RU2323742C1 - Method to increase immunogenicity of inactivated flue vaccine - Google Patents

Abstract

FIELD: medicine, pharmacology.

SUBSTANCE: mixture of chitosonium glutamate obtained from high molecular chitosan with molecular mass 100-500 kD, and restrictedly depolymerised low polymeric chitosan with molecular mass 0.5-20 kD, is added to the inactivated flue vaccine as an adjuvant; the molecules of chitosan and chitosonium glutamate have free aldehydic groups at one of their free end, and have the deacetylation degree within 60-90%; the concentration of high molecular chitosan is brought to 0.5%, and low polymeric chitosan - to 0.05%, and the parenteral vaccination is performed.

EFFECT: method increases the immunogenicity of inactivated flue accine.

4 dwg, 4 ex

Description

The invention relates to the medical industry and relates to the production of viral vaccines, in particular inactivated influenza vaccine.

Existing inactivated influenza vaccines have relatively low immunogenicity and prophylactic efficacy for the elderly and children under 5 years of age and are not able to protect against drift variants of the influenza virus that change antigenicity, which appear in almost every new epidemic season. With a single parenteral administration of existing inactivated influenza vaccines to unprimed individuals (i.e., people who have not had the flu and are not vaccinated), no more than 5-10% of those vaccinated are protected from the flu, which complicates the fight against the flu, especially in the case of a pandemic (Gendon Yu.Z. ., 2004; 2006).

A known method (US 6534065) for producing an intranasal mucosal vaccine when using chitosan deacetylated by 80-90% as a mucoso-adhesive adjuvant) with a molecular weight of from 10,000 to 100,000 Da and with a chitosan concentration in the vaccine of up to 5%. Chitosan salts with the following carboxylic acids were used: lactic, malic, maleic, succinic, fumaric, glutamic and aspartic. However, the intranasal use of an inactivated influenza vaccine with a mucoso-adhesive adjuvant led to serious complications in the form of paralysis of some facial muscles, and therefore the use of such a vaccine was discontinued (Mutsch et al., 2004).

The technical result of the claimed invention is the creation of an inactivated influenza vaccine, administered parenterally, with increased immunogenicity. To achieve the technical result indicated, a method for preparing parenteral inactivated influenza vaccines (whole, split, subunit, etc.) is characterized in that, when preparing an inactivated vaccine, high-polymer chitosan and limited-depolymerized low-polymer chitosan are simultaneously added to it, the molecules of which are carried on one free aldehyde groups from their ends, while the added chitosan preparations have a degree of deacetylation in at 60–90% and a molecular weight in the range of 100–500 kBa (high polymer chitosan) and 0.5–20 kVA (low polymer chitosan).

It is known that preparations of high-polymer chitosan are soluble only at pH below 6.5 (pK protonation of free amino groups of chitosan). In this case, they are polycations that easily form ionic complexes with biopolymers of various origins. In the subcutaneous and muscle tissues of humans and animals, a pH of at least 7.0 is normally maintained, so at the injection site, the complex of chitosan components of the vaccine drops out in the form of a gel. Most low molecular weight fragments of chitosan obtained by limited depolymerization using nitrous acid, at one end, carry an anhydromanose residue having a free aldehyde group. At pH above 7.0, aliphatic aldehyde groups form weak covalent azomethine bonds with primary amino groups, i.e. the so-called Schiff bases are obtained. Therefore, the low polymer chitosan is also immobilized at the injection site.

Due to the slow depolymerization of chitosan molecules under the action of lysozyme and some other hydrolytic enzymes present at the injection site, the complex slowly dissolves and chitosan fragments and viral antigens are gradually released from the gel. This entails a slow and gradual flow of vaccine components into the bloodstream, which leads to an increase in the immune response.

Taking into account the information published in the scientific literature that chitosan with a molecular mass in the range of 0.6-15 kDa stimulates the cellular immunity system, we carried out limited depolymerization of chitosan with nitrous acid and subsequent fractionation so as to obtain a fraction of molecules with a mass of 2-20 kDa ( low molecular weight chitosan - NPH). This fraction also has a bacteriostatic and / or bactericidal effect, which should contribute to the preservation of the vaccine during storage.

