RU2795160C1 - HYBRID GENE CONSISTING OF THE RBD RECEPTOR OF THE SURFACE PROTEIN S OF SARS-CoV-2 CORONAVIRUS, S14P5 AND S21P2 EPITOPES, Fc FRAGMENT, FOR OBTAINING A RECOMBINANT ANTIGEN AND ITS USE AS PART OF A VACCINE COMPOSITION AGAINST CORONAVIRUS INFECTION - Google Patents

Abstract

FIELD: biotechnology; immunology;virology.

SUBSTANCE: following is described: a hybrid gene encoding a recombinant antigen consisting of the RBD receptor of the surface protein S of the SARS-CoV-2 coronavirus, the S14P5 and S21P2 epitopes, the Fc fragment of the IgG1 immunoglobulin, represented as the amino acid sequence of SEQ ID NO: 1. An expression construct is also described in the P-Pharma-Zeo vector represented by SEQ ID NO: 4 containing the nucleotide sequence of SEQ ID NO: 2, consisting of the hybrid gene described above and the sequence of SEQ ID NO: 3 encoding the IL2 leader peptide. A CHO-HP cell line transfected with the expression construct described above to produce the recombinant RBD/S21/S14-Fc antigen is described. A method for obtaining a recombinant RBD/S21/S14-Fc antigen is presented, involving the cultivation of the above-mentioned CHO-HP cell line, under conditions of feeding 4 mM L-alanine-L-glutamine and an anti-cell aggregation reagent, followed by the selection of the culture fluid, its clarification, and filtration, chromatographic purification of the target recombinant antigen, including affinity chromatography, HA sorbent chromatography, hydrophobic and gel permeation chromatography, followed by sterilizing filtration. The recombinant antigen RBD/S21/S14-Fc obtained by the above method is presented, which stimulates the immune response in the body to coronavirus infection. A vaccine composition for the prevention of coronavirus infection is presented, containing the above recombinant antigen and a corpuscular adjuvant based on natural betulin in a buffer solution. A method for the prevention of coronavirus infection is disclosed, which involves immunization of requiring protection patients against coronavirus infection with the above vaccine composition.

EFFECT: invention expands the arsenal of means to combat SARS-CoV-2.

7 cl, 10 dwg, 10 tbl

Description

The field of technology to which the invention belongs.

The invention relates to biotechnology, immunology, virology and concerns the creation of a vaccine against a coronavirus infection, a method for its preparation, a hybrid gene construct and a construct for the expression of the recombinant RBD/S21/S14-Fc antigen, which is a portion of the S-protein of SARS-CoV-2, including RBD, part of SD1 and S2 fused to the Fc fragment of IgG1, a cell line producing a recombinant antigen, a method for producing a recombinant antigen, which, in combination with a particulate adjuvant based on betulin, is able to induce long-term humoral and cellular immunity against coronavirus in animals and humans.

The level of technology.

Vaccination is considered in modern conditions as one of the most effective means of combating infection. Thus, many countries and pharmaceutical companies are involved in the development of vaccines and drugs against SARS-CoV-2, a new RNA-containing virus belonging to the Coronaviridae family to the Beta-CoV lineage.

With the traditional technology for creating vaccines based on the use of a full-sized pathogen, the complexity of production increases sharply, due to the danger of work and the need for strict control over the completeness of inactivation of the pathogen, which makes this option less cost-effective. In this regard, the technology for obtaining vaccines based on subunits of the virus, which has been proven in recent years, has certain advantages, such as safety and the possibility of booster administration of the vaccine. However, some viral subunits have low immunogenicity, which requires the use of adjuvants.

Vaccines based on inactivated whole viruses are generally widely used for immunization, but they have serious drawbacks - from technological, associated with the difficulty of obtaining large quantities of the drug in a short time, to problems regarding their effectiveness - undesirable side effects and problems with the targeting of produced antibodies. on the necessary epitope of viral envelope proteins, as well as contaminant proteins remaining in the vaccine after virus cultivation.

Nucleic acid-based vaccines are based on the principle of introducing genetic information into cells, from which pathogen proteins are produced that cause an immune response. If viral vectors are used for delivery, there is a risk of foreign DNA integrating into the genome of the vaccinated cell, the possible development of autoimmune reactions against cells expressing the viral antigen, or a reaction to foreign DNA. In addition to these shortcomings, there are difficulties with the delivery of such drugs. It should be noted that RNA and DNA vaccines, as well as vaccines based on adenoviral vectors, cause some caution in their use, since the long-term consequences of their use are unknown.

Subunit vaccines are represented primarily by recombinant preparations based on purified proteins. When creating these types of vaccines, an important part is the search for new effective adjuvants. Their use makes it possible to reduce the dose of the antigen, reduce the cost of the final drug, increase the immunogenicity of "weak" or small antigens, which are extremely difficult to develop immunity and obtain a vaccine against this class of pathogens. This makes it possible to prevent the competition of antigens in combined vaccines, increase the rate of development and duration of the immune response in vaccinated people, induce the protective properties of the mucous membranes, and also increase the strength of the immune response. Currently, it is recognized as impossible to create effective and safe subunit or epitope vaccines without the use of adjuvants.

The RBD (receptor-binding domain) of the S-protein, as well as its individual epitopes, is responsible for the function of coronavirus penetration into host cells. In experimental studies, it has been established that most antibodies with virus-neutralizing activity against a type of coronavirus (for example, MERS or SARS) specifically bind to various RBD epitopes. RBD forms a binding site for the ACE2 receptor; therefore, antibodies capable of binding to the RBD domain can block contact with the receptor and, as a result, prevent the virus from entering cells, which has been established in the case of SARS-CoV-2. Based on this, the RBD sequence was chosen as the sequence for creating the antigen. It is worth noting that the choice of the whole S-protein could lead to the production of not only neutralizing antibodies, but also masking ones, increasing the development of an antibody-dependent increase in infection.

The S1 domain consists of the NTD subdomain (N-terminal domain), RBD (residues 319-527), and the SD1 and SD2 subdomains, which are closest to the S2 domain. Thus, the most important task of vaccination is not just the production of antibodies to the S-protein, but specifically neutralizing antibodies to RBD. However, the simple use of RBD as a vaccine antigen is not possible, since the mass of the protein is rather small (less than 40 kDa), and it is also rapidly excreted from the body, so RBD, part of SD1 and S2, conjugated with the Fc fragment of IgG1 became candidates for the antigen. .

Adding an adjuvant will reduce the amount of protein per dose, the cost of the vaccine, and increase the immune response to the antigen. Currently, in experimental and clinical studies, mineral (aluminum hydroxide or phosphate), vegetable (saponins - QuilA, QS21), microbial (killed bacteria, lipopolysaccharide and its derivatives, CpG DNA motifs) and other types of adjuvants are used. Due to the toxicity or lack of efficacy of most adjuvants, only aluminum salts and water-oil emulsions of MF-59, AS03, QS 21, VLP (virus-like particles) and ISCOMs (ImmunoStimulating COMplex) are allowed for general use. The most promising are corpuscular adjuvants - spherical amorphous nanoparticles (SANPs). One of the reasons for their use is their efficient uptake by antigen-presenting cells of the immune system, resulting in an enhanced immune response. These adjuvants include betulin adjuvants based on triterpenoids from birch bark extract (patent RU 2,545,714). Betulin particles, in addition to a pronounced spectrum of biological activity (antimicrobial, antifungal, antiviral action, hepatoprotective, anti-inflammatory, anticancer, antiallergic, membrane stabilizing, immunomodulatory, antioxidant), also have adjuvant properties.

