Hybrid genes for producing recombinant RBD antigens

Abstract

The invention relates to biotechnology, immunology and virology, in particular to a vaccine for preventing coronavirus infection and a preparation method of the vaccine for preventing the coronavirus infection, a fusion gene for coding a recombinant RBD / S21 / S14-Fc antigen is obtained, and a recombinant antigen capable of inducing specific humoral immune response and cellular immune response aiming at epitopes required by SARS-CoV-2 virus envelope protein is obtained. The technical result of the present invention is the development of a recombinant antigen vaccine composition comprising betulin as an adjuvant. The technical result is achieved by a method of creating a gene, a gene construct, producing a cell line (production strain) of a target RBD / S21 / S14-Fc recombinant SARS-CoV-2 antigen fused to an immunoglobulin Fc fragment via a linker, purifying a recombinant protein, obtaining a vaccine composition comprising a recombinant antigen and a particle adjuvant, and inducing an immune response against coronavirus.

Description

Field of the Invention

[0001] The present invention relates to biotechnology, immunology and virology, and to the research and development of a vaccine against coronavirus infection, a method for producing the vaccine, the construction of a fusion gene, and an expression construct of a recombinant RBD / S21 / S14-Fc antigen, which is a region of the SARS-CoV-2 S protein, including a portion of RBD, SD1 and S2, fused to the Fc fragment of IgG1, a cell line for producing the recombinant antigen, a method for obtaining the recombinant antigen, and the antigen is combined with a betulin-based particulate adjuvant, and can induce long-term humoral and cellular immunity against coronavirus in animals and humans. Background Art

[0002] Vaccination is currently recognized as one of the most effective ways to fight infection. Therefore, many countries and pharmaceutical companies have been involved in the development of vaccines and drugs against SARS-CoV-2, a new RNA virus belonging to the Beta-CoV lineage of the Coronaviridae family.

[0003] Using traditional vaccine development techniques that rely on the use of full-sized pathogens, the complexity of production increases dramatically due to safety issues and the need to strictly control the completeness of pathogen inactivation, which makes this option less economically viable. In this regard, vaccine development techniques based on viral subunits have been well demonstrated in recent years and offer certain advantages, such as safety and the possibility of vaccine boosting. However, some viral subunits have low immunogenicity and therefore require the use of adjuvants.

[0004] In general, vaccines based on whole inactivated viruses are widely used for immunization, but they have serious disadvantages: from technical difficulties in obtaining large quantities of drugs in a short period of time, to problems with effectiveness, such as adverse side effects, and whether the antibodies produced can target the necessary epitopes of the viral envelope protein, as well as problems related to protein impurities remaining in the vaccine after virus cultivation.

[0005] Nucleic acid-based vaccines work by delivering genetic information into cells, which use this information to produce pathogen proteins to trigger an immune response. If delivered using viral vectors, the foreign DNA may integrate into the genome of the vaccinee's cells, potentially producing an autoimmune response to cells expressing viral antigens, or a response to the foreign DNA. In addition to these disadvantages, there are also difficulties in the delivery of such drugs. Notably, the use of vaccines based on RNA or DNA, as well as vaccines based on adenovirus vectors, has raised some concerns because their long-term effects are unclear.

[0006] Subunit vaccines are mainly recombinant vaccines based on purified proteins. An important part in the development of such vaccines is the search for new effective adjuvants. The use of such adjuvants can reduce the dose of antigens, reduce the cost of finished products, and improve the immunogenicity of "weak" or small antigens, which poses a major challenge to immune research and development and vaccine manufacturing. The use of adjuvants can increase the speed and duration of the development of vaccine immune responses, induce protective properties of mucosa, and enhance the intensity of immune responses. At present, it is recognized that effective and safe subunit or epitope vaccines cannot be made without the use of adjuvants.

[0007] The RBD (Receptor-binding domain) of the S protein, as well as its various epitopes, are involved in the process of coronavirus entry into host cells. Experimental studies have shown that most antibodies with virus neutralizing activity against a certain class of coronaviruses (e.g., MERS or SARS) specifically bind to various RBD epitopes. RBD forms the ACE2 receptor binding site, so antibodies that bind to the RBD domain can block contact with the receptor, thereby preventing the virus from entering the cell, which has been confirmed in the case of SARS-CoV-2. Based on this, the RBD sequence was selected as the sequence for antigen creation. It is worth noting that in addition to the production of neutralizing antibodies, the selection of the entire S protein may also lead to the production of masking antibodies, thereby increasing the enhancement of antibody-dependent infection.

