RU2804948C2 - Pentavalent subunit vaccine against respiratory infections and method of its preparation - Google Patents

Abstract

FIELD: immunology; medical biotechnology; bioengineering.

SUBSTANCE: method for obtaining a vaccine against respiratory infections is described. Influenza viruses of subtypes A (H1N1), A (H3N2), B/Yamagata lineage, B/Victoria lineage are separately cultivated in developing chicken embryos, followed by separation of the virus-containing allantoic fluid, its concentration and purification. The virus is inactivated and split, followed by centrifugation, ultrafiltration and production of monovalents for each strain, as well as the cultivation of a cell line producing the recombinant protein of the SARS-CoV-2 virus - RBD-Fc with selection and clarification of the culture fluid containing RBD-Fc. Chromatographic purification is carried out to obtain monovalent. An adjuvant is added to each monovalent separately. Betulin is used as an adjuvant at an antigen:adjuvant ratio of 1:1.25 to 1:12.5. Then the monovaccines are combined into a combined vaccine.

EFFECT: invention expands the arsenal of means for the prevention of respiratory infections, in particular influenza and SARS-CoV-2.

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Description

The technical field to which the invention relates.

The present invention relates to immunology, medical biotechnology and bioengineering, in particular to a method for producing a vaccine against respiratory infections.

State of the art. Vaccine prevention is considered in modern conditions as one of the leading mass effective means of combating infections.

Introduction. Acute respiratory viral infections (ARVI) remain a pressing problem of modern healthcare. According to the World Health Organization (WHO), up to 40% of the adult population of the planet falls ill with ARVI every year. Of these, up to 10% of adults and 30% of children fall ill with the flu. ARVI infection, especially in risk groups, can be severe, require hospitalization, and lead to death. At the same time, for example, influenza viruses retain a high pandemic potential, in particular, avian influenza viruses, as well as swine and swine influenza viruses.

If we turn to the pandemic of coronaviruses, in particular SARS-CoV-2, which also cause acute respiratory infections with serious consequences, then by February 2022 about 400 million people had been ill with them, of which death was observed in 6 million cases. Currently, only effective vaccination can prevent ARVI pandemics, which is the main approach to eliminating coronavirus and influenza infections.

At the same time, the inactivated vaccines used, as a rule, are not always effective for a number of population groups: young children, pregnant women, the elderly, people with various chronic diseases, who are considered to be at risk for ARVI, as well as for influenza strains (drift and heterologous), not contained in the vaccine (having a different antigenic composition, primarily hemagglutinin). When using adjuvants, it becomes possible to increase the immunogenicity of influenza vaccines against a set of 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 reduce the dose of antigen, which improves the economics of their production. This is especially important for vaccines in a pandemic because the same production capacity will produce more vaccine doses and, as a result, more people will be immunized.

Currently, to increase the effectiveness of vaccines in experimental and clinical studies, adjuvants of various origins are used: mineral (aluminum hydroxide or phosphate, etc.); plant (saponins - QuilA, QS21); microbial (killed bacteria, lipopolysaccharide and its derivatives, CpG motifs of DNA), etc. Due to the toxicity or insufficient effectiveness of most adjuvants, only aluminum salts and water-oil emulsion MF-59 are allowed for widespread clinical use, and in some countries virus-like particles (VLP - virus-like particles) and immunostimulating complex (ISCOM - immunostimulating complex). Foreign commercial influenza vaccines are produced with only one adjuvant, MF-59, and such vaccines turned out to be more immunogenic when vaccinating older people, however, they had increased reactogenicity.

The development of new classes of adjuvants is a promising area of modern immunobiology. Over the past decades, significant changes have occurred in vaccine production technology. The vaccines currently undergoing clinical trials differ significantly from traditional vaccines. First of all, these are recombinant vaccines based on purified proteins. The creation of new vaccines is, in turn, the reason for the search for new adjuvants. The use of adjuvants makes it possible to reduce the dose of antigen in the vaccine, increase the immunogenicity of “weak” antigens, prevent competition of antigens in combined vaccines, increase the rate of development and duration of the immune response in vaccinated people, induce protective properties of mucous membranes, and also increase the strength of the immune response in children and the elderly age.

Influenza vaccines with adjuvants. Currently, there are two influenza vaccines with adjuvants on the Russian market - the inactivated subunit influenza vaccine Sovigripp® and the quadrivalent polymer single unit vaccine Grippol®.