The technical solution according to the patent RU 2,749,193 is chosen by the authors of the claimed invention as a prototype. Antigen uses are known from this patent, including RBD to induce specific immunity against the SARS-CoV-2 virus as an antigenic component, together with 80-160 nm betulin spherical amorphous nanoparticles (SANP) as an adjuvant. This adjuvant is produced on an industrial scale in accordance with the requirements of the pilot industrial regulations, and was also previously tested in preclinical studies of influenza vaccines, which showed that the presence of a corpuscular adjuvant in the vaccine composition leads to an increase in the immune response and does not affect the safety of the drug.

Disclosure of the essence of the invention.

A significant drawback of the known vaccines against SARS-CoV-2 is the complexity of their production, insufficient immunogenic activity, undesirable side effects, and problems with the targeting of produced antibodies to the necessary epitope of the viral envelope protein. The technical task of the present invention is to create an antigenic composition that allows to cause the induction of a specific humoral and cellular immune response to the required epitope of the SARS-CoV-2 viral envelope protein. The technical result of the disclosed invention is the creation of an antigenic composition with corpuscular betulin as an adjuvant and a recombinant protein as an active component. The technical result is achieved by creating a hybrid RBD/S21/S14-Fc gene encoding a recombinant SARS-CoV-2 antigen fused with an immunoglobulin Fc fragment through a linker, a cell line expressing the recombinant antigen into the culture fluid, isolation and purification of the recombinant RBD antigen /S21/S14-Fc, formulation of a vaccine using a corpuscular adjuvant based on natural betulin, use of a vaccine for immunization of animals and humans in order to obtain an immune response against coronavirus.

Brief description of the drawings

Rice. 1. P-Pharma-Zeo vector map - RBD/S21/S14-Fc.

Rice. 2. Electrophoregrams of sequencing reactions for RBD-Rev 1 primer.

Rice. 3. Electrophoregrams of sequencing reactions for primer RBD-short.

Rice. 4. Electropherograms of sequencing reactions for primer RBD-short (continued).

Rice. 5. Electrophoregrams of sequencing reactions for primer SolR.

Rice. 6. Electropherograms of sequencing reactions for primer SolR (continued).

Rice. 7, Electropherograms of sequencing reactions for primer RBD-Fwd3.

Rice. 8. Results of evaluation of expression and secretion of RBD/S21/S14-Fc protein using Western Blot. 1 - cell lysate; 2 - culture liquid. The column on the left is the molecular weight standard.

Rice. 9. Tryptic peptide coverage of the RBD/S21/S14-Fc sequence.

Rice. 10. Results of size exclusion chromatography on a Superdex 200 Increase column in Na-phosphate buffer pH 7.4.

Implementation of the invention.

RBD/S21/S14-Fc hybrid gene design. At the first stage of the work, the design of the RBD/S21/S14-Fc hybrid gene was developed for expression and production of the antigen-recombinant protein of the SARS-CoV-2 virus. The RBD-based recombinant antigen sequence with additional epitopes was fused to the C-terminal portion of the RBD, wild-type FcIgG1 (no modification), to generate RBD/S21/S14-Fc. RBD modifications were aimed at increasing the immunogenicity of the antigen. Two peptide epitopes were added. The S14P5 epitope is located in the region behind the RBD in the SD1 subdomain; apparently, antibodies to this region sterically prevent binding to the receptor. The S21P2 epitope is located in the S2 region, overlaps with the fusion peptide (FP), and antibodies to this region prevent the virus from fusion with the target cell. The order of the epitopes was changed to improve the stability of the protein. Its amino acid sequence is shown in SEQ ID NO: 1. Estimated molecular weight 107.2 kDa (dimer), 53.6 kDa (reduced monomer).

Obtaining an expression construct and its preparation for transfection. In the first step, an optimized nucleotide sequence was obtained (SEQ ID NO: 2). The nucleotide sequence of the hybrid gene was optimized for expression in CHO-HP cell culture using the GeneArt GeneOptimizer program. In this sequence, the first 60 nucleotides are the leader peptide of IL-2 (SEQ ID NO: 3).

The RBD/S21/S14-Fc plasmid map is shown in Fig. 1. Table 1 shows the main characteristic coding sequences of the plasmid RBD/S21/S14-Fc. The complete nucleotide sequence of the vector is shown by SEQ ID NO: 4.

The RBD/S21/S14-Fc hybrid gene is amplified in a PCR reaction using primers SEQ ID NO: 5 and SEQ ID NO: 6 and a DNA polymerase having corrective 3'-5' exonuclease activity. The reaction product is purified using a DNA spin column purification protocol.

The P-Pharma-Zeo vector is linearized with HindIII and XbaI restriction enzymes, purified by agarose gel electrophoresis, and isolated from the gel using a spin column DNA purification protocol. The ends of the vector are blunted using a DNA polymerase having 3'-5' exonuclease activity, then the vector is purified using a DNA spin column purification protocol. The ligation reaction is carried out using T4-DNA polymerase in the ratio of insert/vector=1/10 using 5% PEG-4000 in the ligation reaction. The resulting plasmid DNA is purified using a spin column DNA purification protocol. The ligation mixture is transformed into chemically competent E. Coli cells (strain 10beta), after transformation, cells are seeded in LB-agar on a petri dish containing 100 μg/ml ampicillin. Cells were incubated at 37°C for 18 hours until individual colonies (bacterial clones) appeared.

Next, the presence of insertion and orientation were checked using primers, SEQ ID NO: 7 and SEQ ID NO: 8 (to check the presence of the gene in the plasmid) and SEQ ID NO: 9 SEQ ID NO: 10 (to check the correct orientation of the gene) in the selected bacterial clones. The volumes of reagents for the PCR reaction are presented in table. 2.

After the PCR reaction, the reaction product was analyzed by agarose gel electrophoresis (1% agarose). Bacterial clones, PCR products from which have a PCR product size ~ 1700 bp) and correctly oriented (PCR product size ~ 400 bp), are transferred into 50 ml of LB medium containing 100 μg/ml ampicillin, cultured with stirring within 24 hours. The resulting suspension is centrifuged and the supernatant is removed without affecting the cell sediment. Plasmid DNA is isolated from the cell pellet using a spin column purification protocol.

