SU1730144A1 - Method of viruses reproduction suppression - Google Patents

Abstract

Use: medical biochemistry. Summary of the Invention: In the method for suppressing the reproduction of viruses, antibodies with covalently fatty acid residues attached to them are used as antiviral agents. At the same time, the effectiveness of suppressing the reproduction of viruses is 1.5-2 times. The yield of antiviral agents is 90-100% of the original antibodies, the preparations are stable for a year. 3 tab. cl

Description

This invention relates to medical biochemistry and concerns a method for suppressing viral reproduction with antibodies specific to antigenic determinants of this virus. The method opens new perspectives in medicine, in particular in the field of antiviral therapy as well as in basic research: the study of the mechanisms of viral replication, the action of components of the immune system, etc.

A known method for suppressing viral activity with antibodies specific to antigenic determinants of a virus.

However, due to the impermeability of cell membranes for antibodies, the latter cannot interact with intracellular viral particles and, therefore, are not capable of influencing the reproduction of the virus, only activating extracellular viral particles.

The closest to the proposed and achieved effect is a method of suppressing viral reproduction with antibodies by exposing virus-infected cells to antibodies specific for the antigenic determinants of the virus incorporated into the liposomes, since liposomes can ensure the delivery of antibodies into the cell. ML cell culture is incubated with sphingomyelin, cholesterol, and stearylamine liposomes containing immunoglobulins G (lgG) with a high titer to Coxsackie A-21 virus and infect with this virus. After two days, the infectious activity of the resulting virus is determined. For cells incubated with liposomes, a 23-74% decrease in infectious activity is observed compared to cells infected with a virus but not incubated with liposomes.

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The disadvantages of this method are the low efficiency of the antiviral effect, as well as the complexity of the procedure for obtaining liposomes containing antibodies, and the low efficiency of incorporation of antibodies into liposomes, which leads to a significant loss of antibodies in the preparation of an antiviral drug. long-term storage of antiviral drug.

The purpose of the invention is to increase the antiviral effect and simplify the method

The goal is achieved in that according to the method specific antigenic determinants of a virus with covalently attached fatty acid residues are used as antiviral agents.

The effectiveness of the method is proved by examples of suppressing the reproduction of model viruses, namely, influenza viruses of various serotypes and respiratory syncytial virus. When exposed to virus-infected cells, antibodies specific to the antigenic determinants of this virus, with fatty acid residues covalently attached to them, provide a significant suppression (by 1.5-2 times, see Tables 1-3) of virus reproduction.

As antibodies, you can use immunoglobulins of various classes and their total fractions. As modifying antibodies reagents can use various lipids - derivatives of synthetic and natural fatty acids.

PRI me R 1. Production of antibodies modified by fatty acid residues.

To 10 nm of a 0.1 M solution of sodium salt of sulfo-succinic acid di-2-ethylhexyl ester in octane, 450 µl of 1 mM antibody solution in 0.1 M borate buffer, pH 9.5, are added. The system is vigorously stirred for 1-2 minutes until the appearance of optical transparency, and then 450 µl of a 5 mM solution of the acid chloride or N-oxysuccinimide fatty acid ester (stearic or palmitic or myristic, etc.) in octane is added to it. After 2 h, the protein is precipitated from the reaction system in the cold (0 ° C) with 30 ml of acetone. The precipitation is separated and washed 4-5 times with 30 ml of cold (4 ° C) acetone.

The acetone residue is removed on a rotary evaporator.

Modified antibodies are fractionated by hydrophobic chromatography on

phenyl-sepharose and determine the yield of the modified protein. The degree of modification of immunoglobulins is determined using radioactively labeled fatty acids for modification.

The affinity of antibodies is determined by ELISA and radioimmunoassay.

Modified antibodies are stored in a dry state at a low temperature (-10 - (- 1 ° C) ° C).

The yield of codified antibodies per protein is 95-100%. Modified antibodies contain 1 fatty acid residue per protein molecule. They retain 80-100% of specific activity (affinity) compared to the original (unmodified) antibodies. When stored in a dry state for a long time (more than a year), the activity of the modified antibodies is reduced by no more than 20%.

PRI me R 2, Reproduction of the influenza virus (strain A // Chile, serotype H1N1) in the permissive cells of MDSC.

A monolayer of permissive MDSK cells is infected with an influenza virus (strain A / chili, serotype H1N1) with a multiplicity of 1-10 plaque-forming units per cell. After 3.5 h after infection, antibodies (normal rabbit IgG; normal rabbit IgG modified with fatty acid residues; specific rabbit IgG against H1N1 serotype influenza virus; specific rabbit H1N1 influenza virus modified with fatty acid residues) were added to the cells, equal to their titer in the hemagglutination inhibition reaction. 8.5 hours after infection, the cells are washed 2 times with a 2-fold volume of medium, incubated for 2 hours with a 2-fold volume of medium, washed with a 5-fold volume of C | Leda, and fresh medium containing no antibodies is added to them. | After 24 h after infection, the culture medium is removed, the cells are precipitated and the cells are detrised by 2-fold by centrifugation at 8000 rpm for 20 minutes. In the supernatant, infectious activity in embryonic infectious diseases (EIDs / ml) and hemagglutinating activity in the hemagglutination reaction with 0.7:% chicken erythrocytes (GAU / ml) are determined,

The results are shown in table 1.