The Sovigripp vaccine (RU 2446824) contains a copolymer of 2-methyl-5-vinylpyridine and N-vinyl-pyrrolidone as an adjuvant and carrier. The method for obtaining it involves cultivating the influenza virus in developing chicken embryos, followed by separation of the virus-containing allantoic fluid, concentration, purification, inactivation and cleavage of the virus. Concentration and purification are carried out by sorption of the virus on chicken erythrocytes, followed by ultracentrifugation in a sucrose density gradient. Inactivation and breakdown of the virus is carried out by any approved means. The digested virus concentrate is centrifuged and subjected to ultrafiltration. The resulting concentrate of purified antigens of each influenza virus strain is considered a monovaccine. After mixing the monovaccines, an adjuvant is added to the polyvaccine - a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone. The resulting polymeric trivalent vaccine contains strains of influenza virus type A, B with an antigen-adjuvant mass ratio of 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 considered at risk for influenza, as well as against antigenically different strains (drift and heterologous ), not contained in the vaccine. To increase the effectiveness of inactivated influenza vaccines, strains of two lines of influenza B (Yamagata and Victorian) are used instead of one, and immunoadjuvants are also added.

The closest in technical essence to the claimed invention is the technology for producing the tetravalent Grippol® vaccine, which contains antigens of the influenza virus subtypes A (H1N1), A (H3N2), B/Yamagata lineage, B/Victoria lineage, as an adjuvant and carrier - a poly derivative -1,4-ethylenepiperazine - Polyoxidonium, and as excipients phosphate-buffered saline solution, thiomersal, manitol, povidone, Triton X-100. The method for producing Grippol® vaccines involves cultivating 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, splitting the viral concentrate with detergent, isolating influenza virus antigens, combining monovalents into a polyvalent vaccine with subsequent addition adjuvant poly-1,4-ethylenepiperazine "Polyoxidonium". This vaccine is not effective enough, which is due to the polymer adjuvant used, which is not capable of forming globular structures (corpuscles), thereby strengthening the cellular component of the immune response.

Promising developments of adjuvants. Currently, in world and domestic practice, adjuvants are considered in the form of various forms: liposomes, virosomes, immunostimulating complexes, nanoemulsions (MF59, SAF, Montanide), polymer nanospheres, virus-like particles, etc. The most promising adjuvants, according to researchers, are nanoparticles (LF). One of the reasons for using NPs as adjuvants is the known fact that they are efficiently taken up by antigen-presenting cells. Thus, if an antigen is bound to NPs, it will be targetedly absorbed by macrophages, which will lead to an enhanced immune response.

Among nanoparticles, the most interesting are adjuvants based on birch bark triterpenoids, in particular, nanoparticles based on the natural pentacyclic triterpene substance - betulin (betulenol, betulinol, lupendiol). Its use (RU 232209), unlike known adjuvants, provides, along with adjuvant properties, a wide range of biological activity, in particular antibacterial, antifungal, antiviral, hepatoprotective, anti-inflammatory, anticancer, antiallergic, membrane-stabilizing, immunomodulatory, antioxidant effects. When creating such adjuvants, there are difficulties associated with the extremely low solubility of triterpenoids in water. Thus, the solubility of betulin has been determined to be less than 1 μg/ml. Therefore, the creation of a water-soluble form is very important, as it should lead to an increase in the concentration of particles and an increase in the effectiveness of the adjuvant. The search for such forms led to the creation of spherical amorphous nanoparticles based on birch bark.

Patent - betulin. The problem solved by the invention RU 2545714 C is to improve the quality of betulin adjuvant, making it possible to obtain it in accordance with the requirements for drugs administered parenterally: sterility, non-pyrogenicity, atoxicity, as well as ease of scaling and storage stability.