Examples of the method

Example 1. Obtaining chitosan adjuvant

High-polymer chitosan (VPH) preparation (Sonat company, Russian Federation, Moscow), having an average molecular weight of 300 kDa, a deacylation degree of 85% and containing only trace amounts of heavy metals (Pb, Hg, Cu, Cd) -4ppm, ash - less than 0, 01%, protein less than 0.02%, was subjected to limited depolymerization using the methodology described in US patent No. 5312908. The process was carried out for 4 hours at a temperature of 5 ° C. The reaction was stopped by adding an aqueous solution of NaOH to a final pH of 9.0, which led to the precipitation of fragments of chitosan in the sediment. To completely remove the low molecular weight reaction products, three cycles of drug dissolution were carried out by acidification to pH 4, followed by alkaline precipitation. Then the NPH solution in a minimum amount of 0.05 m acetic acid was dialyzed 2 times against 0.05 m AcOH and 1 time against deionized water using an Sigma ultrafiltration membrane (USA), which allowed molecules to pass only less than 0.5 kDa. Subsequently, the preparation was subjected to exhaustive dialysis using a cellulose dialysis membrane retaining molecules with a mass of more than 20 kDa. The solution containing the molecules passing through the membrane was freeze-dried to obtain a dry NPH preparation (Fig. 1).

To prepare the VPH / NPH adjuvant concentrate, a weighed portion of the dry VPH preparation was mixed with a weighed portion of crystalline glutamic acid in a ratio of 0.99 mol of acid per 1 mol of chitosan amino groups. Then, with constant stirring, deionized water was added in the amount necessary to obtain a 2% solution of chitosonium glutamate. Upon reaching maximum dissolution, the solution was filtered off from trace amounts of undissolved chitosan and freeze-dried. After dissolution of the necessary sample of chitosonium glutamate in deionized water (pH of the resulting solution = 6.1-6.4), the solution was sterilized by autoclaving. To the resulting solution (after cooling), a 0.2 μm NPH solution in deionized water and a sterile solution of 3 mol / liter sodium chloride, pre-sterilized by filtration through a pore membrane, were added so as to obtain 1% VPH containing 0.1% NPH and 0 15 mol / liter NaCl (VPH / NPH concentrate). Thus, the VPH / NPH preparation is a mixture of two chitosonium salts: 90% glutamate and 10% acetate.

Example 2

An integrated VPH / NPH concentrate diluted in a sterile sodium chloride solution was added to split or subunit inactivated influenza vaccines to obtain a final concentration of chitosonium salt with glutamic acid - VPH = 0.5%, NPH = 0.05%, sodium chloride = 0, 9% and 3 μg of the vaccine in total = 0.2 ml. Mice were vaccinated twice by intramuscular injection of 3 μg of the vaccine with or without chitosan. There was an increase in the formation of antibodies that delay hemagglutination, for the influenza virus serotype A / H1 by 4-16 times, serotype A / H3 - 4-8 times and serotype B - 4-8 times, and neutralizing antibodies to serotype A / H1 - 8-32 times, serotype A / H3 - 8-16 times and serotype B - 8-32 times. This effect is manifested both in immunization with split and subunit inactivated vaccines (figure 2).

Example 3

Chitosonium glutamic acid (HPV) + NPH salt preparations (as described in Example 1) were added to inactivated influenza vaccines and mice were vaccinated twice, injecting 3 μg of the vaccine intramuscularly. In control experiments, mice were vaccinated without chitosan. The presence of antibodies in sera was investigated in the hemagglutination inhibition reaction and in the neutralization reaction with influenza virus strains contained in the vaccine, as well as with drift antigenic variants of serotypes A and B. In the sera of mice vaccinated without chitosan, the titer of antibodies to drift variants of the virus was 2-4 times lower than for vaccine strains. When vaccinated with drugs containing HPV / NPH, there was not only a significant increase in antibody titers to strains of viruses contained in vaccines, but also a significant formation of antibodies that inhibit hemagglutination, as well as neutralizing antibodies to drift variants of the influenza virus of serotypes A and B that exceeded the titers antibodies that inhibit hemagglutination and neutralizing antibodies for strains A / H3 2-8 times, and for strains of influenza B virus 2-4 times in comparison with the corresponding titers of antibodies to drift variants, iruemyh vaccines without chitosan and were equal to or higher than titers induced by vaccines without chitosan to viruses contained in the vaccines (Figure 3).

Example 4

Chitosonium salts with glutamic acid (HPV) up to 0.5% and NPH up to 0.05% were added to the inactivated influenza vaccine (split) and twice vaccinated intramuscularly with different doses of the vaccine (3.0 μg, 1.5 μg and 0.75 μg ) with and without chitosan. Chitosan is able to increase antibody titers by 4 times, even with a significant reduction in the dose of antigen. Compared to vaccination without chitosan, antibody titers when vaccinated with chitosan at a dose of 3 μg vaccine increase by 4-5 times, with 1.5 μg 3-5 times and at 0.75 μg 3-5 times, depending on the serotype of the virus (figure 4).