[0008] The S1 domain is composed of the NTD subdomain (N-terminal domain), the RBD subdomain (amino acid residues 319-527), and the SD1 and SD2 subdomains adjacent to the S2 domain. Therefore, the most important goal of vaccination is not only to produce antibodies to the S protein, but also to produce neutralizing antibodies to the RBD. However, it is impossible to use the RBD itself as a vaccine antigen because the molecular weight of this protein is very small (less than 40kDa) and it is quickly excreted from the body, so the RBD and a part of SD1 and S2 that bind to the Fc fragment of IgG1 become candidate antigens.

[0009] Adding adjuvants can reduce the protein content of each dose of vaccine, reduce vaccine costs, and improve the immune response to antigens. At present, the adjuvants used in experiments or clinical studies are as follows: minerals (aluminum hydroxide or phosphate), plant sources (saponin-QuilA, QS21), microorganisms (inactivated bacteria, lipopolysaccharides and their derivatives, CpG motifs of DNA) and other types of adjuvants. Due to the toxicity or lack of effectiveness of most adjuvants, only aluminum salts and water-oil emulsions MF-59, AS03, QS21, VLP (virus-like particles) and ISCOMs (Immunostimulating complexes) are generally allowed to be used. The most promising is the particulate adjuvant-spherical amorphous nanoparticles (SANP). One of the reasons for using this adjuvant is that it can be effectively absorbed by the antigen-presenting cells of the immune system, thereby enhancing the immune response. This type of adjuvant includes betulin adjuvant based on triterpenoids in birch bark extract (patent RU2545714). In addition to significant biological activities (antibacterial, antifungal, antiviral, hepatoprotective, anti-inflammatory, anticancer, antiallergic, membrane stabilizing, immunomodulatory and antioxidant), betulin granules also possess adjuvant properties.

[0010] From patent RU 2749193, variants of antigen use are known, including RBD as an antigenic component to induce specific immunity against SARS-CoV-2, and betulin spherical amorphous 80-160 nm nanoparticles (SANPs) as an adjuvant. This adjuvant is produced on a commercial scale in accordance with the requirements of pilot production regulations and was tested earlier in non-clinical studies of influenza vaccines, which showed that the inclusion of particulate adjuvants in the vaccine leads to an enhanced immune response and does not affect the safety of the drug. Summary of the invention

[0011] A significant drawback of known vaccines against SARS-CoV-2 is the complexity of their production, insufficient immunogenic activity, adverse side effects, and problems associated with the production of antibodies targeting the desired epitopes of the viral envelope protein. The technical purpose of the present invention is to prepare an antigenic composition capable of inducing specific humoral and cellular immune responses to the desired epitopes of the SARS-CoV-2 viral envelope protein. The technical result of the present invention is the preparation of an antigenic composition in which betulin particles are used as adjuvants and recombinant proteins are used as active components. The technical result is achieved by creating a fusion RBD / S21 / S14-Fc gene encoding a recombinant SARS-CoV-2 antigen, which is fused to the Fc fragment of an immunoglobulin via a linker; a cell line that produces the recombinant antigen and secretes it into the culture medium; isolation and purification of the recombinant RBD / S21 / S14-Fc antigen; formulating a vaccine using a particle adjuvant based on natural betulin; and immunizing animals and humans with the vaccine to obtain an immune response to the coronavirus.

[0012] Description of the drawings

[0013] Figure 1 P-Pharma-Zeo-RBD / S21 / S14-Fc vector map.

[0014] Figure 2 Sequencing reaction peak diagram of RBD-Rev 1 primer.

[0015] Figure 3 Sequencing reaction peak diagram of RBD-short primer.

[0016] Figure 4 Sequencing reaction peak diagram of RBD-short primer (continued).

[0017] Figure 5 Sequencing reaction peaks of SolR primers.

[0018] Figure 6 Sequencing reaction peak diagram of SolR primer (continued).

[0019] Figure 7 Sequencing reaction peak diagram of RBD-Fwd3 primer.

[0020] Figure 8 Western blot was used to evaluate the expression and secretion of RBD / S21 / S14-Fc protein (lane 1 - cell lysate; lane 2 - culture medium. The left lane is a molecular weight standard).

[0021] Fig. 9 The RBD / S21 / S14-Fc sequence was overlaid with a tryptic peptide.

[0022] Fig.10 Results of gel filtration chromatography using a Superdex 200 Increase column in sodium phosphate buffer, pH 7.4.

[0023] Embodiments of the present invention

[0024] RBD / S21 / S14-Fc fusion gene design. In the first phase of this study, the fusion gene RBD / S21 / S14-Fc was developed for the expression and production of the antigen, a recombinant SARS-CoV-2 protein. The recombinant sequence of the RBD-based antibody with additional epitopes was fused to wild-type FcIgG1 (unmodified) to obtain RBD / S21 / S14-Fc. The purpose of modifying the RBD was to improve the immunogenicity of the antigen. Two peptide epitopes were added. The epitope S14P5 is located in the SD1 subdomain in the posterior region of the RBD, and it seems that antibodies targeting this region sterically prevent binding to the receptor. The S21P2 epitope is located in the S2 region, overlapping with the fusion polypeptide (FP), and antibodies targeting this region prevent the virus from fusing with the target cell. The order of the epitopes was changed to increase the stability of the protein. Its amino acid sequence is shown in SEQ ID NO:1. The estimated molecular weight is 107.2 kDa (dimer) and 53.6 kDa (reduced monomer).