The technical result is achieved by the fact that in the method for producing an adjuvant including dissolving a mixture of birch bark triterpenoids in tetrahydrofuran, adding a cryoprotectant and lyophilization, according to the invention, a mixture of birch bark triterpenoids in tetrahydrofuran with a concentration of 5-10 g/l is obtained, followed by dissolving oleic acid in an amount of 5-10 g/l. 10% by weight of birch bark triterpenoids, carry out sterilizing filtration of the mixture, add a 25-fold excess of 0.01 M Tris buffer pH = 9.0 ± 0.2 to form spherical amorphous nanoparticles and mix, carry out ultrasonic treatment for 5-10 minutes, remove organic solvent using ultrafiltration at a rate of 1.0-1.2 l/minute, at a pressure of 0.6-0.8 atm and preferably on membranes with an exclusion threshold of 300 KDA, freeze the resulting concentrated mixture containing a mixture of terpenoids 1 mg/ml when adding a cryoprotector below a temperature of (-350) ° C, maintain at this temperature for 4-6 hours and lyophilize. The following can be used as a cryoprotectant: sucrose, mannitol, maltose, trehalose, mannose, sorbitol, etc. Preferably, the lyophilization method uses sucrose as the cheapest carbohydrate.

One of the advantages of this adjuvant is the presence of oleic acid in its composition in an amount of 5-10% by weight of birch bark triterpenoids, which ensures storage stability. The introduction of a sterilizing filtration step ensured that the adjuvant was sterile and pyrogen-free, and the use of ultrafiltration purification helped eliminate toxicity and remove tetrahydrofuran. The inclusion of an ultrasonication step allowed for greater stabilization of the adjuvant. The combination of distinctive features ensures easy scaling of the technological process from laboratory to industrial.

Currently, betulin in the form of spherical amorphous nanoparticles is proposed to be used as part of mono-vaccines against hepatitis B, diphtheria, Newcastle disease virus, and also as an adjuvant for quadrivalent influenza vaccines (RU 2355423, RU 2546861, RU 2545714, RU 2740751). Its use for polyvaccines is not described in the available literature. A spherical nanoadjuvant based on betulin is currently used in the recombinant coronavirus vaccine Betuvax CoV-2 (RU 2749193 C1, RU 2021139955 A). This vaccine was developed during the SARS-CoV-2 coronavirus pandemic and has proven itself to be an effective vaccine with a reduced content of the recombinant antigen, which is the RBD region of the S protein of the SARS-Cov-2 virus.

List of drawings:

Figure 1. Scheme of SARS-CoV-2 S protein and recombinant protein based on RBD-SD1-Fc. a) diagram of the S-protein on the surface of SARS-CoV-2, b) diagram of the structure of the recombinant RBD-SD1-Fc antigen, c) diagram of the S-protein of SARS-CoV-2, with the indicated RBD-SD1 part taken as the basis for recombinant proteins .

Fig. 2. Map of the P-Pharma-RBD-Fc plasmid.

The objective of the present invention is to expand the range of used vaccines against respiratory infections, using a combined vaccine containing antigens of both influenza viruses and the SARS-CoV-2 virus and a new corpuscular adjuvant based on birch bark triterpenoids. Such a combined vaccine seems to be a strategically important aspect of protecting the population of the Russian Federation from the severe social and economic consequences of the pandemic, which can only be effectively controlled through vaccination. The technical result is achieved through a method for obtaining variants of influenza virus antigens of subtypes A (H1N1), A (H3N2), B/Yamagata lineage, B/Victoria lineage and variants of recombinant protein antigens based on a section of the SARS-CoV-2 S protein, including RBD and SD1 fused to the Fc fragment of IgG, followed by the addition of an adjuvant in the form of spherical amorphous nanoparticles to each antigen and their combination into a pentavalent vaccine.

Disclosure of the invention

The vaccine production technology includes separate 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 allantoic fluid, concentration, purification, inactivation and cleavage of the virus, its subsequent centrifugation, ultrafiltration and obtaining monovalents for each strain, as well as cultivating a cell line producing RBD-Fc of the recombinant protein of the SARS-CoV-2 virus (antigen) with selection and clarification of the culture liquid containing the antigen, subsequent chromatographic purification of the antigen and obtaining a monovalent. The addition of an adjuvant to each type of virus is carried out separately - betulin is introduced into each of the monopreparations as an adjuvant at an antigen-adjuvant ratio of 1:1.25 to 1:12.5, after which the monovaccines are combined into a combined vaccine with thorough mixing.

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

Description of the invention. To obtain a pentavalent subunit vaccine against respiratory infections containing a corpuscular adjuvant based on natural betulin, antigens from two strains of the SARS-CoV-2 virus and strains of influenza virus type A (H1N1), A (H3H2), B1 and B2 were used.

