RU2740751C1 - Method of producing tetravalent subunit influenza vaccine - Google Patents

Abstract

FIELD: medical biotechnology.

SUBSTANCE: invention relates to a method for producing influenza vaccine. Method for producing a tetravalent subunit influenza vaccine involves culturing influenza viruses of subtypes A (H₁N₁), A (H₃N₂), B/Yamagata lineage, B/Victoria lineage in developing chicken embryos with subsequent separation of virus-containing allantoic liquid, concentration, purification, inactivation and splitting of virus, its subsequent centrifugation and ultrafiltration, bringing monovaccine into polyvaccine with the adjuvant, wherein the virus is cultured separately, and after ultrafiltration in each of the monopreparations is introduced as an adjuvant betulin in ratio of antigen: carrier 1:1.25–12.5.

EFFECT: use of the given invention enables to obtain a stable and effective tetravalent subunit anti-influenza vaccine when included as an adjuvant of betulin in a weight ratio of the antigen: carrier of 1: 1.25–12.5.

1 cl, 4 tbl, 8 ex

Description

The invention relates to medical biotechnology, in particular to a method for producing an influenza vaccine.

Influenza remains an urgent problem of modern healthcare. According to the World Health Organization (WHO), up to 10% of adults and 30% of children fall ill with influenza every year. Influenza infection, especially in risk groups, can be severe, require hospitalization, and be fatal. At the same time, influenza viruses retain a high pandemic potential, in particular, influenza viruses of birds, as well as swine and swine origin.

Currently, the main approach in the fight against influenza is vaccine prophylaxis using inactivated anti-influenza vaccines administered intramuscularly. [Results and prospects for the development of vaccine prevention in the XXI century., IV. Feldblum, http://www.rusmedserv.com/epidinf/m-dokl/].

At the same time, the usually used trivalent inactivated influenza vaccines are not always effective for a number of population groups: young children, pregnant women, the elderly, people with various chronic diseases who are classified as influenza risk groups, as well as against antigenic different strains (drift and heterologous) not contained in the vaccine.

It is known to use adjuvants of various origins to increase the effectiveness of vaccines: mineral (aluminum hydroxide or phosphate, etc.); vegetable (saponins - QuilA, QS21); microbial (killed bacteria, lipopolysaccharide and its derivatives, DNA CpG motifs), etc. When using adjuvants, it becomes possible to increase the immunogenicity of influenza vaccines against a set of antigenically different strains, as well as for immunization of various population groups, including risk groups. In addition, with a significant increase in immunogenicity due to the addition of an adjuvant to the vaccine, it becomes possible to switch to simple (single) immunization regimens, as well as to reduce the dose of antigen (hemagglutinin), which improves the economics of their production.

However, due to the toxicity or insufficient effectiveness of most adjuvants, only aluminum salts and MF-59 water-oil emulsion are allowed for wide clinical use, and in some countries virus-like particles (VLP - virus-likeparticles) and an immuno-stimulating complex (ISCOM).

Currently, there are two adjuvanted influenza vaccines on the Russian market - the inactivated subunit influenza vaccine Sovigripp and the Grippol® tetravalent polymer-subunit vaccine.

The vaccine "Sovigripp" (RU 2446824, 2012) contains a copolymer of 2-methyl-5-vinylpyridine and N-vinyl-pyrrolidone as an adjuvant and a carrier. The method for its production involves the cultivation of the influenza virus in developing chicken embryos, followed by the separation of the virus-containing allantoic fluid, concentration, purification, inactivation and splitting of the virus. Concentration and purification is carried out by sorption of the virus on chicken erythrocytes, followed by ultracentrifugation in a sucrose density gradient. Inactivation and cleavage of the virus is carried out by any means permitted for use. The split virus concentrate is centrifuged and ultrafiltered. The obtained concentrate of purified antigens of each strain of influenza virus is considered as a monovaccine. After mixing monovaccines, an adjuvant is added to the polyvaccine - a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone. The obtained polymeric trivalent vaccine contains strains of influenza virus type A, B with a mass ratio of antigen-adjuvant from 1: 5 to 1:30.

The disadvantage of this vaccine is the limited use of trivalent inactivated influenza vaccines for a number of population groups: young children, pregnant women, the elderly, people with various chronic diseases who are classified as influenza risk groups, as well as with respect to antigenically different strains (drift and heterologous ) not contained in the vaccine.