Example 3: Reproduction of influenza virus (strain A / Texas, serotype H3N2) in permissive MDSC cells.

Same as in example 2, but using influenza viruses (strain A / Texas, serotype

H3N2) and antibodies specific to the H3N2 serotype influenza virus.

The results are shown in table 2.

PRI me R 4. Reproduction of PC virus (Long strain) in Hela permissive cells.

A monolayer of permissive Hela cells infect with a respiratory syncytial virus (PC virus) (Long strain) with a multiplicity of 1-10 cytopathic units (CPUs) per cell. 6 h after infection, antibodies (normal rabbit IgG; normal rabbit IgG modified with fatty acid residues; specific rabbit IgG against PC virus; specific rabbit IgG against PC virus modified with residues of fatty acid) are added to the cells at a concentration equal to the titer in the reaction of complement fixation with the purified virus. Thirteen hours after infection, the cells are washed (as described in Example 2) and fresh medium containing no antibodies is added to them.

After 28 h after infection, the culture medium is removed, the cells and cell derbies are precipitated by 2-fold centrifugation at 2000 rpm for 25 minutes. In the supernatant, infectious activity is determined by the cytopathic effect on the culture of Hela cells (CpdbO / ml).

The results are shown in table 3.

As shown in the examples (Tables 1-3), the reproduction of the virus is suppressed by exposing virus-infected cells to antibodies specific for antigenic

the determinants of this virus are 1.5–2 orders of magnitude, while in the known method the suppression does not exceed 1 order. Comparison of the data given in the examples and the known method suggests that the suppression of the reproduction of the virus requires in this method at least 2 orders of lower concentrations of antibodies.

Claims (1)

When antiviral agents are obtained by modifying antibodies specific for antigenic determinants of the virus with fatty acid residues, the yield of modified antibodies is 95-100%, while in a known method no more than 10% of the antibodies taken are included in the liposomes. In addition, antibodies with fatty acid residues covalently attached to them can be stored in a dry state for a long time (more than 1 year) without significant loss of specific activity, while antiviral agents used in a known method (antibodies included in liposomes ) cannot be stored for more than a few days. DETAILED DESCRIPTION OF THE INVENTION Method of suppressing viral reproduction by exposing virus-infected cells to immobilized antibodies specific for antigenic determinants of the virus, characterized in that, in order to increase the antiviral effect and simplify the method, antibodies with 1-2 fatty acid residues are covalently attached to them. Table 1 Reproduction of influenza virus (strain A / Chile, serotype H1N1) in permissive MDSC cells (example 2) Experimental conditions Infectious activity after 24 hours. Ig (EIDaO / ml) Infected cells do not incubate with antibodies. Infected cells. incubated with: normal rabbit IgG normal rabbit IgG modified with residues stearic acid normal rabbit IgG, modified residues palmitic acid rabbit IgG specific against eerotin flu virus H1N1 Hemagglutinating activity after 24 h (GAU / ml) 5.3 8 8 8 8 rabbit IgGs against the H1N1 serotin influenza virus, modified with residues of stearin acids specific rabbit IgGs against serotin H1N1 influenza virus modified by palmitic acid residues table 2 Reproduction of influenza virus (strain A / Texas, serotype H3N2) in permissive cells MDSC (example 3). Experimental conditions Infectious activity after 24 hours. Ig (EIDzo / ml) Infected cells do not incubate with antibodies. Infected cells. incubated with: normal rabbit IgG normal rabbit modified IgG residues stearic acid normal rabbit IgG, modified residues myristic acid rabbit IgG specific against serotina flu virus H3N2 specific rabbit IgG against the H3N2 serotin influenza virus modified by stearic acid residues specific rabbit IgG against the H3N2 serotin influenza virus modified by myristic acid residues Table 3 Reproduction of PC virus (Long strain) in permissive Hela cells (Example 4) Experimental conditions Infected cells do not incubate. with antibodies Infected cells are incubated with: normal rabbit IgG normal rabbit IgG modified with residues of stearic acid specific rabbit IgG against PC-virus Continued table. one 3.5 3.5 Hemagglutination activity after 24 h (GAU / ml) 512 512 512 512 512 128,128 Infectious activity after 28 h, Ig (TsDPVO / ml) 5.4 5.5 5.5 5.3 Continued table. 3