Preparation of an adjuvant based on spherical amorphous nanoparticles. The adjuvant was obtained using the technology presented in patents RU 2545714 C1 and RU 2749193 C1. Briefly, a sterile solution of betulin in tetrahydrofuran was supplied to a container with a sterile buffer solution with ultrasound turned on at 35 KHz for 10 minutes. After feeding and mixing the betulin solution, the resulting suspension was subjected to ultrafiltration to remove tetrahydrofuran, and upon completion of ultrafiltration, the suspension was treated with ultrasound at 35 kHz for 3 minutes.

Method for obtaining influenza virus antigens. Chicken embryos 10-12 days old were infected with 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 duration of incubation was 48 hours, for type B virus strains - 72 hours. At the end of incubation, the embryos were checked using an ovoscope. Viable embryos were placed in a refrigerator at a temperature of (4±2)°C for (18±2) hours for cooling. Collection of virus-containing 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 virus-containing allantoic fluid, mixed for 10 minutes on a shaker, and microfiltration was carried out through a cascade of filter elements with pore sizes, respectively: 2.3-2.6-1.2- 0.8-0.44 microns. A beta-propiolactone solution was added to the contents of the bottle (filtrate) to a final concentration of 0.1%, mixed thoroughly and left in the refrigerator on a shaker for 17-18 hours.

Next, concentration and partial purification of the inactivated IMPORTANT were carried out: ultrafiltration was carried out on a tangential flow ultrafiltration unit with 4 cassette modules with a nominal cut-off molecular weight of 300 kDa. The resulting IMPORTANT concentrate was washed with 3-4 volumes of phosphate buffer solution and further purified by ultracentrifugation in a density gradient using two sucrose solutions of 50% and 20% at a rotor speed of 30,000 rpm. The contents of the rotor were dispensed into fractions of 20 ml, after which they were titrated in RGA to determine the peak of the virus and collected into a single volume with the maximum hemagglutinin content. To purify the ultracentifugate concentrate, gel chromatography on a polymer carrier was used. The volume of carrier for chromatography was determined at the rate of 5 volumes of carrier to one volume of sample. The column was equilibrated with Tris buffer, the sucrose concentrate was applied to the column through a pump, and then eluted with Tris buffer. The target eluate was collected by optical density at a wavelength of 280 nm.

To inactivate the virus, a device containing a source of ultraviolet rays with an ultraviolet irradiation flux of at least 90 lumens was used. To destroy the inactivated viral concentrate, octyl-D-Glucopyranoside was used in a mass ratio of detergent to total protein of 10:1. Incubation was carried out for 1 hour in a refrigerator at a temperature of 5±3°C. Next, the split viral preparation was concentrated in an ultrafiltration unit with 2 cassette modules with a pore diameter of 10 kDa.

The concentrated digest was further purified from detergent by gel filtration on an agarose-based chromatographic support with an exclusion limit of 150 kDa. The eluate was collected in a sterile container. To separate lipoproteins, ultracentrifugation was used at 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.

Influenza virus antigens and adjuvant. The resulting semi-finished products (monovalent substances) were controlled according to the following indicators: sterility, residual detergent content, specific activity according to ARID (hemagglutinin content), hemagglutinating activity, specific safety, authenticity, protein according to the Lowry method with precipitation. After the control, a pre-prepared corpuscular adjuvant based on natural betulin was added to the receiver bottle in a mass ratio of monovalent hemagglutinin and adjuvant of 1:5 in a phosphate buffer solution pH 7.5. The bottle was thoroughly mixed and placed in a shaker in the refrigerator for 3 hours. Then the bottles with monovalent substance and adjuvant were stored in a refrigerator at a temperature of 4±2°C. The final concentration of NA was 25 μg/ml, and the adjuvant concentration was 400 μg/ml in phosphate buffer solution pH 7.5.

Method for producing recombinant antigen proteins based on the SARS-CoV-2 S protein. According to application RU 2021139955 A, at the first stage of work, the design of the recombinant RBD-Fc gene was developed for the expression and production of the antigen-recombinant protein of the SARS-CoV-2 virus. A region of the S protein of the SARS-CoV-2 strain Wuhan-Hu-1 (hereinafter referred to as Wuhan), including RBD and SD1, fused with the Fc fragment of human IgG was selected (Fig. 1). The region of the S protein (amino acids 302-638) including RBD and SD1 is shown in Fig. 1 as "RBD-SD1 antigen".