To increase the effectiveness of inactivated vaccines against the composition of two lines of influenza B (Yamagata and Victorian), as well as add immunoadjuvants.

The closest in technical essence to the claimed invention is a technology for producing a tetravalent vaccine "Grippol®" (https://www.rlsnet.ru/tn_index_id_96550.htm), which contains antigens of the influenza virus subtypes A (H1N1), A (H3N2), B / Yamagata lineage, B / Victoria lineage, as an adjuvant and carrier - a derivative of poly-1,4-ethylenepiperazine - Polyoxidonium, and as auxiliary substances - phosphate-buffered saline, thiomersal, mannitol, povidone, Triton X-100. The method of obtaining vaccines "Grippol®" provides for the cultivation of the influenza virus in developing chicken embryos, obtaining a viral concentrate, purifying the viral concentrate by ultracentrifugation in a sucrose density gradient, inactivating the viral material, cleaving the viral concentrate with a detergent, isolating influenza virus antigens, converting monovalent the subsequent addition of the adjuvant poly-1,4-ethylenepiperazine "Polyoxidonium".

The disadvantage of this vaccine is insufficient effectiveness due to the fact that the used adjuvant does not sufficiently increase immunogenicity when added to influenza vaccines, in particular, in terms of effectiveness, it corresponds to trivalent reference vaccines for three matching strains, although it surpasses them in relation to the fourth strain - influenza B virus, not included in trivalent analogs (https://www.rlsnet.ru/tn_index_id_96550.htm).

At the same time, new adjuvants have now been developed that can improve the effectiveness of vaccines. One of the most promising adjuvants are nanoparticles (NPs), the advantage of which is that they are efficiently absorbed by antigen-presenting cells. As a result, if an antigen is bound to NPs, then it will be targetedly absorbed by macrophages, which will lead to an increase in the immune response. Among the nanoparticles, the most interesting are adjuvants based on birch bark triterpenoids, in particular, nanoparticles based on a natural pentacyclic triterpene substance, betulin. Its use (RU 2322091, 2008), in contrast to the known adjuvants, provides, along with adjuvant properties, a wide range of biological activity, in particular, antibacterial, antifungal, antiviral, hepatoprotective, anti-inflammatory, anti-cancer, anti-allergic, membrane-stabilizing, immunomodulatory, antioxidant effects.

At present, it is proposed to use betulin in the form of spherical amorphous nanoparticles as a part of monovaccines against hepatitis B, diphtheria, Newcastle disease virus, avian reovirus, influenza A / California / 7/09 (H1N1) strain (RU 2355423, 2009; RU 2546861, 2015; RU 2545714, 2015). Its use for polyvaccines is not described in the reviewed literature.

The problem solved by the authors was to expand the range of vaccines used through the use of tetravalent vaccines that are more effective, in particular, possessing biocidal, antioxidant and other positive properties.

The technical result was achieved by those in the technology of obtaining a vaccine, including the cultivation of influenza viruses of subtypes A (H1N1), A (H3N2), B / Yamagata lineage, B / Victoria lineage in developing chicken embryos with subsequent separation of virus-containing allantois fluid, concentration , purification, inactivation and cleavage of the virus, its subsequent centrifugation and ultrafiltration, mixing of monovaccines into a polyvaccine with the addition of an adjuvant, in which the viruses are cultivated separately, after ultrafiltration, betulin is introduced as an adjuvant as an adjuvant in the antigen-to-carrier ratio of 1 in mass : 1.25 to 1: 12.5, and then the monopreparations are combined.

The introduction of betulin in a concentration of less than 1: 1.25 does not allow achieving the required level of the immune response, and its use in a concentration of more than 1: 12.5 does not improve the vaccine and leads to a decrease in the concentration of the active principle and is economically inexpedient.

The essence and advantages of the invention are illustrated by the following examples.

Example 1. To obtain a tetravalent subunit influenza vaccine containing a corpuscular adjuvant based on natural betulin, influenza virus strains of type A (H1N1), A (H3H2), B1 and B2 were used.

