RU2427385C1 - Biologically active complex exhibiting influenza virus protective activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology, immunology, virology. What is described is a biologically active complex which can be used for preparing vaccine and therapeutic preparations for viral and other infectious diseases and other purposes.

EFFECT: invention allows extending the range of biologically active complexes and improves biological activity of the complex in 1,6-1,7 times.

3 tbl, 5 ex

Description

The invention relates to the field of molecular biology, immunology, virology and relates to a biologically active complex having protective activity against influenza virus. This complex can be used, for example, to obtain vaccine preparations.

A biologically active complex is known consisting of viral particles carrying epitopes (antigenic determinants) of various animal viruses on the surface (M. Bendahmane, M. Koo, E. Karrer, RN Beachy, Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions, J. Mol. Biol, 1999, 290, 9-20).

Known biologically active complex consisting of a rod-like particle of the tobacco mosaic virus (TMV), connected to at least one polypeptide - the antigenic polypeptide of the human immunodeficiency virus (AAMcCormic, KEPalmer, Genetically engineered tobacco mosaic virus as nanoparticle vaccines, Expert Rev Vaccines, 2008 , 7 (1), 33-41).

Closest to the claimed is a known biologically active complex, which is a conjugate consisting of a modified rod-shaped virus particle coupled to a water-soluble polypeptide (US patent 20090053261) - a prototype. In this complex, a rod-shaped particle with covalently bound biotin is used as a modified rod-shaped virus particle. In this case, a polypeptide containing covalently bound avidin is used as a polypeptide. When the above components are mixed in an aqueous medium, a single biologically active complex is formed due to the interaction of biotin and avidin grafted groups in the complex components.

A disadvantage of the known vaccine preparation is its relatively low protective activity against influenza virus.

An object of the invention is to expand the range of biologically active complexes and increase the protective activity of the complex against influenza virus.

The specified technical result is achieved due to the fact that in a biologically active complex that has protective activity against the influenza virus and is a conjugate consisting of a modified rod-shaped virus particle connected to a water-soluble polypeptide, the modified rod-shaped virus particle is obtained by the interaction of 1 wt. parts of rod-shaped viral particles and 0.01-0.1 wt. parts of at least one water-soluble polymer with cationic groups in the following ratio of the starting components in the conjugate (wt. parts):

modified one rod-shaped viral particle polypeptide 0.1-25

The proposed biologically active complex is not described in the literature and is new, which significantly expands the range of biologically active complexes.

In the proposed technical solution, viruses of various biological genera, for example, the genus Tobamovirus, the genus Tobravirus, the genus Pomovirus, etc., can be used as a rod-shaped viral particle. These rod-shaped viral particles can be isolated from the cells of living organisms by known methods or purchased from commercial organizations, for example, from the company AGDIA (USA).

Various polymers can be used as a water-soluble polymer with cationic groups, for example, such as polylysine, chitosan, polyhexamethylene guanidinium phosphate, polydimethyldiallylammonium chloride (PDMDAAH), poly-N-ethyl-4-vinylpyridinium bromide (HDPE), etc. In addition to cationic groups, the polymers used may contain hydrophilic electroneutral and / or anionic and / or hydrophobic groups (units). You can use both an individual polymer with cationic groups, and mixtures of two or more different polymers with cationic groups. The molecular weight of the polymers can vary within wide limits, for example, from one to several thousand kilodaltons. Water-insoluble polymers cannot be used in this technical solution.

Polypeptides do not form biologically active complexes with an unmodified rod-shaped viral particle.

The product of the interaction of a rod-shaped viral particle and at least one polymer with cationic groups is formed only in an aqueous medium. You can use both distilled water and an aqueous solution of salts at a concentration of not more than 1%, for example, physiological saline. Upon receipt of such a product per 1 wt. part of the rod-shaped viral particles should be 0.01-0.1 wt. parts of at least one water-soluble polymer with cationic groups. The optimal above ratio of rod-shaped viral particles and at least one water-soluble polymer with cationic groups was established experimentally. With a smaller than the stated number of mass parts of a water-soluble polymer with cationic groups, the biological activity of the final complex decreases, while with a higher polymer content the product inevitably requires additional purification from the unreacted polymer, which complicates the proposed method. In this case, the pH of the aqueous medium can vary widely and is selected experimentally depending on the nature of the viral particle and the chemical structure of the water-soluble polymer.