The developed gene construct containing the RBD-Fc sequence was synthesized at Evrogen JSC. Thus, the pAL-TA-RBD-Fc construct was obtained. Next, the recombinant RBD-Fc gene was amplified in a PCR reaction using flanking primers, phosphorylated, and cloned into the P-Pharma plasmid vector for expression in mammalian cell lines. The resulting plasmid was named P-Pharma-RBD-Fc.

The P-Pharma-RBD-Fc gene construct was derived from the pCBG99 vector (GenBank, AY258596). The pCBG99 vectors were modified as follows: the SV-40 promoter was replaced with a hybrid promoter construct containing the hCMV MIE enhancer (pcDNA3.1-GFP, GenBank, MH325108) and the hEF-1 promoter (pEXPR5, GenBank, M34017), the SV-40 polyadenylation signal was replaced bGH Poly(A) signal (pcDNA3.1-GFP, GenBank, MH325108). In addition, the matrix attachment region (MAR) of the human beta interferon gene locus and the Epstein-Barr virus terminal repeat (EBVTR) protein domains were added. Promoters of eukaryotic selection markers were added - hPGK (GenBank, M34017) for resistance to zeocin and mPGK (pHPmF1, GenBank, JN195815) for the expression of mouse DHFR (GenBank, BC005796).

The plasmid was digested with HindIIIl/XbaI restriction enzyme mixture in CutSmart buffer (NEB), and the resulting product was dephosphorylated and blunted with CIP (NEB) and Phusion DNA polymerase (NEB), respectively. The RBD-Fc sequence was amplified with flanking primers containing the Kozak sequence and a transcription termination signal. The resulting product was phosphorylated with PNK T4 (NEB), ligated into a vector and transformed into E. coli 10 beta (NEB) and isolated using the commercial Plasmid Midiprep 2.0 kit (Eurogen). The design diagram is shown in Fig. 2.

To produce RBD-Fc recombinant protein, Chinese hamster ovary cells CHO-HP (Pharmapark LLC, Russia) were used. The method for producing RBD-Fc recombinant protein includes the following steps:

1) cultivation of a strain producing a recombinant protein,

2) selection and clarification filtration of the culture fluid,

3) chromatographic purification of the recombinant protein,

4) confirmation of the authenticity of the resulting recombinant protein.

RBD-Fc recombinant protein was obtained using technology according to application RU 2021139955 A. Briefly, CHO-S cells were cultured in a shaker-incubator at 5% CO2, 80% humidity and a shaking speed of 120 rpm in the HyClone HyCell TransFx- nutrient medium. C transfection media (GE Healthcare, USA) with the addition of 8 mM glutamine (PanEco, Russia), to achieve a density of 2*106 cells/ml 1 liter of suspension. The resulting suspension was transfected with plasmid vectors using 1 mg/ml Polyethylenimine reagent (Polyscience, USA), according to the manufacturer's instructions.

In CHO cells, the RBD-Fc recombinant protein was biosynthesized in a transient expression mode. Then, transfected cells were selected for 2 weeks on the antibiotic hygromycin B. This antibiotic was chosen because the plasmid vectors contain the resistance gene to the antibiotic Hygromycin B. The production of this protein ranged from 5 mg/l to 10 mg/l.

Isolation of RBD-Fc recombinant protein was carried out from the culture liquid at the end of the cultivation of CHO cells. Cells were pelleted by centrifugation for 5 min at 1000 rpm, the supernatant was transferred to clean tubes and pelleted by centrifugation for 30 min at 4000 rpm. The supernatant was again transferred to clean tubes, 1/10 volume of 10× Tris-HCl buffer (20 mM Tris-HCl, 150 mM NaCl pH 7.2 1× buffer) was added and applied to a pre-equilibrated 1 ml column with HiTrap MabSelect SuRe carrier (Cytiva, USA). Isolation of protein preparations was carried out on an Acta Pure chromatograph (GE Healthcare, USA) at a liquid flow rate of 2-4 ml/min and a pressure of no more than 0.5 MPa. After application, the column was washed with 10 column volumes of Tris-HCl buffer. The recombinant protein was eluted with citrate buffer (20 mM citric acid-NaOH, 200 mM NaCl, pH 3.0), and detection of proteins was carried out in real time by optical absorption at 280 nm. The fraction containing the desired product was neutralized by adding 1/10 of the volume of the fraction of neutralization buffer (1M Tris-HCl pH 8.0). The eluate was dialyzed against two changes of phosphate buffer (10 mM NaH2PO4, 1.7 mM K2HPO4, 137 mM NaCl, KCl 2.7 mM pH = 7.4), sterilizing filtration was performed and stored at +4°C.