Chicken embryos of 10-12 days of age were infected with the influenza virus into the allantoic cavity. Infected embryos were placed in a thermal room at a temperature of 32-35 ° C and humidity (60 ± 10)%. For type A influenza strains, the incubation time was 48 hours, for type B virus strains - 72 hours. After incubation, the embryos were checked using ovoscopes. Viable embryos were placed in a refrigerator at a temperature of (4 ± 2) ° С for (18 ± 2) h for cooling. The collection of vaccinated allantoic fluid (VAF) was carried out by pouring into a bottle. An equal amount of phosphate buffer solution was added to bottles containing 5-6 liters of vaccinated allantoic fluid, mixed for 10 minutes on a shaker and microfiltration was carried out through a cascade of filter elements with pore sizes, respectively: 2.3; 1.6; 1.2; 0.8; 0.44 μm. A solution of beta-propiolactone was added to the contents of the bottle (filtrate) to a final concentration of 0.1%, mixed thoroughly and left in a refrigerator chamber on a shaker for 17-18 hours. Then, the concentration and partial purification of the inactivated VAF were carried out: ultrafiltration was carried out on a tangential flow ultrafiltration unit with 4 cassette modules with a pore size of 300 kD. The resulting VFA concentrate was purified by ultracentrifugation in a density gradient using two solutions of sucrose 50% and 20% at a rotor speed of 30,000 rpm. The contents of the rotor were dispensed into 20 ml fractions. Then titrated in RHA to determine the peak of the virus and collected in a single volume with the maximum hemagglutinin content. To purify the ultracentifugate concentrate, gel chromatography on a polymer carrier was used. The volume of the support for chromatography was determined at the rate of 5 volumes of the support to one volume of the sample. After the sucrose concentrate was applied to the column, it was fed with Tris buffer through a pump. The collection of the target eluate was carried out by optical density at a wavelength of 280 nm. To inactivate the virus, we used an installation containing a source of ultraviolet rays with an ultraviolet radiation flux of at least 90 lumens. To destroy the inactivated viral concentrate, octyl glucoside (Octil-D-Glucopyranoside) was used in a detergent to total protein mass ratio of 10: 1. Incubation was carried out for 1 hour in a refrigerator at a temperature of 5 ± 3 ° C. Then, the concentration of the digested viral preparation was carried out on an ultrafiltration unit with 2 cassette modules with a pore diameter of 10 kDa. The concentrated cleaved preparation was further purified from the detergent by gel filtration on a chromatographic carrier based on agarose with an exclusion limit of 150 kD. The eluate was collected in a sterile container. To separate lipoproteins, ultracentrifugation was used at an acceleration of 90,000 g for 2 hours at a temperature of 2 to 8 ° C. The centrifugate was transferred to sterilizing filtration through a set of filters with pore diameters of 0.45 and 0.22 μm. The resulting semi-finished product (monovalent substance) was monitored according to the following parameters: sterility, residual detergent content, specific activity according to ORID (hemagglutinin content), hemagglutinating activity, specific safety, authenticity, protein according to the Lowry method with precipitation. After the control, the previously prepared adjuvant-betulin in the mass ratio of hemagglutinin monovalent-adjuvant 1: 5 and phosphate buffered saline were added to the receiving bottle to pH 6.8. The bottle was thoroughly mixed and placed in a shaker in the refrigerator for 3 hours. Then the bottles with the monovalent substance and the adjuvant were stored in a refrigerator at a temperature of 4 ± 2 ° C. Semi-finished products of four different strains of influenza virus: type A (H1N1; H3N2) and type B1 and B2, which passed all controls, were combined in a sterile bottle in an equal ratio (1: 1: 1: 1). The resulting combined bulk was placed in a refrigerator at + 4C and stirred on a shaker for 2 hours. Then it was stored at the same temperature and, if necessary, was connected to a filling machine and, when using a dispensing device, was filled into vials, syringes or ampoules.

Example 2. The vaccine was obtained according to the technology of example 1 with the introduction of an adjuvant at a ratio in the mass ratio of hemagglutinin monovalent-adjuvant 1: 1.25.

Example 3. The vaccine was obtained according to the technology of example 1 with the introduction of an adjuvant at a ratio in the mass ratio of hemagglutinin monovalent-adjuvant 1: 12.5

Example 4. The vaccine was obtained according to the technology of example 1 with the introduction of an adjuvant at a ratio in the mass ratio of hemagglutinin adjuvant 1: 5, and the adjuvant was administered after mixing monovaccines.

Example 5. Study of the safety of a tetravalent subunit influenza vaccine containing a corpuscular adjuvant based on natural betulin in animal experiments.

The safety study was carried out in acute and chronic toxicity tests.

In the study of acute toxicity, the vaccine according to example 1, the adjuvant and placebo were administered to the animals intramuscularly once in a dose equivalent to the human course - 1 ml per animal weighing 20 g (mice) and 200 g (rats). Additionally, the mice were injected with the vaccine according to example 1 and placebo intravenously at a dose of 0.5 ml per animal weighing 20 g.

