.05 M phosphate buffer containing 0.05 M NaCl and pH 7.5. The yield of the conjugate was controlled by flow spectrometer at λ = 226 nm. Antigen content was determined and the conjugates were analyzed by electrophoresis on fluorescence spectroscopy and polyacrylamide gels (PAAG). The protein / CPL conjugate molar ratio was 1: 5. The yield of the conjugate was 97%.

The copolymer composition was determined by NMR-spectroscopy and IR-spectroscopy techniques, and the molecular weight and molecular weight distribution were determined by the small-angle laser radiation scattering method.

Example 2 Preparation of a Vaccine Based CPL Complex with Surface Proteins HA and NA of Influenza Virus

100 mg of CPL (n = 1000, q = 0.4, z = 0.4, MW = 145000, MWD = 1.7) were dissolved in 10 ml of 0.05M phosphate buffer at pH 5.8. A 10 mg solution of HA and NA mixture in 2 ml of the same buffer was added at a temperature of 2-4 ° C. At this pH value the macromolecules of the polymer carrier and antigens are oppositely charged.

The complexation reaction was carried out by slowly adding a solution of antigens to the solution of the polymer while preventing precipitation. The reaction mixture was maintained at a temperature of 2-4 ° C. for 24 hours. At the completion of the reaction, the pH of the solution was adjusted to 7.1 by adding a solution of sodium hydroxide. The protein / CPL molar ratio was 1:10. The yield of the target product was 96%. Protein content was determined and compounds were analyzed using electrophoresis on fluorescence spectroscopy and PAAG.

Example 3.

Using CPL having the following properties as an antigen carrier: n = 400, q = 0.3, z = 0.7, MW = 60000, MWD = 1.1, M-protein and NP which are internal proteins of influenza virus as vaccine antigens of influenza virus Using the protein, it was carried out in the same manner as in Example 1. The protein / CPL molar ratio was 1: 6. Proteins in the conjugates were analyzed by the Lowry method.

Example 4.

Using CPL with the following properties as a carrier: n = 900, q = 0.35, z = 0.65, MW = 100000, MWD = 1.6, with the surface HA and NA proteins of the influenza virus and the internal M-protein as a vaccine antigen Using a mixture of NP-proteins, it was carried out in the same manner as in Example 1. The protein / CPL molar ratio was 1: 5. Protein content was determined by the Bradford method.

Example 5.

Using a CPL with the following properties as a carrier: n = 540, q = 0.25, z = 0.75, MW = 78000, MWD = 1.8, using the internal M-protein and NP-protein of the influenza virus as the vaccine antigen, It was carried out in the same manner as in Example 2. The protein / CPL molar ratio was 1:30. Protein content was determined by the Bradford method.

Example 6.

Using a CPL having the following properties as a carrier: n = 900, q = 0.3, z = 0.7, MW = 135000, MWD = 1.7, with the surface HA and NA proteins of the influenza virus and the internal M-protein as a vaccine antigen The mixture was performed in the same manner as in Example 2, using a mixture of NP-proteins. The protein / CPL molar ratio was 1:25.

Example 7.

Copolymer of 1,2-ethylenepiperidine N-oxide with (N-carboxyethyl) -1,2-ethylenepiperidinium bromide (CPL-A) having the following properties as a carrier of the vaccine antigen of influenza virus Using: n = 100, q = 0.4, z = 0.4, MW = 145000, MWD = 1.7, using HA and NA as antigen, in the same manner as in Example 1. The protein / carrier molar ratio was 1:10. Proteins were analyzed by Lowry method.

Example 8.

Using CPL-A with the following properties as a carrier: n = 400, q = 0.3, z = 0.7, MW = 60000, MWD = 1.1, substrate M-protein and NP- which are internal proteins of influenza virus as a vaccine antigen Using the protein, it was carried out in the same manner as in Example 1. The antigen-carrier molar ratio was 1: 8. Proteins were analyzed by Lowry method.

Example 9.