The sterile recombinant RBD-Fc protein obtained in accordance with the above steps, dissolved in a Tris-HCl buffer solution, was then added to a container with a previously prepared sterile adjuvant in a 0.05 M Tris-HCl buffer solution with the addition of 0.15 M NaCl (pH 7.5 ) was thoroughly mixed for two hours at +4°C. The RBD-Fc semi-finished product, which has passed all controls, is adjusted to a protein concentration of 100 μg/ml and an adjuvant concentration of 400 μg/ml in a phosphate buffer solution pH 7.5. The container was placed in a shaker in the refrigerator for 3 hours. Then the bottles with the recombinant protein and adjuvant were stored in a refrigerator at a temperature of 2 to 8°C. The resulting vaccine bulk, after carrying out controls, was poured into 0.5 ml bottles and used as a candidate vaccine for preclinical and clinical studies.

An association. After stirring on a shaker for 0.5 hours in a refrigerator at +4°C, the monovalent semi-finished products are combined in a sterile bottle by adding equal parts of the semi-finished products (1:1:1:1:1). The resulting combined beam is placed in a refrigerator at +4°C and mixed on a shaker for 2 hours. Then stored at the same temperature. The resulting semi-finished vaccine with adjuvant is controlled according to the following indicators: sterility, adjuvant content, specific safety, authenticity, protein using the Lowry method (Lowry-M) with precipitation. In the combined preparation, the content of each antigen should be as follows: H1N1: 5±1.4 μg/ml, H3N2: 5±1.4 μg/ml, B1: 5±1.4 μg/ml, B2: 5±1, 4 µg/ml, RBD-Fc: 18±2 µg/ml. If necessary, connect to a filling machine and, using a dosing device, fill into vials, syringes or ampoules.

Thus, the development of a domestic safe and effective drug, a vaccine against coronavirus infection containing a corpuscular adjuvant, was carried out using a betulin-based adjuvant. Also, each of the created vaccines made it possible to control separately the prevention of influenza or a new coronavirus infection.

The essence of the invention is illustrated by the following examples:

Example 1. The vaccine was prepared using the technology described above with a mass ratio of antigens (RBD-Fc recombinant protein and hemagglutinins) and adjuvant of 1:1.25.

Example 2. The vaccine was produced according to the technology described above with a mass ratio of antigens (RBD-Fc recombinant protein and hemagglutinins) and adjuvant of 1:12.5.

Example 3. The vaccine was prepared according to the technology described above with a mass ratio of antigens (RBD-Fc recombinant protein and hemagglutinins) and adjuvant of 1:5, and the adjuvant was introduced after mixing the monovalents.

Immunogenicity study. In the study of the immunogenicity of vaccine preparations, outbred female mice were used (Rappolovo, Leningrad Region). The animals were kept under standard conditions (18-22°C, relative air humidity 50-70%, 12 hours dark/12 hours light), in accordance with Directive 2010/63/EU of the European Parliament and Council of the European Union of September 22, 2010. for the protection of animals used for scientific purposes and in accordance with sanitary and epidemiological rules SP 2.2.1.3218-14 “Sanitary and epidemiological requirements for the design, equipment and maintenance of experimental biological clinics (vivariums)” (approved by the resolution of the Chief State Sanitary Doctor of the Russian Federation dated August 29, 2014 No. 51). We used granulated complete extruded food for mice, rats, and hamsters (Laboratorkorm LLC, Moscow) ad libitum. All drugs (Table 1) were stored in a refrigerator (5±3°C) in the dark before use.

Immunization of animals was carried out intraperitoneally in a volume of 125-250 μl/animal. Sera were collected within the time periods specified in the study design (Table 2). Blood was obtained from the mandibular vein, after which the blood samples were left at room temperature for 2 hours, after which they were centrifuged for 10 minutes at 5000 rpm and the resulting whole serum was collected. Until use, the serum was stored at -20°C.