Plasmid DNA from the selected colonies was isolated from the bacterial culture using the Plasmid Midiprep 2.0 kit according to the manufacturer's recommendations. 50 ml of culture was centrifuged at a speed of 3000 g and a temperature of 4±2°C for 20 minutes. The supernatant was completely removed, 5 ml of the resuspending solution with RNase A was added to the sediment. The contents of the tube were carefully inverted 10 times. Then 5 ml of a neutralizing solution, pre-cooled to 4±2°C, were added and mixed by inverting. The lysate was precipitated at a speed of 3000 g for 45 min on an Allegra 25R centrifuge. Before applying the lysate, 1.0 ml of a solution to remove endotoxins was applied to the membrane of the spin column, completely wetting the entire surface of the sorbent. Incubated for 1 minute and applied to the membrane the entire volume of the bacterial lysate. The spin column was centrifuged at 3000 g for 2 minutes. Then, 1 ml of endotoxin removal solution was applied to the membrane and centrifuged at 3000 g for 1 minute. 15 ml of washing solution with ethanol was added and centrifuged at 3000 g for 1 minute, washing was repeated with the same volume of washing solution. After the second wash, the spin column was centrifuged at 3000 g for 5 minutes to remove residual liquid. The spin column was placed in a new 50 ml tube and left open under a laminar flow hood for 5 minutes to remove residual ethanol. After that, 4 ml of the eluent solution, preheated in a TW-2 water bath to 50±2°C, was applied to the membrane and incubated at room temperature for 2 minutes. The spin column was centrifuged at 3000 g for 2 minutes. The eluate was concentrated using VivaSpin6 ultrafiltration concentrators (cut-off 10 kDa) to 500 µl at a speed of 3000 g.

To 500 µl of plasmid DNA solution was added 50 µl of 3 M sodium acetate. 1400 µl of ethanol pre-cooled to minus 20°C was added to the mixture, mixed and incubated at minus 20±2°C for 20 minutes. The resulting solution was centrifuged at a speed of 14,000 g for 20 minutes at a temperature of 4±2°C, the supernatant was completely removed, and 500 μl of 70% ethanol was carefully added to the plasmid DNA pellet. The sample was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed. The precipitate was dried under a laminar flow of sterile air for 10 minutes and dissolved in 20 µl of sterile water free of DNase and RNase.

1 μl is taken to assess the concentration of DNA, as well as to determine the sequence of the RBD/S21/S14-Fc gene. For sequencing the introduced gene by the Sanger method, primers SEQ ID N0:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 are used.

Figures 2-7 show electropherograms of sequencing reactions. The final results of sequencing using primers covering the entire sequence of the RBD/S21/S14-Fc gene confirmed the sequence identity of the hybrid RBD/S21/S14-Fc gene with the reference sequence.

Transfection and selection of transfectants. Creation of a producer strain.

For transfection of CHO cells, 8.6 mg/ml of pure ("transfection grade") plasmid DNA P-Pharma-Zeo-RBD/S21/S14-Fc was obtained. As a result of transfection with the P-Pharma-Zeo-RBD/S21/S14-Fc expression vector, one cell pool was obtained, in which the viability of the cell culture 48 h after transfection was 92%. Control transfection with a vector containing the gene for the fluorescent green protein GFP demonstrated a transfection efficiency of 50±20%. After 72 hours, the selection of transfectants was started by culturing with the addition of 400 μg/ml Zeocin and 500 nM MTX. Within 10 days under selective pressure, viability reached a minimum of 55±5%. Restoration of culture viability in the pool to 90±5% occurred on the 16th day, after which some of the pool cells were frozen, and limiting dilutions were made with a density of 0.5 cells per well. The total number of tablets was 100 pcs. From the 96-well plates from the stage of cloning by the method of limiting dilutions, 105 clones were selected and transferred to 24-well plates. All clones grown in 24-well (77) plates were transferred to 6-well plates with Anti Clumping Agent B increased to 1.4% to destroy cell aggregates. After scaling for adaptation to cultivation with stirring in flasks, 10 subclones were selected that had the best characteristics in terms of parameters: specific productivity (pcd) ≥20 pg/cell, viable cell content ≥0.9 million/ml, protein content in culture fluid ≥30 mcg/ml. The reseeding of subclones was carried out within 2 passages (1 passage-4 days), after the second passage, the cells were frozen with a cell content of 10±1.5 million cells/cryotube. Table 3 shows data on the productivity of clones in the second passage, counting from passage from 96-well plates.

Based on the CHO-HP cell line transfected with the P-Pharma-Zeo-RBD/S21/S14-Fc vector, four primary clone-producers of the RBD/S21/S14-Fc fusion protein were obtained, at least 17±2 pg/(cell*day ). The clone with the highest productivity was chosen as the producer strain (Table 3, clone number 10).

Thus, the CHO-HP cell line was created, obtained from the CHO-K1 cell line (Cricetulus griseus species) by adaptation to suspension cultivation, containing the gene for the RBD/S21/S14 fusion protein of the S protein domain from SARS-CoV-2, connected with the Fc fragment of human IgG1 as part of the P-Pharma-Zeo vector. Thus, the assembly and testing of RBD/S21/S14-Fc was carried out and a producer strain was created for the production of the RBD/S21/S14-FC protein.

Secretion analysis and confirmation of protein identity. On fig. 8 shows the results of evaluation of RBD/S21/S14-Fc expression by Western blotting.

For mass spectrometric identification of the protein, after SDS-PAGE electrophoresis, Coomassie-stained bands were cut out and trypsinolysis was performed in the gel. Separation of tryptic peptides was performed on an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Analysis of MS/MS data was performed using the PEAKS Studio 8 software. Primary peptide structures generated by the PEAKS Studio software were analyzed against a database of corresponding protein sequences. The assay was run with the following settings: Met oxidation, Asn/Gln deamidation, and Cys carbamide methylation. On fig. 9 shows tryptic peptide coverage of the RBD/S21/S14-Fc sequence. On fig. 10 shows the results of size exclusion chromatography on a Superdex 200 Increase column in Na-phosphate buffer pH 7.4.

A method for obtaining a recombinant antigen from the cells of a producer strain. The developed method for obtaining a recombinant antigen includes the following steps: 1) cultivation of the strain producing the recombinant RBD protein of the SARS-CoV-2 virus; 2) selection and clarifying filtration of the culture liquid; 3) chromatographic purification of the recombinant RBD protein of the SARS-CoV-2 virus; 4) confirmation of the authenticity of the obtained recombinant RBD protein of the SARS-CoV-2 virus.

Cultivation. A test tube from the main cell bank containing 10 ⁷ cells of the strain producing the recombinant RBD/S21/S14-Fc antigen was thawed at 37°C in a water bath for 10 minutes; 4 ml of ActiPro medium (Hyclone, USA) containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% anti-cell aggregation reagent were mixed and centrifuged at 200 g for 5 minutes, the supernatant was removed, the cell pellet was resuspended in 5 ml of ActiPro medium (Hyclone, USA), containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% reagent against cell aggregation, and incubated for a day in an incubator at 37°C and a humidity of at least 70%. For cultivation in a bioreactor, the inoculum is increased in the required amount for the initial inoculation into the reactor with a cell density of 0.5×10 ⁶ cells/ml, i.e. in an amount of at least 400×10 ⁶ cells in flasks with stirring, the cell culture is subcultured with an increase in the volume of the flasks. The cell culture was inoculated into a 125 ml flask with 20 ml of medium and cultivated for 4 days in a CO2 shaker incubator at a platform rotation speed of 120 ± 5 rpm, a temperature of 37 ± 1°C, a CO2 content of 5 ± 1%, and a relative humidity 70±5%. The entire culture volume was transferred into a 500 ml flask with 125 ml of medium and cultivated for 3 days under the same conditions. The required volume of cells is transferred into a BioFlo 320 bioreactor with a working volume of 1 liter (Eppendorf, Germany) to a cell density of 0.5×10 ⁶ cells/ml with a final culture volume of 800 ml. Cell cultivation of the producer strain is carried out in a BioFlo 320 bioreactor with a working volume of 1 liter (Eppendorf, Germany) on ActiPro medium (Hyclone, USA) containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% reagent against cell aggregation (Lonza, Switzerland). Every day from the third day of cultivation, a complex of additives is introduced: Cell Boost 7A (Cytiva, USA) 1.5-3.5% of the cultivation volume and a mixture of amino acids L-cystine (152 mM), L-tryptophan (90 mM), L-tyrosine (250 mM) (AppliChem, USA for three amino acids).