Using CPL-A with the following properties as a carrier: n = 900, q = 0.35, z = 0.65, MW = 110000, MWD = 1.6, surface HA and NA proteins of the influenza virus as the vaccine antigen and internal M- Using a mixture of protein and NP-protein, it was carried out in the same manner as in Example 1. The antigen-carrier molar ratio was 1: 5. Protein content was determined by the Bradford method.

Example 10.

Using CPL-A with the following properties as a carrier: n = 540, q = 0.25, z = 0.75, MW = 78000, MWD = 1.8, HA-protein and NA-protein, surface proteins of influenza virus as vaccine antigens Was carried out in the same manner as in Example 2. The antigen-carrier molar ratio was 1:30. Proteins were analyzed by Lowry method.

Example 11.

Use CPL-A with the following properties as a carrier: n = 950, q = 0.3, z = 0.7, MW = 120000, MWD = 1.7, using the internal M-protein and NP-protein of influenza virus as vaccine antigens Thus, the same procedure as in Example 2 was carried out. The antigen / carrier molar ratio was 1:25. Protein content in the formulation was determined by the Lowry method.

Example 12.

Using CPL-A with the following properties as a carrier: n = 600, q = 0.4, z = 0.4, MW = 90000, MWD = 1.7, surface HA and NA proteins of the influenza virus as vaccine antigen and internal M- Using a mixture of protein and NP-protein, it was carried out in the same manner as in Example 2. The antigen / carrier molar ratio was 1:20. Protein content was determined by Lowry method.

Example 13. Study of efficacy of vaccine against influenza virus.

To establish specificity and determine the elevation of specific antibody titers, antigen activity is performed on the blood serum of mice and the blood serum of volunteers by a hemagglutination-inhibition reaction (HAIR). Was evaluated.

After removal of nonspecific inhibitors, 2-fold dilutions of serum were prepared in a volume of 0.2 ml in wells of Plexiglass plate. 0.2 ml of homologous antigen was added to each serum dilution in an amount of 4 AU. The mixture was stirred and left at a temperature of 20 ± 2 ° C. for 30 minutes. Each well was then added 0.4 ml of a 1% suspension of chicken red blood cells. The mixture was stirred again and left at a temperature of 20 ± 2 ° C. for 40 to 45 minutes (until red blood cells precipitated in the control). The reaction results were then calculated according to the 4-cross system. The maximum antigen dilution resulting in apparently significant hemagglutination was assumed to be antigen titer or 1 aggregation unit (1 AU). If certain antibodies are present in the serum, a delay occurs in the aggregation of red blood cells. Antibody titers in serum were adopted as the limiting dilution of the antigen resulting in complete inhibition of hemagglutination.

Sexually mature mice in the (CBA × C57B1 / 6) F1 strain with a body weight of 18-20 g were used for the experiment. Preparations based on the HA, NA, M-protein and NP-proteins of influenza viruses isolated from the corresponding H3N2, H1N1, B-type influenza virus lines, as well as their compounds with polymer carriers CPL and CPL-A Was used as the antigen.

Antigen doses were selected in preliminary experiments on mice. Each group consisted of 8 to 10 mice. All formulations were administered intraperitoneally in a volume of 0.5 ml. Mice in the control group received 0.5 ml of physiological solution. A double vaccination method was used that was revaccinated after 2 weeks. After 12 days after the second immunization, the immunogenicity of the agent under investigation was analyzed by hemagglutination-inhibiting response (HAIR).

The results obtained are shown in Table 1, and the antigenic activity of the control group is shown in position 13 in the following table.

Formulation One 2 3 4 5 6 7 8 9 10 11 12 13 HAIR titer 640 160-320 80 2560 20-40 640 1250 40 2560 160-320 20 1280 Less than 10

The data shown in Table 1 show that in all test groups to which the agent of the present invention was administered, antibody titers determined from hemagglutination-inhibition response exceed that of the control, ie, a pronounced antigenic response to agent administration is observed.

Example 14. Immunogenicity Analysis of Vaccines Against Influenza Viruses

Inbred (CBA × C51B1) F1 mice were administered in varying amounts according to various schemes. Thereafter, multiparametric investigations were performed for antigen specific immune responses, including determining the content of HA-specific antibodies in the serum of immunized animals.