To perform the hemagggutination inhibition test (HIT), mouse sera were treated with RDE according to the manufacturer's instructions (RDE, Receptor destroying enzyme, Denka Seiken, Japan). To remove nonspecific agglutinins, the sera were additionally treated with an equal volume of 10% chicken erythrocytes. Then a series of two-fold dilutions were prepared 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. Influenza diagnosticums were used as antigens for staging the hemagglutination inhibition reaction (PPDP LLC, St. Petersburg), containing influenza virus strains: A/Victoria/2570/19 (H1N1pdm09), A/Cambodia/e0826360/20 (H3N2), B/Washington/02/19 (line B/Victoria/2/87), B/Phuket/3073/2013 (line B/Yamagata/16/88). The antigen-containing sera were incubated for an hour at room temperature, after which 50 μl of 0.5% chicken erythrocytes dissolved in 0.9% NaCl was added to all wells. The maximum dilution of serum that completely inhibited hemagglutination of chicken erythrocytes was taken as the serum titer. The RTGA titer was the reciprocal value of the serum dilution in the last well with the absence of hemagglutination.

Double immunization of outbred mice led to a significant increase in titers of specific antibodies to all four influenza monocomponents of vaccine preparations. For influenza monocomponents H1N1pdm09 and H3N2, in the case of using the pentavalent vaccine, a significantly higher value of geometric mean titers was observed compared with the use of the tetravalent vaccine TetraFluBet (p <0.05). Double immunization with vaccine preparations at the dosage used led to the pronounced formation of virus-neutralizing antibodies.

Microneutralization reaction. A TCID50 (Tissue Culture Infectious Dose)-based microneutralization reaction was used to detect neutralizing antibodies. Mouse sera were heated for 60 min at 56°C to avoid complement-associated reduction in viral activity. Then a series of two-fold dilutions of the serum was prepared in MEM + 2% HI-FBS medium in a volume of 60 μl, starting from 1:10 (in terms of whole serum). Each serum dilution was mixed in an equal volume with live SARS-CoV-2 virus (hCoV-19/St_Petersburg-3524V/2020 virus, GISAID EPI_ISL_415710, 60 μl containing 25 TCID50 / 50 μl) and incubated for 1 hour at 37°C. 100 μl dilutions were then transferred into 96-well Vero cell monolayer plates (ATCC CCL-81). The plates were incubated at 37°C in an atmosphere of 5% CO2 for 4 days, after which they were checked for the presence of CPE (cytopathic effect of the virus). Neutralization was recorded if 100% of the cells in the well were retained (no visible plaque or cytopathic effect). The neutralizing serum titer was expressed as the reciprocal of the highest serum dilution at which neutralizing activity was shown (Barchuk A, 2021). All experiments were performed in the BSL3 laboratory.

Statistical analysis of primary data was carried out in GraphPad Prism v9. The following statistical indicators were used to present the data: geometric mean, standard deviation. To determine the significance of differences between group means, one-way ANOVA was used for group comparisons, followed by Dunnett's test for post hoc pairwise group comparisons. The group significance level was taken equal to α=0.05. Differences were considered significant at p<α.

The results of the neutralization reaction against the SARS-CoV-2 coronavirus in the blood sera of mice after double immunization showed that in the experimental groups vaccinated with the pentavalent vaccine, neutralization of the virus was observed, which was significantly different from the group vaccinated with the tetravalent influenza vaccine TetraFluBet and from the control (placebo). The results are presented in tables 3-7.

Claims (2)

1. A method for producing a pentavalent subunit vaccine against influenza and SARS-CoV-2 related to ARVI, including separate cultivation of influenza viruses of subtypes A (H1N1), A (H3N2), B/Yamagata lineage, B/Victoria lineage in developing chicken embryos with subsequent separation of the virus-containing allantoic fluid, concentration, purification, inactivation and cleavage of the virus, its subsequent centrifugation, ultrafiltration and production of monovalents for each strain, as well as cultivation of a cell line producing the recombinant protein of the SARS-CoV-2 virus - RBD-Fc with selection and clarification culture liquid containing RBD-Fc, subsequent chromatographic purification and production of a monovalent, the addition of an adjuvant to each monovalent is carried out separately, betulin is used as an adjuvant at an antigen:adjuvant ratio from 1:1.25 to 1:12.5, after which the reduction is carried out monovaccines into a combination vaccine. 2. A method for producing a pentavalent subunit vaccine against influenza and SARS-CoV-2, related to ARVI, according to claim 1, characterized in that recombinant RBD-Fc is obtained by expression in CHO cells using transient transfection.