The formation of just the product of the interaction, and not just a mechanical mixture of a rod-shaped virus particle and at least one water-soluble polymer containing cationic groups, is confirmed by the results of the following experiments. The above product is prepared, an aqueous dispersion of the obtained product is placed in an ultracentrifuge cell, and the rotation speed of the centrifuge rotor is selected in such a way as to ensure quantitative (complete) precipitation of the product. Make sure that the supernatant (supernatant) does not contain either a free water-soluble polymer or free viral rod-shaped particles. After that, two control experiments are carried out: in the first, an aqueous solution of the cationic polymer is centrifuged, in the second, an aqueous suspension of viral particles. The concentration of the polymer and viral particles in the control experiments is taken equal to the concentrations of these components during product formation. Centrifugation of the polymer solution and the suspension of viral particles is carried out at the same speed at which complete precipitation of the product was observed. At the same time, it is ascertained that under the indicated conditions neither a single polymer, nor individual virus particles are precipitated from a solution (suspension). The totality of the results clearly indicates that the mixing of a polymer solution and a suspension of viral particles in an aqueous medium is accompanied by the interaction of components (polymer and viral particles) with each other and the formation of a new product, which quantitatively includes both components of the initial mixture. The resulting product does not have significant biological activity.

As the polypeptide, any biologically active water-soluble polypeptide can be used, for example, influenza virus vaccine proteins, for example, such as hemagglutinin, neuraminidase, M2E. The biological activity of any proposed complex will be determined by the nature of the polypeptide used in its creation. Water-insoluble polypeptide cannot be used in the proposed technical solution. The polypeptide can be obtained by various methods, for example, by biosynthesis in cells of bacteria, yeast, plants, etc. This product can be purified and isolated as an individual preparation.

Upon receipt of the biologically active complex of one mass. part of the modified rod-shaped viral particles are mixed with 0.1-25 wt. parts of the polypeptide. With less than the stated number of mass parts of the polypeptide, the biological activity of the above complexes is significantly reduced. With a higher content of the polypeptide, the biological activity of the complex does not increase, but the resulting complex requires additional purification from free excess polypeptide, which complicates the proposed method. Upon receipt of the complex, the pH of the aqueous medium can vary widely and is selected experimentally depending on the nature of the viral particle and the chemical structure of the water-soluble polymer. It is possible to obtain a biologically active complex in a wide range of temperatures traditional for producing biologically active complexes, for example, from +4 to + 30 ° C.

This complex is a conjugate and is a fairly stable formation consisting of a modified viral particle and a polypeptide that can be isolated from the solution in the form of a gel-like precipitate. Such a precipitate may be resuspended in an aqueous medium. Depending on the characteristics of the modified viral particles, the nature of the polypeptide, and the concentrations of both components, the aqueous suspension of the biologically active complex can be practically transparent, slightly opalescent, or very cloudy.

The formation of a conjugate (complex) upon mixing an aqueous suspension of the aforementioned modified viral particles and an aqueous solution of the polypeptide, rather than their simple mechanical mixture, can be proved by ultracentrifugation in the same way as was done above for the reaction product of viral particles and a water-soluble polymer containing cationic groups. At the same time, they are convinced that the supernatant (supernatant) does not contain a free polypeptide, and the sediment has high biological activity, which was absent in the modified viral particles.

The biological activity of the complexes was demonstrated using traditional experiments on laboratory animals.

The advantages of the proposed biologically active complex are illustrated by the following examples.