The amount of the mixture of amino acids used is 10 times less relative to the volume of Cell Boost 7A. During the whole process, 200 g/L concentrated glucose solution (Panreac, USA) is added daily to maintain the glucose concentration in the culture liquid at about 5 g/L. All these additives are added daily through the bioreactor controller pump in such a way that the calculated volume per day does not affect the sharp change in pH and osmolality. On the first day of cultivation, the prepared biomass is transferred to a bioreactor to obtain an initial concentration of living cells of 0.5±0.1×10 ⁶ cells/ml. Cultivation is carried out at pH 7.0±0.2 and dissolved oxygen concentration of 40±5%. The pH is adjusted by supplying CO2 and 0.5 M NaHCO ₃ (Sigma-Aldrich, USA). The regulation of the concentration of dissolved oxygen at the beginning of the process is carried out with nitrogen and air, then with air and oxygen as the concentration of viable cells and the rate of oxygen consumption increase. The mixture of gases is fed through the macrobubbler at a rate of 0.2 to 1.5 l/min. Cultivation is carried out at a speed of rotation of the stirrer type "sea propeller" from 60 to 100 rpm (cascade control) and a temperature of 37°C, followed by a decrease to 35°C in the exponential growth phase and to 33°C on the next day of cultivation.

Selection and clarifying filtration of culture liquid. Starting from the third day of cultivation, a daily sampling of the culture fluid is carried out to determine the number of viable cells in it, the concentration of glucose and lactate. Cultivation is stopped when viability drops below 90%. The culture fluid is clarified by centrifugation in two stages: 1000 g for 30 minutes and 3270 g for 15 minutes. To sterilize the culture fluid, use a filter with a rating of 0.22 µm. The resulting volume of sterile culture fluid was 1.05 L. 375 mg of the recombinant RBD/S21/S14-FC antigen was obtained from the obtained culture liquid by purification according to the protocol described below.

Chromatographic protein purification. Chromatographic purification of the protein from the clarified cultural liquid is carried out on a preparative chromatograph. The first purification stage is the affinity sorbent Mab Select Shure LX (Cytiva), as well as MabSelect Prisma (Cytiva), Mab Select (Cytiva) with a volume of 4.7 ml. Equilibrate with buffer 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 for 10 column volumes at a rate of 2.5 ml/min. Applying the culture liquid to the column, contact time 1 min. After completion of the loading, the column was washed with buffer 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 for 5 column volumes at a rate of 5 ml/min. After washing is completed, elution is carried out with a buffer of 20 mm sodium citrate, pH 3.0 for 4 column volumes at a rate of 1 ml/min. Bringing the pH in the intermediate with a 1M Tris solution to a value of 7.0. The second stage of purification is the sorbent of mixed action HA Ultrogel (Sartorius), as well as CNT Ceramic Hydroxyapatite (Biorad), Ca ++ Pure-HA (Tosoh Biosciences) with a volume of 10 ml. Equilibrate with buffer 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 for 8 column volumes at a rate of 2 ml/min. The application of the intermediate obtained from the first stage of purification to the column occurs in a 50% gradient of the buffer 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0, contact time 5 min. After loading, the column was washed with buffer 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 for 4 column volumes at a rate of 2 ml/min. After washing is completed, elution is carried out with a buffer of 20 mm sodium phosphate, 1 M sodium chloride, pH 7.0 for 4 column volumes at a rate of 1 ml/min. Adding NaCl to the intermediate to a final content of 2 M. Bringing the pH in the intermediate with a 1M solution of phosphoric acid to a value of 6.0. The third stage of purification is the hydrophobic sorbent Butyl Sepharose HP (Cytiva), as well as Capto Butyl ImpRes (Cytiva), Phenyl Sepharose HP ( Cytiva), Capto Phenyl ImpRes (Cytiva) 5 ml. Equilibrate with buffer 20 mM sodium phosphate, 2 M sodium chloride, pH 6.0 for 10 column volumes at a rate of 2.5 ml/min. Application of the intermediate obtained from the second stage of purification on the column, contact time 5 min. After completion of the loading, the column is washed with 20 mM sodium phosphate, 2 M sodium chloride, pH 6.0 buffer for 5 column volumes at a rate of 2.5 ml/min. After washing is completed, elution is carried out in a gradient of 100% buffer 20 mm sodium phosphate, pH 6.0 for 3 column volumes at a rate of 2 ml/min. The fourth stage of purification is transfer to the GLF buffer using Sephadex G25 sorbent with a volume of 53 ml. Before loading, the column is equilibrated with buffer 20 mM sodium phosphate, pH 7.0, loading takes place at a rate of 1 ml/min; . The fifth stage is the normalization of protein concentration. The concentration is adjusted with a buffer of 20 mM sodium phosphate, pH 5.0-7.0 to a value of 2 mg/ml and sterile filtered through a filter nozzle on a Steritop 0.22 mkm vial (Merck - Millipore, Germany). Received 180 ml of sterile purified recombinant antigen RBD/S21/S14-Fc. The recombinant antigen is stored in a refrigerator at +4°C.

Protein authentication. Confirmation of the identity of the obtained recombinant RBD/S21/S14-Fc antigen is carried out using polyacrylamide gel electrophoresis followed by immunoblotting. For electrophoresis, a gradient polyacrylamide gel 4-20% (Bio-Rad, Cat No. 4561093) is used. Analysis of samples by electrophoresis is carried out under reducing conditions. 100 ng of the protein solution is added to the wells of the gel. Separation is carried out at a voltage of 200 V for 50 minutes. Transfer to the PVDF membrane was carried out using the Trans Blot Turbo system (Biorad) according to the manufacturer's recommendations. After plugging the membrane with 3% bovine serum albumin, conjugation is carried out with an alkaline phosphatase-labeled anti-IgG1 antibody (Abcam, cat no. ab6760). After washing the membrane, the conjugate will be detected using a BCIP/NBT solution.

Obtaining a homogeneous dispersion of corpuscular adjuvant based on betulin (KA). In order to obtain a homogeneous dispersion of corpuscular adjuvant (CA) based on betulin, 25 volumes of sterile 0.01 M Tris -buffer pH-9.0±0.1 using a peristaltic pump with constant stirring for 15 minutes with a propeller-type magnetic stirrer. After adding the buffer, the content of THF in the solution was 3.85%. Next, the resulting suspension was treated with ultrasound at 35 kHz for 10 minutes. To do this, a glass container with the obtained suspension of corpuscular adjuvant was placed in an ultrasonic bath. And processed in the above mode. As a result of processing, a white uniform suspension was obtained without conglomerates and inclusions. The homogeneity of the suspension was monitored by light scattering, sequentially processed for 30 seconds until two identical particle distribution spectra were obtained in the range of 100-150 nanometers. At the next stage, the KA dispersion was purified from tetrahydrofuran. Ultrafiltration was applied on hollow fibers with a nominal cut-off molecular weight of 100 kDa in filtration mode. To prevent sorption, a 2–3 fold dilution of the initial KA dispersion was used. The ultrafiltration rate was 1.0-1.2 l/min, the pressure was set within 0.6-0.8 atm. A 10-fold volume (25 liters) of 0.01 M Tris buffer was used for diafiltration. Then the dispersion was concentrated to the content of betulin 1.0-2.0 mg/ml (4.5 l of buffer). The concentration of betulin was determined by HPLC. The container with corpuscular adjuvant was stored in a refrigerator at a temperature of about +4 C.