Determination of "chronic" toxicity was carried out on two species of animals: b / n rats and guinea pigs. Sexually mature b / n rats were injected with the vaccine at a dose of 8 μl for 10 days (total 80 μl), adjuvant at a dose of 3.2 μg for 10 days (total 32 μg) and placebo. Sexually mature guinea pigs were injected with the vaccine at a dose of 13 μl for 10 days (130 μl in total), an adjuvant at a dose of 5.2 μg for 10 days (52 μg in total), and placebo. The drugs were administered in a volume of 0.5 ml.

When conducting a study of "acute" and "chronic" toxicity after administration of drugs and a placebo solution, the animals were observed for 24 days. Subject to daily registration: the death of animals, their weight, behavior and external condition, as well as the presence of clinical symptoms of intoxication. On days 11, 17 and 24 of the study, the animals of the experimental and control groups were euthanized. Complete necropsy of bodies and macroexamination of organs, histopathological studies were performed.

To assess the hematological and biochemical parameters of the blood, blood was taken before the drug was administered, after 11, 17 and 24 days in rats from the tail vein, in guinea pigs - from the heart.

The results of the study showed that with a single and repeated administration, the vaccine did not cause death of animals, changes in behavior, decreased physical activity, decreased appetite, changes in hairline, and a decrease in body weight, which indicates the absence of a toxic effect of the drug on the systems and organs of laboratory animals.

The conducted macroscopic and histopathomorphological examination of the systems and organs of laboratory animals showed that the administration of the vaccine and the adjuvant does not cause pathological, including pronounced dystrophic and necrobiotic changes in the internal organs of animals, which indicates the absence of toxicity of the vaccine.

The results of the analysis of hematological and biochemical blood parameters of laboratory animals did not reveal significant differences before and after administration of the vaccine and adjuvant, the values of all blood parameters were within the physiological norm for these animal species, which confirms the absence of the toxic effect of the drugs.

Example 6. Study of the immunogenicity of a tetravalent subunit influenza vaccine.

Immunogenicity was determined by the content of specific antibodies in the blood serum of animals in relation to each antigen included in the vaccine in comparison with a similar complex of monovalents included in its composition, without an adjuvant (hereinafter, the vaccine without adjuvant). The study was carried out "in vivo" on Balb / c mice.

The test preparations were injected once and twice (according to the scheme 0-14) intraperitoneally, 0.5 ml with a syringe with a capacity of 1 ml. Similarly to the control groups of animals, an adjuvant solution and sodium chloride, 0.9% solution for infusion were administered. Blood sampling was carried out on the 14th day after a single immunization and on the 14th day after a double immunization. Antibodies were determined in sera using the ELISA method.

Data on the number of animals, vaccines and their dosage during immunization are presented in Table 1.

Serum obtained from the blood of mice (individually from each animal) was monitored for the presence of antibodies to each monovalent by enzyme immunoassay using an experimental test system (IPTS). When constructing the IPTS, commercial reagents were used: a / mouse IgG (H + L) Strong Zyme HRP Conjugate (SDT, Germany); chromogen - 3,3,5,5-tetramethylbenzidine (Hema, Russia); a 5% sulfuric acid solution was used as a stop reagent. The enzyme-linked immunosorbent assay was performed on polystyrene plates from Greiner bio-one, Germany. The results of enzyme-linked immunosorbent assay were recorded on a PR 2100 spectrophotometer manufactured by Sanofi Diagnostiks Pasteur (France) at two wavelengths 450/620 nm.

To calculate the immunogenic activity of the vaccines, the arithmetic mean of the optical density (OD) of the sera of mice from the group administered with sodium chloride solution 0.9% and an adjuvant (negative control group) was calculated. Sera were considered positive if their OD was equal to or higher than the arithmetic mean OD of sera from mice in negative control groups plus the coefficient of the test system (0.075).

Based on the results obtained, the ED50 values were calculated using the Kerber formula:

log ED50 = log DN - s (S Li - 0.5), where

DN is the maximum of the tested doses;

s is the logarithm of the ratio of each subsequent dose to the previous one (the logarithm of the dilution factor);

Li is the ratio of the number of seropositive animals to the total number of animals that received a given dose;

S Li is the sum of Li values found for all doses during control.

Next, the anti lg of the obtained value was calculated.