The preparations were diluted in buffer and administered intraperitoneally or subcutaneously to the tail basal region of mice in an amount of 0.1-10 mg / mouse in terms of protein. To induce a secondary immune response, 21 days after the first administration, a second injection was performed. Mice were decapitate, blood was collected, and serum was obtained within a defined period after immunization.

The content of HA-specific antibodies in serum was determined by immuno-enzyme analysis (IEA) method. Analysis was performed by following conventional methods in 96-well polystyrene plates.

The IEA-titers of the antibodies were determined after the first and second immunizations in the test and control, and the specific immune memory factor K _im and the specific immunostimulation factor K _ss were calculated. No immune memory was formed in mice in the control group.

These factors do not depend on the absolute value of titers, but are objective properties of the immunogenic properties of the vaccine formulation. The results of the analysis are shown in Table 2.

Formulation Immune memory factor Non-stimulating factor One 65 80 2 40 35 4 70 100 6 45 50 7 60 70 9 70 95 10 30 40 12 35 30

The results shown in Table 2 show that all agents prepared according to the invention have immunogenic properties and form immune memory.

Example 15.

In accordance with approved test record sheets, volunteers were analyzed for the immunogenicity of the vaccines according to the invention. Volunteers for vaccination were selected from serum-negative individuals of age 30-40 (HAIR titers below 10). The test groups consisted of 10-12 people. Vaccination was performed in a single dose of 0.5 ml, using a disposable syringe, intramuscularly, to the left arm deltoid. Each injection included a hemagglutinin (5 ± 1) μg of influenza A virus (H1N1) and a carrier CPL-1 corresponding to the amount described in Examples 1, 2, 4, 6. Individuals in the control group received 0.5 ml of physiological solution.

To analyze the immunogenicity of the agents under investigation, a paired sera of vaccinated individuals, taken before vaccination and one month after vaccination, was subjected to hemagglutination-inhibition reaction using a vaccine strain of influenza virus (HAIR). ). The efficiency of the formulation was assessed from the percentage of individuals with protective antibody titers. Individuals were considered immunoprotected if the antibody titer in HAIR exceeded 1:40.

The results of immunogenicity studies of the vaccines are shown in Table 3 below.

Formulation HAIR titer 1 month after vaccination Effectiveness of vaccination (% of individuals with protective antibody titers) One 80-1280 95 2 80-320 91 4 160-1280 100 6 160-640 97 Physiological solution below 10 -

The experimental data shown in Table 3 indicate that high levels of immune responses with high protective antibody titers are formed in individuals vaccinated with the formulations according to the invention.

Example 16. Analysis of the effect of the vaccine against influenza virus using virus neutralization test.

The titers of "protective" antibodies were determined according to the method for determining virus-neutralizing activity of mouse serum immunized with the aforementioned agents.

The experiment was performed as in Example 14. After dual doses (every 21 days) of the optimal immunogenic dose of the vaccine to their mice, their blood serum was collected and their activity was examined by a live influenza virus inactivation test. To this end, various serum dilutions were mixed with suspensions of influenza virus and the titers of virus-neutralizing antibodies were analyzed by following commonly adopted methods. Virus-neutralizing titers of rodent sera were considered their maximum dilution, which abolished the hemagglutination activity of the capsular space fluid in 50% of embryos. The results of the analysis are shown in Table 4.

Formulation Virus-neutralizing titer One 1: 400 2 1:80 4 1: 400 6 1: 160 7 1: 160 9 1: 400 10 1: 160 12 1: 320 13 1:10

A titer of 1: 400 and a seroconversion factor of 40 in influenza virus neutralization tests are known to be reliable characteristics in the effectiveness of vaccines against influenza viruses and are considered to be "protective" titers. The data shown in Table 4 show that the serum of mice immunized with the formulations according to the invention contains at least 40-fold amounts of virus-neutralizing antibodies as compared to normal rodent serum.

The present invention may find application in the manufacture of next generation anti-influenza vaccines, which, due to polymer immunoregulatory carriers, have outstanding immunogenic activity, high levels of safety and preventive effects, which It expands the possibilities of using next-generation vaccines to prevent influenza for a diverse population, including children and people at high risk.