Obtaining a candidate vaccine for immunization of animals and volunteers. The sterile recombinant RBD/S21/S14-FC antigen obtained in accordance with the above steps, dissolved in a 100 ml Tris-HCl buffer solution, was added to a container with a 1-liter pre-prepared sterile adjuvant in a 0.05 M Tris-HCl buffer solution with the addition of 0 15M NaCl (pH 7.5) was thoroughly mixed for two hours at +4°C. The resulting semi-finished product containing RBD/S21/S14-Fc, which passed all controls, was adjusted to a protein concentration of 40 μg/ml and an adjuvant concentration of 400 μg/ml in a phosphate buffer solution pH 7.5. The components were thoroughly mixed and placed in a shaker in the refrigerator at a temperature of 2 to 8°C for 3 hours. Then the containers with the "bulk" of the candidate vaccine were poured into 0.5 ml bottles and stored in a refrigerator at 2-8°C. The content of antigen and adjuvant in one dose (0.5 ml) was 20 μg and 200 μg, respectively. According to the same scheme, a candidate vaccine sample was prepared containing the antigen and adjuvant at a dose (0.5 ml): 5 μg of the RBD/S21/S14-Fc recombinant antigen and 200 μg of the corpuscular adjuvant.

The resulting vaccine was used as a candidate vaccine for preclinical and clinical studies.

Determination of the content of corpuscular adjuvant (CA). The determination was carried out by reverse-phase HPLC. The study was carried out using a Shimadzu LC-2010 liquid chromatograph (Japan) and a Kromasil 300-5C8 250 x 4.6 mm analytical column with a particle size of 5 μm, AkzoNobel (Holland) in the isocratic elution mode and detection at 210 nm. The results are presented in table. 4.

Evaluation of the specific activity of the vaccine. The aim of the study was a comparative study of the immunogenicity of the vaccine in animal experiments. The study compared the production of antibodies in Balb/c mice immunized with experimental vaccines weighing 12-14 g of both sexes. The test preparations were administered twice (according to the scheme 0-14) intraperitoneally, 0.5 ml each, with a syringe with a capacity of 1 ml. Similarly, the control groups of animals were injected with sodium chloride, solution for infusion 0.9%. 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. The specific activity of the vaccine containing only RBD (in two doses: 5 and 20 µg of recombinant protein per dose (0.5 ml), N+RBD vaccine at a dose of 5 µg of antigen per dose (0.5 ml), and the vaccine without data on the number of animals, vaccines and their dosage during immunization are presented in Table 5.

Serum obtained from the blood of mice (individually from each animal) was monitored for the presence of antibodies to the SARS-CoV-2 antigen by enzyme immunoassay using an experimental test system (IFTS). When designing IPTS, commercial reagents were used: a/mouse IgG (H+L) Strong Zyme HRP Conjugate (SDT, Germany); chromogen - 3,3,5,5-tetramethylbenzidine (Khema, Russia); the blocker was a protein-salt solution; a 5% solution of sulfuric acid was used as a stop agent. The enzyme immunoassay was performed on polystyrene plates from Greiner bio-one, Germany. 1 μg of the SARS-CoV-2 antigen was applied to the substrate and adsorbed during the day. The results of enzyme immunoassay were recorded on a PR 2100 spectrophotometer manufactured by Sanofi Diagnostiks Pasteur (France) at two wavelengths of 450/620 nm and are presented in Table 6.

Study of the immune response (antibody titer) of a candidate vaccine based on the recombinant RBD/S21/S14-Fc protein and corpuscular adjuvant based on natural betulin. In the study, females of the BALB/c line (Stolbovaya Branch of the Federal State Budgetary Institution of Science and Technology of the National Center for Biomedical and Biotechnological Medicine of the Federal Medical and Biological Agency of Russia) were used. Animals were kept under standard conditions (18-22°C and relative air humidity 50-70%, NH ₃ =0.001 mg/m ³ , CO ₂ =0.1%, 12 h dark/12 h light), fed with granular full ration extruded food for mice, rats, and hamsters (OOO Laboratorkorm, Moscow) ad libitum in accordance with Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals used for scientific purposes and in accordance with the sanitary and epidemiological rules SP 2.2.1.3218-14 "Sanitary and epidemiological requirements for the arrangement, equipment and maintenance of experimental biological clinics (vivariums)" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of August 29, 2014 No. 51).

In table. Figure 7 shows preparations of a candidate vaccine based on the recombinant RBD/S21/S14-Fc protein with a corpuscular adjuvant for animal immunization and study design. Physiological saline (0.9% NaCl) was used as the placebo preparation.

BALB/c mice weighing 18-20 g were injected intraperitoneally with 0.5 ml syringes with a capacity of 1 ml, twice, on the 1st and 14th day, according to the design of the study. Before each filling of the syringe, the drug was stirred 5-6 times. The control group of mice of the same batch was injected with a solution of 0.9% sodium chloride. 28 days after the first immunization, mice were bled from the mandibular vein. The blood samples were kept at room temperature for 2 hours, centrifuged for 10 min at 5000 rpm, and the resulting whole serum was collected. Until the moment of use, the serum was stored at -20°C. Serum obtained from the blood of mice (individually from each animal) was monitored for the presence of antibodies by enzyme immunoassay (ELISA) and RMN. Statistical analysis of primary data was carried out in GraphPad Prism v9. The following statistical indicators were used to represent the data: geometric mean, standard deviation, arithmetic mean, error of the mean. To determine the significance of differences between group means, one-way ANOVA analysis of variance was used, followed by Tukey's test for post hoc pairwise comparisons of groups with each other. The group significance level was taken equal to α=0.05. Differences were considered significant at p<α.

Linked immunosorbent assay. The presence of specific class G immunoglobulins (IgG) was determined in mouse sera samples after double immunization. Sorption of the antigen diluted in sterile DPBS to a concentration of 1.0 μg/ml was carried out overnight at +4°C in 96-well plates Maxisorp (Nunc) (100 μl/well). The plates were then washed twice with 0.1% TWEEN20 (Serva) in PBS using an ELx50 automatic washer (BioTek) at 500 µl/well. After washing, the plates were incubated with blocking buffer (PBS-T containing 5% dry milk, TF Ditol) for 2 hours at room temperature and washed again. After that, a series of two-fold dilutions of sera was added to the plate, starting from a 1:400 dilution for the groups of animals that received vaccine preparations and from a 1:50 dilution for the placebo group. The plates with mouse sera were incubated for 1 h at room temperature, then washed three times. Horseradish peroxidase-labeled Goat anti mouse IgG (H+L) HRP (Abcam) diluted 1:7500, 100 µl/well, was used as secondary antibodies. After incubation with secondary antibodies, the plates were washed four times. A TMB solution (OOO Imtek, Russia) was used as a color substrate at a rate of 100 µl/well for 10 min at room temperature, then the reaction was stopped by adding IN H2SO4 (Vekton, Russia). Optical density (OD) at a wavelength of 450 nm was measured on a multiplate reader with LVF monochromators CLARIOstar (BMG LABTECH). -Fc in the sera of immunized animals using an indirect ELISA using an experimental test system. The results of determining the level of specific antibodies are presented in the table. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by pairwise comparison of groups using Tukey's test. The study showed the formation of a pronounced level of serum antibodies specific to the RBD/S21/S14-Fc protein after double immunization with vaccine preparations in both doses used. The results are presented in table.8.