Drawing captions

Figure 1. The depolymerization scheme of chitosan with sodium nitrite at a pH of 3.0-4.0:

A - a preparation of high-polymer chitosan dissolved in diluted acetic acid (chitosonium acetate);

GlcN — glucosamine residue;

GlcNAc - acetylglucosamine residue;

B - the product of the depolymerization of chitosonium acetate with nitrous acid.

Figure 2. The effect of chitosan on the formation of antibodies to the three serotypes of the influenza virus. Mice were immunized twice with subunit and cleaved with inactivated trivalent vaccines with or without chitosan. Analysis of antibody titers was performed with virus strains corresponding to those contained in the vaccines:

A - titers of antibodies that inhibit hemagglutination; B - neutralizing antibody titers;

H1, H3, B - serotypes of influenza virus; X - titers with the separate introduction of the vaccine and chitosan;

a - subunit vaccine MG; b - Vaxigrip vaccine; c - Influvac subunit vaccine.

- вакцина без хитозана,

- вакцина с хитозаном.

- vaccine without chitosan,

- vaccine with chitosan.

The ordinate axis is the dilution of serums.

All observed differences between antibody titers induced by vaccines with and without chitosan were statistically significant (P <0.01).

Figure 3. The formation of antibodies to drift variants of the influenza virus during vaccination with chitosan vaccines.

Mice were immunized twice with Vaxigrip vaccines for the 2004/05 epidemic season and for the 2005/06 epidemic season with or without chitosan. Analysis of antibody titers was performed with strains of viruses contained in vaccines, and with drift variants of influenza viruses A / H3N2 and B;

A - titers of antibodies that inhibit hemagglutination, B - titers of neutralizing antibodies;

a - A / H3N2 strain corresponding to the vaccine strain for the 2004/05 epidemic season, b - A / H3N2 drift variant for the 2004/05 epidemic vaccine, recommended for 2005/06 epidemic season, c - A / H3N2 drift variant for the 2004/05 epidemic vaccine recommended for epidemiological season 2003/2004, d - strain B, corresponding to the vaccine strain for epidemic season 2004/05, e - drift variant of virus B for the vaccine epidemic season 2004/05, recommended for epidemic season 2003/04, f - strain A / H3N2, corresponding vaccine strain for the 2005/06 epidemic season, g - A / H3N2 drift variant for the 2005/06 epidemic vaccine recommended for epidemic Zones 2004/05, h - drift variant A / H3N2 for the epidemiological vaccine 2005/06, recommended for the epidemic season 2003/04, i - strain B corresponding to the vaccine strain for the epidemic season 2005/06, j - drift variant of virus B for the vaccine epidemic season 2005 / 06, recommended for the epidemic season 2003/04;

- вакцина без хитозана;

- вакцина с хитозаном.

- vaccine without chitosan;

- vaccine with chitosan.

The ordinate axis is the dilution of serums.

All observed differences between antibody titers to drift variants of viruses during vaccination with chitosan and without it were statistically significant (P <0.05-0.01).

Figure 4. The effect of chitosan on the formation of antibodies that inhibit hemagglutination with a decrease in the dose of antigen.

Mice were immunized with a double-split Vaxigrip vaccine, injecting intramuscularly with 3.0, 1.5 and 0.75 μg of antigen with or without chitosan:

I - vaccine without chitosan, II - vaccine with chitosan;

 a - 3.0 μg of antigen, b - 1.5 μg of antigen, c - 0.75 μg of antigen.

Literature

1.Gendon Yu.Z. Advantages and disadvantages of inactivated and live flu vaccines. Q. virusol. 2004, 49, No. 4, pp. 4-12.

2.Gendon Yu.Z. Problems of influenza prevention in young children. Q. virusol. 2006, 51, No. 2, pp. 4-10.

3.Mutsch M., Zhou W., Rhodes P. at al. Use of the inactivated intranasal influenza vaccine and the risk of Bell's - Palsy in Switzerland. N. Engl. G.Med. 2004, 350, p. 896-903.

4.US 6534065 - Influenza vaccine composition with chitosan adjuvant.

Claims (1)

A method of increasing the immunogenicity of an inactivated influenza vaccine, comprising adding chitosonium high polymer glutamate to the vaccine as an adjuvant, characterized in that a mixture of chitosonium glutamate obtained from high molecular weight chitosan with a molecular weight of 100-500 kDa and a limited depolymerized low copolymer low molecular weight is used as adjuvant 5-20 kDa; while the molecules of chitosan and chitosonium glutamate carry free aldehyde groups at one of their ends and have a degree of deacetylation in the range of 60-90%; the concentration of high molecular weight chitosan in the vaccine is adjusted to 0.5% and low polymer chitosan to 0.05%, and vaccination is carried out by the parenteral method.

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