Example 1

From 100 g of tobacco plants infected with TMV, the preparation of rod-shaped virus particles is isolated by centrifugation in a sucrose concentration gradient. Prepare 10 ml of a suspension of rod-shaped viral particles in distilled water containing 100 mg of viral particles (concentration of viral particles is 10 mg / ml).

From 100 g of E.coli cells (E. coli) on a Ni-containing agarose, a water-soluble polypeptide containing the influenza hemagglutinin fragment is isolated. Prepare 2 ml of a solution of the polypeptide in distilled water containing 8 mg of the polypeptide.

To obtain modified rod-shaped particles using the following procedure. A solution of HDPE with a degree of polymerization of 600 in water at a concentration of 0.3 mg / ml and 10 ^-2 M TRIS-HCl buffer solution with a pH of 7.5 is prepared. Mix 0.04 ml of HDPE solution (0.012 mg of a polymer containing cationic groups, 0.02 wt. Parts), 1.76 ml of a buffer solution and 0.06 ml of a TMV suspension with a concentration of 10 mg / ml (0.06 mg rod-shaped viral particles, 1 wt. part). The mixture is incubated for 1 hour at 4 ° C. Modified rod-shaped particles are isolated by centrifugation of the resulting mixture for 30 min at 60,000 g and 4 ° C. The precipitated complex is resuspended in physiological saline.

Product formation — modified rod-shaped particles, rather than a mechanical mixture of rod-shaped viral particles and free polymer — is proved as follows. The resulting sample is placed in an ultracentrifuge cell and the centrifuge rotor rotational speed is selected in such a way as to ensure quantitative (complete) deposition of the product. Make sure that the supernatant (supernatant) contains neither free virus particles, nor polymer. After this, two control experiments are carried out: in the first, an aqueous suspension of viral particles is centrifuged, in the second, an aqueous polymer solution. The concentration of viral particles and polymer in control experiments is taken equal to the concentrations of these components during product formation. Centrifugation of the suspension of viral particles and the polymer solution is carried out at the same speed at which complete precipitation of the product is observed. At the same time, it is ascertained that under the indicated conditions, neither individual virus particles nor a single polymer are precipitated from a suspension (solution). The totality of the results clearly indicates that the mixing of the solution of viral particles and the polymer in the above ratio is accompanied by the interaction of the components - virus particles and polymer - with each other and the formation of a new product, which quantitatively includes both components of the initial mixture.

The conjugate (complex) of the rod-shaped particle and the polypeptide is obtained by mixing 1 ml of a suspension of modified rod-shaped viral particles in physiological solution containing 1 mg of particles (1 wt. Part) and 0.25 ml of an aqueous solution of the above polypeptide containing 1 mg of the polypeptide (1 wt. part). The mixture is incubated for 1 hour at 4 ° C. The formation of a complex, and not their simple mechanical mixture of the above components, is proved by ultracentrifugation in the same way as was done above for the product of the interaction of viral particles and a water-soluble polymer containing cationic groups. At the same time, they are convinced that the supernatant (supernatant) does not contain an unbound polypeptide, which is originally taken in a large molar (but not mass) excess with respect to the rod-shaped particle. After that make sure that the sediment has a high biological activity, which is absent in the modified viral particles. The complex is isolated by centrifuging the resulting mixture for 30 min at 60,000 g and 4 ° C. The precipitated complex is resuspended in physiological saline. The method of electrophoresis in denaturing polyacrylamide gel determines the composition of the complex: rod-shaped particle - 1 wt. part, polypeptide - 1 wt. part.

The resulting complex does not require additional purification.

The biological activity of the resulting complex is evaluated by its protective activity. Laboratory animals (mice) are immunized by subcutaneous injection, injecting 50 μg of the drug in the presence of Freund's adjuvant into the mouse three times with an interval of 7 days. Two weeks after the third immunization, the animals are infected with the influenza virus using an A / PR / 8/34 (H1N1) strain of influenza virus adapted to mice. Before infection, animals are titrated in mice to determine the lethal dose (LD) of the virus. To infect mice, lethal doses of LD70 and LD90 are used, at which 70 and 90% of non-immunized mice die, respectively.