The number of positive responses to the administration of vaccines with an adjuvant and vaccines without an adjuvant are presented in Table 2.

Vaccine doses (HA concentration, μg / ml) inducing antibody production in 50% of mice (ED50) are presented in Table 3.

Thus, the immunization of animals with influenza vaccines contributes to an increase in the antibody titer, while it should be noted that the drug, which contains an adjuvant-betulin in a mass ratio of hemagglutinin monovalent-adjuvant 1: 5, has a more pronounced effect.

Example 7. Study of the antigenic activity of a tetravalent subunit influenza vaccine containing a corpuscular adjuvant based on natural betulin.

Antigenic activity was assessed by the level of the antibody response in the blood serum of mice after single and double immunization with vaccine samples in the hemagglutination inhibition reaction, which allows one to determine the immune response to each of the vaccine components separately.

Mice were immunized intraperitoneally with a 250 μl volume of vaccine or adjuvant.

On day 28 after the first immunization, blood samples were collected from the mice from the mandibular vein. Blood samples were centrifuged (6000 rpm for 10 min), the serum was transferred into plastic Eppendorf tubes and stored at -20 ° C until RTGA was placed. The sera of mice vaccinated with placebo were used as a negative control.

For the production of RTGA, the sera of mice were treated with the RDE reagent according to the manufacturer's instructions (RDE, Receptor destroying enzyme, Denka Seiken, Japan). To remove nonspecific agglutinins, sera were additionally treated with an equal volume of 10% chicken erythrocytes. After sedimentation of chicken erythrocytes to the bottom of the sera were prepared a series of two-fold dilutions in 96-well panels for serological reactions ("Medpolymer") in 25 μl of 0.9% NaCl solution. Then, 25 μl of antigen containing 4 HAU (hemagglutinating units) was added to the serum dilutions. The sera with the antigen were incubated for an hour at room temperature, after which 50 μl of 0.5% chicken erythrocytes dissolved in 0.9% NaCl were added to all wells. The maximum serum dilution, which completely inhibited hemagglutination of chicken erythrocytes, was taken as the serum titer. RTGA titer was the reciprocal of the serum dilution in the last well with no hemagglutination.

To present the data obtained, we used descriptive statistics such as geometric mean titers (GST) calculated using the Excel computer program (version 10). The statistical significance of the differences was assessed using the computer program Statistica (version 6.0) using the test of nonparametric Mann-Whitney tests used for small biological samples, the normal distribution of which has not been proven. Differences were considered significant at p <0.05.

The results are presented in Table 4. As a result of the study, it was shown that the most pronounced antigenic activity was in the H1N1pdm and H3N2 components of the tetravaccine. The components of the influenza B virus tetravaccines caused the formation of a less pronounced specific immunity in mice after double immunization. All animals of the group receiving the adjuvant did not have a specific immune response to the viral antigens included in the vaccine.

Example 8. The effect of long-term storage on immunogenic vaccine preparations were studied by evaluating antigenic activity and humoral immune response (immunogenicity).

When determining the antigenic activity of the vaccine variants according to examples 1-3, the formation of antibodies in the blood sera of animals was studied. The experiments were carried out on white outbred mice weighing 10-12 g. After a two-fold intraperitoneal injection of the vaccine with an interval of 7 days, the sera of immunized mice were studied in the hemagglutination inhibition reaction (RTGA) with homologous vaccine antigens. The study of the stability of antigenic activity during storage of the claimed preparation was carried out by the "accelerated aging" method. Samples of the declared vaccines and the closest analogue were laid for accelerated storage at 37 ° C and 22 ° C. Indicators of antigenic activity of the claimed vaccine are shown in table. 4.

The results of the study of the stability of antigenic activity showed that the developed drug has high stability, maintaining the indicators that meet the requirements of regulatory documents.

Claims (1)

A method of obtaining a tetravalent subunit influenza vaccine, including the cultivation of influenza viruses of subtypes A (H ₁ N ₁ ), A (H ₃ N ₂ ), B / Yamagata lineage, B / Victoria lineage in developing chicken embryos, followed by separation of virus-containing allantoic fluid, concentration, purification, inactivation and cleavage of the virus, its subsequent centrifugation and ultrafiltration, mixing monovaccines into a polyvaccine with the addition of an adjuvant, characterized in that the viruses are cultivated separately, and after ultrafiltration, betulin is injected as an adjuvant in a mass ratio of 1: antigen as an adjuvant 1.25-12.5.