Microneutralization reaction (MNT). To carry out the neutralization reaction, the sera of mice were diluted with sterile DPBS in a ratio of 1:4 and heated for 30 min at 56°C. Then dilutions of sera were prepared in MEM + 2% FBS medium in a volume of 50 μl, starting from 1:10 (in terms of whole serum). An equivalent volume of the SARS-CoV-2 virus (infecting dose of 25 TCID50) was added to the dilutions of sera and incubated for 1 hour at 37°C. The dilutions were then transferred to Vero cells in 96-well plates. The plates were incubated for 4 days at 37°C, after which they were checked for the presence of CPE. The neutralizing titer was considered the highest dilution of serum, at which there is no CPP. All studies with infectious coronavirus were carried out in the laboratory of immunology and prevention of viral infections of the Federal State Budgetary Scientific Institution "IEM" (Sanitary and epidemiological conclusion of Rospotrebnadzor for St. IV group of pathogenicity). The results of determining the level of neutralizing antibodies against the SARS-CoV-2 coronavirus in the blood sera of mice after a double immunization showed that virus-neutralizing antibodies in an amount significantly different from the placebo group are formed in the group of animals that received a candidate vaccine containing a recombinant protein as in a dose 5 mcg, and at a dose of 20 mcg. The level of virus-neutralizing antibodies did not significantly differ from that in both groups. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by pairwise comparison of groups using Tukey's test. The results are presented in table.8. The study showed that double immunization led to the formation of virus-neutralizing antibodies in an amount significantly different from the placebo group only in the group of animals vaccinated with a candidate vaccine containing a recombinant protein at a dose of 5 or 20 μg. Thus, it has been established that the vaccine containing a corpuscular adjuvant based on natural betulin has a high antigenic activity.

Study of the immunogenic properties (assessment of the cellular immune response) of a candidate vaccine based on the recombinant RBD/S21/S14-Fc protein and corpuscular adjuvant based on natural betulin. To assess the T-cell immune response, RBD1 was administered to C57/black SPF mice (n=5 per group) intraperitoneally at 5 or 20 μg, twice, on the 1st and 22nd day. Spleens were collected 28 days after the second immunization. The unvaccinated group (n=3) served as controls. The study design is presented in Table 9.

Spleen homogenates were filtered (pores 70 μm), washed with DPBS containing 2.5% FCS (Gibco), stripped red blood cells with RBC lysis buffer (BioLegend), washed with DPBS (2.5% FCS). Spleen cells were seeded in 96-well flat bottom plates at a density of 2x106 cells and stimulated with a pool of SARS-CoV-2 S protein peptide (PepTivator® SARS-CoV-2 Prot S) for 6 hours in the presence of anti-CD28 (BioLegend) and Brefeldin A (BD Biosciences). The cell phenotype was studied using flow cytometry and CD8-PE/Cy7, CD4-PerCP/Cy5.5, CD44-BV510, CD62L-APC/Cy7, IFNγ-BV780, TNFα-BV421, IL2-PE (BioLegend). Dead cells were detected using Zombie Red (BioLegend). True Stain containing anti-CD16/CD32 (BioLegend) was used to block non-specific binding. Cells were stained with Cytofix/Cytoperm (BD Biosciences) according to instructions. The relative ratio of CD8+ and CD4+ T-lymphocytes with the phenotype of effector memory cells (CD44+CD62L-) was determined. A significant increase in S-protein specific CD4+ and CD8+Tem cells was measured after two immunizations of 20 µg, while immunization with 5 µg of antigen resulted in a statistically significant increase in CD4+Tem alone (p<0.05). There was a trend towards dose-dependent growth of cells producing Th1-cytokine (IFNγ, IL-2, TNFα) among both Tem populations. The effect reached significance for Tem producing IL-2 (IFNγ-IL-2+TNFα-) and for multifunctional CD8+ T cells (IFNγ+IL-2+TNFα+) in both dosing groups. In the high dose group, the increase in IL-2 produced by CD8+Tem was also significant.

Study of the immunogenic properties (assessment of the cellular immune response) of a candidate vaccine based on the recombinant RBD/S21/S14-Fc protein and corpuscular adjuvant based on natural betulin on volunteers.

In order to strictly control the occurrence of adverse events and any post-vaccination complications, the volunteers were admitted to the hospital twice for 3 full days (4 days) for each of the two vaccine/placebo administrations. Volunteers were admitted to the hospital one day before the introduction of the vaccine. Two days after the first and second vaccine/placebo injections, in the absence of adverse events (AEs) and serious adverse events (SAEs), the volunteers were discharged from the hospital. Further follow-up was carried out on an outpatient basis. The study was carried out in two stages.

The first phase of the first phase study. The first phase of the clinical trial is an open randomized study of the safety, tolerability (reactogenicity) and immunogenicity of the vaccine when administered twice intramuscularly at doses of 20 μg + 5 μg and 20 μg + 20 μg. Taking into account the estimated number of subjects found by screening to be ineligible and/or eligible for non-inclusion, the study was conducted on 20 healthy volunteers in parallel groups aged 18 to 60 years. Group 1 (10 people) - received the study vaccine preparation according to the following scheme: the first injection of 20 μg - 0.5 ml of suspension for intramuscular injection (40 μg / ml), the second injection of 5 μg - 0.5 ml of suspension for intramuscular injection (10 mcg / ml), after 28 days (hereinafter referred to as "double immunization according to the scheme 20 mcg + 5 mcg"). Group 2 (10 people) - received the study drug according to the following scheme: the first and second injections of 20 μg each - 0.5 ml of a solution for intramuscular injection (40 μg / ml), after 28 days (hereinafter referred to as “double immunization according to scheme 20 mcg+20 mcg"). Volunteers were admitted to the hospital one day before each of the two vaccine injections. The length of stay in the hospital was two periods of 4 days each (4 Visits).

The second stage of the study of the first phase. The second stage of the clinical study is a double-blind, randomized, parallel group clinical study of the safety, tolerability and immunogenicity of the vaccine when administered twice intramuscularly at doses of 20 μg + 5 μg and 20 μg + 20 μg. Taking into account the estimated number of subjects found by the results of screening not meeting the inclusion criteria and / or meeting the exclusion criteria, the study was conducted in 96 healthy volunteers aged 18 to 60 years. Group 3 (32 people) - received the study drug according to the following scheme: the first injection of 20 μg - 0.5 ml of suspension for intramuscular injection (40 μg / ml), the second injection of 5 μg - 0.5 ml of suspension for intramuscular injection (10 μg /ml), intramuscularly, after 28 days; Group 4 (32 people) - received the study drug according to the following scheme: the first and second injections of 20 μg each - 0.5 ml of a solution for intramuscular injection (40 μg / ml), intramuscularly, after 28 days; Group 5 (32 people) received a placebo according to the following scheme: the first and second injections - 0.5 ml of sodium chloride, 0.9% injection solution, intramuscularly, after 28 days.