The biological activity of the complex, determined by this method, is shown in Table 1. The obtained result is compared with the biological activity of a known biologically active drug selected as a prototype.

Table 1 Groups of mice Injectable drug The number of surviving mice
on the 14th day Surviving animals,% Infection dose Infection dose LD70 LD90 LD70 LD90 Experienced The inventive complex 20/14 * 20/8 70 40 Control Prototype 20/8 20/5 40 25 Note: * in the numerator, the total number of mice in the experiment, in the denominator the number of surviving mice.

Example 2

The experiment is carried out analogously to example 1, however, as a modified rod-shaped viral particles using the interaction product of 1 wt. parts of the streaky mosaic virus of barley (GSHM) and 0.01 wt. parts of a water-soluble polymer with cationic groups of polylysine, a protein fragment of the neuraminidase influenza virus protein is used as a water-soluble polypeptide in the ratio of components in the complex: modified rod-shaped virus particle 1 wt. part, polypeptide 0.1 wt. part.

The resulting complex does not require additional purification.

The biological activity of the complex is shown in Table 2.

table 2 Groups of mice Injectable drug The number of surviving mice
on the 14th day Surviving animals,% Infection dose Infection dose LD70 LD90 LD70 LD90 Experienced The inventive complex 20/15 * 20/9 75 45 Control Modified rod-shaped particle VSHMYA 20/5 20/1 25 5 Note: * in the numerator, the total number of mice in the experiment, in the denominator the number of surviving mice.

Example 3

The experiment is carried out analogously to example 1, however, as a modified rod-shaped viral particles using the interaction product of 0.05 wt. parts of a water-soluble polymer with cationic groups PDMDAAH, 0.05 wt. parts of a water-soluble polymer with cationic groups of chitosan and 1 wt. parts of TMV, a hemagglutenin fragment is used as a water-soluble polypeptide with a ratio of components in the complex: a modified rod-shaped virus particle of 1 wt. part, polypeptide 25 wt. parts.

The resulting complex does not require additional purification.

The biological activity of the complex is shown in Table 3.

Table 3 Groups of mice Injectable drug The number of surviving mice
on the 14th day Surviving animals,% Infection dose Infection dose LD70 LD90 LD70 LD90 Experienced The inventive complex 20/13 * 20/7 65 35 Control The modified rod-shaped particle TMV 20/4 20/2 twenty 10 Note: * in the numerator, the total number of mice in the experiment, in the denominator the number of surviving mice.

Example 4

The experiment is carried out similarly to Example 1, however, as a cationic polymer, instead of HDPE, a mixture of 0.008 mg of polylysine and 0.004 mg of chitosan is used. The biological activity of the obtained complex, up to an experimental error, coincides with the biological activity of the proposed complex described in Example 1.

Example 5

The experiment is carried out similarly to Example 2, however, as a cationic polymer, instead of polylysine, a mixture of 0.003 wt. parts of HDPE, 0.003 wt. parts of chitosan and 0.004 wt. parts of PDMDAAH. The biological activity of the obtained complex, up to an experimental error, coincides with the biological activity of the proposed complex described in Example 2.

Thus, from the above examples it is seen that the proposed complex has protective activity against the influenza virus and its biological activity is 1.6-1.7 times higher than the biological activity of the known complex, selected as a prototype. The proposed complex is new, which expands the range of biologically active complexes.

Claims (1)

A biologically active complex having protective activity against the influenza virus, which is a conjugate consisting of a modified rod-shaped virus particle connected to a water-soluble polypeptide, characterized in that the modified rod-shaped virus particle is obtained by the interaction of 1 part by weight rod-shaped viral particles and 0.01-0.1 wt.h. at least one water-soluble polymer with cationic groups in the following ratio of the starting components in the conjugate, parts by weight:
modified rod-shaped viral particle one polypeptide 0.1-25