Claims (21)

In a vaccine against an influenza virus comprising a compound of a polymer carrier with a vaccine antigen of an influenza virus, a derivative comprising hetero-chain polyamines having the following formula (1) is used as the polymer carrier: <Formula 1> Wherein n is the number of basic units; q is the number of alkylation units; z is the number of oxidized units; A is CH or N. The vaccine of claim 1, wherein the derivatives of the heterochain polyamines have a molecular weight of 50000 to 150000 Daltons. The compound of claim 1 or 2, wherein n is 350 to 1000, q is (0.2 to 0.4) n, z is (0.4 to 0.8) n, x is 2 or 4, and y is 1 or 2 The vaccine characterized in that. The vaccine of claim 1 or 2, wherein the compound has an antigen / carrier ratio of 1: 5 to 30. The vaccine according to claim 1, wherein the compound comprises a conjugate of a vaccine antigen of influenza virus covalently linked with derivatives of heterochain polyamines, and has the following formula (2): <Formula 2> In which R represents the vaccine antigens of influenza virus. The vaccine of claim 1, wherein the compound comprises a complex of a vaccine antigen of influenza virus and derivatives of heterochain polyamines obtained by formation of an electrostatic bond. The copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide according to claim 1, wherein the derivatives of the heterochain polyamines have the following formula (3): A vaccine comprising the following: <Formula 3> The method of claim 1, wherein the derivatives of the heterochain polyamines, which have the following formula (4), are air of 1,4-ethylenepiperidine N-oxide and (N-carboxyethyl) -1,4-ethylenepiperidinium bromide. Vaccines characterized by the use of coalescing: <Formula 4> The vaccine of claim 1, wherein the surface proteins of the influenza virus are used as the vaccine antigen of the influenza virus. The vaccine according to claim 1, wherein hemagglutinin and / or neuraminidase are used as the vaccine antigen of influenza virus. The vaccine according to claim 1, wherein the vaccine of the influenza virus uses an internal protein, a matrix protein (M-protein) and a nucleic acid protein (NP-protein) of the influenza virus. The vaccine according to claim 1, wherein the vaccine of the influenza virus is used as the surface and internal proteins of the influenza virus. In the method for producing a vaccine against influenza virus, comprising reacting a vaccine carrier with a vaccine antigen of an influenza virus, derivatives of heterochain polyamines are used as the polymer carrier, and the vaccine against influenza virus of the polymer carrier is used. The reaction with the antigen is carried out by the azide method. The method according to claim 13, wherein derivatives of heterochain polyamines having a molecular weight of 50000 to 150000 Daltons are used. The method according to claim 13, wherein the reaction between the derivatives of the heterochain polyamines and the vaccine antigens of the influenza virus is performed at an antigen / carrier ratio of 1: 5 to 30. The method according to claim 13, wherein the derivatives of the heterochain polyamines are pre-activated. The method according to claim 16, wherein the activation of the derivatives of the heterochain polyamines is by introducing hydrazide groups. In the method for producing a vaccine against influenza virus, comprising reacting a vaccine carrier with a vaccine antigen of an influenza virus, derivatives of heterochain polyamines are used as the polymer carrier, and the vaccine against influenza virus of the polymer carrier is used. The reaction with the antigen is carried out by a complexation reaction. 19. The method according to claim 18, wherein the complexing reaction of the derivatives of the heterochain polyamines with the vaccine antigens of the influenza virus is performed at an antigen / carrier ratio of 1: 5 to 30. 20. The method according to claim 18 or 19, wherein the complexing reaction is carried out between the derivatives of the oppositely charged heterochain polyamines and the macromolecules of the vaccine antigens of the influenza virus. The copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide according to claim 18 is used as derivatives of the heterochain polyamines. For example, hemagglutinin and neuraminidase, which are the surface proteins of influenza viruses, are used, and the complexing reaction is carried out under phosphate buffer conditions of pH 5.8 at a temperature of 2 to 4 ° C.