96 volunteers (Groups 3, 4 and 5) were hospitalized 1 day prior to the first administration of study drug/placebo. The duration of stay in the hospital was 4 days (3 days). All volunteers in Groups 3, 4 and 5 were followed up by the investigator for 6 months (180±5 days) after the first administration of the study drug/placebo in order to identify possible late adverse reactions, assess immunogenicity. A final safety and tolerability assessment was performed at Visit 20 (180±5 days from the first administration of study drug/placebo).

Data analysis showed that for all the studied systems, AEs in the analyzed groups did not significantly differ from each other. One volunteer with randomization number R004 (from G1) at Visit 4 was diagnosed with COVID-19 coronavirus infection based on confirmation of the presence of SARS-CoV-2 virus RNA by GAD and clinical manifestations (fever, cough), which was the criterion for exclusion from the study . This AE was mild and ongoing at the time of the interim data analysis. Evaluation of the differences between G1 and G2 using the Fisher exact test showed no statistical significance (p=1.000) for the criterion "Proportion of volunteers with symptoms of COVID-19 and confirmed infection with SARS-CoV-2 infection by the CFS method." Comparative characteristics of primary and secondary criteria for safety and tolerability showed no statistically significant differences between the study groups.

In addition to the safety of the candidate vaccine, the volunteers in Groups 1-4 were found to have specific antibodies to the SARS-CoV-2 virus in their blood. The results of the determination of specific antibodies in vaccinated volunteers are shown in table.10.

The results of the determination of specific antibodies after immunization of volunteers with two doses of the Betuvax-CoV-2 vaccine showed that when the vaccine was administered intramuscularly with an interval of 28 days and the test regimens (20 μg + 5 μg and 20 μg + 20 μg) were used, the majority of volunteers observed the synthesis of specific antibodies in high titer.

SEQUENCE LIST

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<110> Betuvaks LLC

Krasilnikov, Igor V

Isaev, Artur A

Kudriavtsev, Aleksandr V

Frolova, Maria E

Vakhrusheva, Anna V

Ivanov, Aleksandr V

Djonovic, Milana

Ivanishin, Taras V

Askretkov, Aleksandr D

Voronina, Yekaterina V

Zyryanov, Dmitry A

Serogin, Yuriy A

Stukova, Marina A

Romanovskaya-Romanko, Yekaterina A

Smirnov, Ivan V

Mokrushina, Yuliana A

Kryuchkov, Nikolay A

Blagodatskikh, Konstantin A

<120> Gene composition for expression of a recombinant antigen,

cell line producing recombinant antigen, method

obtaining a recombinant antigen, a vaccine against coronavirus

infection, how to get it

<130> KF

<160> 14

<170>PatentIn version 3.5

<210> 1

<211> 479

<212> PRT

<213> Artificial Sequence

<220>

<223> Recombinant fusion protein

<400> 1

Ala Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

1 5 10 15

Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn

20 25 30

Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr

35 40 45

Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe

50 55 60

Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg

65 70 75 80

Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys

85 90 95

Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn

100 105 110

Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe

115 120 125

Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile

130 135 140

Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys

145 150 155 160

Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly

165 170 175

Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala

180 185 190

Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gln Ile Leu Pro Asp

195 200 205

Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn

210 215 220

Lys Val Thr Leu Ala Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln

225 230 235 240

Gln Phe Gly Arg Asp Ile Ala Glu Pro Lys Ser Cys Asp Lys Thr His

245 250 255

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 2

<211> 1500

<212> DNA

<213> Artificial Sequence

<220>

<223> Hybrid RBD-Fc codon-optimized sequence

<400> 2

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60

gcacccaata tcaccaatct gtgccccttc ggcgaggtgt tcaatgccac cagattcgcc 120

tctgtgtacg cctggaaccg gaagcggatc agcaattgcg tggccgacta ctccgtgctg 180

tacaactccg ccagcttcag caccttcaag tgctacggcg tgtcccctac caagctgaac 240

gacctgtgct tcacaaacgt gtacgccgac agcttcgtga tccggggaga tgaagtgcgg 300

cagattgccc ctggacagac aggcaagatc gccgactaca actacaagct gcccgacgac 360

ttcaccggct gtgtgattgc ctggaacagc aacaacctgg actccaaagt cggcggcaac 420

tacaattacc tgtaccggct gttccggaag tccaatctga agcccttcga gcgggacatc 480

tccaccgaga tctatcaggc cggcagcacc ccttgtaacg gcgtggaagg cttcaactgc 540

tacttcccac tgcagtccta cggctttcag cccacaaatg gcgtgggcta tcagccctac 600

agagtggtgg tgctgagctt cgaactgctg catgcccctg ccacagtgtg cggccctaag 660

aaaagcaccc agattctgcc cgatcctagc aagcccagca agcggagctt catcgaggac 720

ctgctgttca acaaagtgac actggccaca gagagcaaca agaagttcct gccattccag 780

cagtttggcc gggatatcgc cgagcccaaa tcttgtgaca aaactcacac atgcccaccg 840

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1200

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1260

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1320

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1380

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1440

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1500

<210> 3

<211> 60

<212> DNA

<213> Homo sapiens

<400> 3

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60

<210> 4

<211> 9290

<212> DNA

<213> Artificial Sequence

<220>

<223> Plasmid DNA

<400> 4

ctagagtcgg ggcggccggc cgctcgctga tcagcctcga ctgtgccttc tagttgccag 60

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 120

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 180

ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 240

gctggggatt aattaagatc gcatgcttct atattatttt ctaaaagatt taaagttttg 300

ccttctccat ttagacttat aattcactgg aatttttttg tgtgtatggt atgacatatg 360

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 420

tcgcatgctt ctatattatt ttctaaaaga tttaaagttt tgccttctcc atttagactt 480

ataattcact ggaatttttt tgtgtgtatg gtatgacata tgggttccct tttatttttt 540

acatataaat atatttccct gtttttctaa aaaagaaaaa gatccgtacg catttaattc 600

taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg 660

ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt 720

aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta 780

gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta 840

gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc 900

ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga 960

aggggtggggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg 1020

tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc 1080

tcttcctcat ctccgggcct ttctcgagct caggcttggg gggggggaca gctcaggggct 1140

gcgatttcgc gccaaacttg acggcaatcc tagcgtgaag gctggtagga ttttatcccc 1200

gctgccatca tggttcgacc gctgaactgc atcgtcgccg tgtcccagaa tatgggcatc 1260

ggcaagaacg gagattttcc ctggccaatg ctcaggaacg aattcaagta cttccaaaga 1320

1380

tggttctcca ttcctgagaa gaatcgacct ttaaaggaca gaattaatat agttctcagt 1440

agagagctca aggaaccacc acaaggagct cattttcttg ccaaaagttt ggatgatgcc 1500

1560

ggcagttctg tttaccagga agccatgaat caaccaggcc acctcagact ctttgtgaca 1620

aggatcatgc aggaatttga aagtgacacg tttttcccag aaattgattt ggggaaatat 1680

aaacttctcc cagaataccc aggcgtcctc tctgaggtcc aggaggaaaa aggcatcaag 1740

tataagtttg aagtctacga gaagaaagac taacaggaag atgctttcaa gttctctgct 1800

cccctcctaa agctatgcat ttttataaga cccatgggact tttgctggct ttagatcgta 1860

caagtaaagc ggccgcgacg ttcgatcata atcagccata ccacatttgt agaggtttta 1920

cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 1980

gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 2040

aatttcacaa ataaagcatt ttttcactg cattctagtt gtggtttgtc caaactcatc 2100

aatgtatctt aaggcgtaaa ttgtaagcgt taataggatc cgtcgatcga ccgatgccct 2160

tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg 2220

cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc 2280

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2340

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 2400

2460

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2520

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2580

ccctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 2640

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 2700

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2760

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 2820

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2880

tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2940

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3000

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3060

3120

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3180

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3240

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3300

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagaa 3360

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 3420

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 3480

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 3540

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 3600

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 3660

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 3720

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 3780

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3840

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3900

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 3960

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 4020

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4080

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4140

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4200

ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 4260

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 4320

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 4380

cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 4440

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 4500

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 4560

gatttataag ggattttgcc gattcggcc tattggttaa aaaatgagct gatttaacaa 4620

aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg 4680

ctgcgcaact gttgggaagg gcgatcggtc gacgatccac tagttctagc tagaatggtg 4740

gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt 4800

ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc 4860

agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg 4920

cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc 4980

gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga 5040

5100

gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg 5160

cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttctc 5220

gagttgagac gaccttccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 5280

gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 5340

gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 5400

gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 5460

tacgccgagt ggtcgggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 5520

accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 5580

tgcgtgcact tcgtggccga ggagcaggac tgagcggccg caataaaata tctttatttt 5640

cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac gctctccatc 5700

aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca agtgcaggtg 5760

ccagaacatt tctctatcga taacgcgtct catcacggtc acgcatggct gcgcccgcgc 5820

agcgcccccc gacccccccc cgatccccgg aacgccagcc gccatccacc gcccgcagcc 5880

cccggagccc gcggacccg accccccgcc gctcgcccgc cgttgcgccg ctctctgcgg 5940

gggggctagg gggccgcggg gggaaggcca cgccccctcc actttttcca ggaatgcgcg 6000

gcatgacccg ccccccccat gacccgccaa cacccccccg atgccccccg ggggtctttc 6060

ctggggggcc tttgtcaggg ttgcctgtgt cacccacgggg ccccttggcc acgcttgcct 6120

gggtcacccc ggagccccgg gtcacgtttg ccggggtcag ccctggggcg cttcttcacg 6180

ctccattgcc ggtacctagt tattaatagt aatcaattac ggggtcatta gttcatagcc 6240

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 6300

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 6360

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 6420

6480

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 6540

tagtcatcgc tattaccatg ggtgaggctc cggtgcccgt cagtgggcag agcgcacatc 6600

6660

gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg 6720

tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt 6780

tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg 6840

ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc 6900

cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc 6960

gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg 7020

gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg 7080

acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca 7140

cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac 7200

atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca 7260

agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc 7320

ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc 7380

tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc 7440

cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta 7500

ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg 7560

ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt 7620

taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc 7680

ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc 7740

gtgaggaatt agcttggtac taatacgact cacaagctgc cgccaccatg tacaggatgc 7800

aactcctgtc ttgcattgca ctaagtcttg cacttgtcac gaattccgca cccaatatca 7860

ccaatctgtg ccccttcggc gaggtgttca atgccaccag attcgcctct gtgtacgcct 7920

ggaaccggaa gcggatcagc aattgcgtgg ccgactactc cgtgctgtac aactccgcca 7980

gcttcagcac cttcaagtgc tacggcgtgt cccctaccaa gctgaacgac ctgtgcttca 8040

caaacgtgta cgccgacagc ttcgtgatcc ggggagatga agtgcggcag attgcccctg 8100

gacagacagg caagatcgcc gactacaact acaagctgcc cgacgacttc accggctgtg 8160

tgattgcctg gaacagcaac aacctggact ccaaagtcgg cggcaactac aattacctgt 8220

accggctgtt ccggaagtcc aatctgaagc ccttcgagcg ggacatctcc accgagatct 8280

atcaggccgg cagcacccct tgtaacggcg tggaaggctt caactgctac ttcccactgc 8340

agtcctacgg ctttcagccc acaaatggcg tgggctatca gccctacaga gtggtggtgc 8400

tgagcttcga actgctgcat gcccctgcca cagtgtgcgg ccctaagaaa agcacccaga 8460

ttctgcccga tcctagcaag cccagcaagc ggagcttcat cgaggacctg ctgttcaaca 8520

8580

atatcgccga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg 8640

aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 8700

tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 8760

tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 8820

aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 8880

ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 8940

agaaaaccat ctccaaagcc aaagggcagc ccgagaacc acaggtgtac accctgcccc 9000

catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 9060

atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 9120

ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 9180

acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 9240

acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgataa 9290

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 5

gccgccacca tgtacaggat gcaactcc 28

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 6

ttatcattta cccggagaca g 21

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7

ctttttgagt ttggatcttg gt 22

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8

caaacaacag atggctgg 18

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 9

ctttttgagt ttggatcttg gt 22

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 10

acttcatctc cccggatcac 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 11

acttcatctc cccggatcac 20

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 12

aattgcgtgg ccgactactc c 21

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 13

caaacaacag atggctgg 18

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 14

cagcacgtac cgtgtggtca gc 22

<---

Claims (7)

1. A hybrid gene encoding a recombinant antigen consisting of the SARS-CoV-2 coronavirus surface protein S receptor RBD, S14P5 and S21P2 epitopes, an IgG1 immunoglobulin Fc fragment, represented as the amino acid sequence of SEQ ID NO: 1. 2. Expression construct in the P-Pharma-Zeo vector represented by SEQ ID NO: 4, containing the nucleotide sequence of SEQ ID NO: 2, consisting of the hybrid gene according to claim 1 and the sequence of SEQ ID NO: 3 encoding the leader peptide of IL2. 3. A CHO-HP cell line transfected with the expression construct of claim 2 capable of producing the recombinant RBD/S21/S14-FC antigen. 4. A method for obtaining a recombinant RBD/S21/S14-Fc antigen, involving the cultivation of the CHO-HP cell line according to claim 3, under conditions of feeding 4 mM L-alanine-L-glutamine and a reagent against cell aggregation, followed by the selection of the culture liquid, its clarification, filtration, chromatographic purification of the target recombinant antigen, including affinity chromatography, HA sorbent chromatography, hydrophobic and gel permeation chromatography followed by sterilizing filtration. 5. Recombinant antigen RBD/S21/S14-Fc, obtained by the method according to p. 4, stimulating the immune response in the body to coronavirus infection. 6. Vaccine composition for the prevention of coronavirus infection, containing a recombinant antigen according to claim 5 and a corpuscular adjuvant based on natural betulin in a buffer solution. 7. A method for the prevention of coronavirus infection, which involves immunization of those in need of protection against coronavirus infection with a vaccine composition according to claim 6.

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