[0025] Obtain expression construct and prepare it for transfection.In the first step, obtain optimized nucleotide sequence (SEQ ID NO:2).Use GeneArt GeneOptimizer software to optimize the nucleotide sequence of fusion gene to express in CHO-HP cell culture.The first 60 nucleotides of this sequence encode IL-2 leader peptide (SEQ ID NO:3).

[0026] The map of RBD / S21 / S14-Fc plasmid is as follows Figure 1 Table 1 shows the main characteristic coding sequence of the RBD / S21 / S14-Fc plasmid. The complete nucleotide sequence of the vector is shown in SEQ ID NO:4.

[0027]

[0028]

[0029]

[0030] The RBD / S21 / S14-Fc fusion gene was amplified in a PCR reaction using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 and a DNA polymerase with 3′-5′ exonuclease proofreading activity. The reaction product was purified using a spin column according to the DNA purification protocol.

[0031] The P-Pharma-Zeo vector was linearized using HindIII and XbaI restriction endonucleases, purified by agarose gel electrophoresis, and separated from the gel using a centrifugal column according to the DNA purification protocol. The vector ends were blunted using a DNA polymerase with 3′-5′ exonuclease activity, and the vector was purified using a centrifugal column according to the DNA purification protocol. T4-DNA polymerase was used for ligation under the conditions of insert / vector ratio = 1 / 10 and 5% PEG-4000 was added to the ligation reaction. The resulting plasmid DNA was purified using a centrifugal column according to the DNA purification protocol. The ligase mixture was transformed into chemically competent Escherichia coli cells (E. coli) (10beta strain), and after transformation, the cells were inoculated into LB-agar culture dishes containing 100 μg / mL ampicillin. The cells were cultured at 37°C for 18 hours until single colonies (bacterial clones) appeared.

[0032] Next, the selected bacterial clones were checked for the presence and orientation of the insert using primers SEQ ID NO: 7 and SEQ ID NO: 8 (to detect the presence of the gene in the plasmid) and primers SEQ ID NO: 9 and SEQ ID NO: 10 (to detect the correct orientation of the gene). The volumes of reagents used for the PCR reactions are shown in Table 2.

[0033]

[0034]

[0035] After the PCR reaction, the reaction products were analyzed by agarose gel electrophoresis (1% agarose). The bacterial clone (PCR product size of about 1700 bp, PCR product size of about 400 bp in the correct direction) was transferred to 50 mL LB medium containing 100 μg / mL ampicillin and cultured with stirring for 24 hours. The obtained suspension was centrifuged and the supernatant was discarded without affecting the cell pellet. Plasmid DNA was isolated from the cell pellet using a spin column according to the DNA purification protocol.

[0036] Plasmid DNA of selected colonies was isolated from bacterial culture using the Plasmid Midiprep 2.0 Kit according to the manufacturer's instructions. 50 mL of culture was centrifuged at 3000 g and 4 ± 2 °C for 20 min. The supernatant was completely discarded, and 5 mL of resuspension solution containing RNase A was added to the pellet. The mixture was thoroughly stirred using a vortex until the pellet was completely resuspended, and then 5 mL of lysis solution was added. The tube was gently inverted 10 times. Then 5 mL of neutralization solution precooled to 4 ± 2 °C was added and the contents of the test tube were mixed by inversion. The lysate was pelleted by centrifugation at 3000 g for 45 min using an Allegra 25R centrifuge. Before applying the lysate, 1.0 mL of endotoxin removal solution was applied to the spin column membrane to completely wet the entire surface of the resin. The spin column was incubated for 1 min, and then the entire volume of bacterial lysate was applied to the membrane. The spin column was centrifuged at 3000 g for 2 min. Next, 1 mL of endotoxin removal solution was applied to the membrane and the spin column was centrifuged at 3000g for 1 minute. An ethanol wash (15 mL) was added and the spin column was centrifuged at 3000g for 1 minute, repeating the wash step with the same volume of wash solution. After the second wash, the spin column was centrifuged at 3000g for 5 minutes to remove any residual liquid. The spin column was placed in a new 50 mL tube and opened in the clean bench for 5 minutes to remove any ethanol residue. Subsequently, 4 mL of eluate preheated to 50 ± 2 ° C in a TW-2 water bath was applied to the membrane and the spin column was incubated at room temperature for 2 minutes. The spin column was centrifuged at 3000g for 2 minutes. The eluate was concentrated to 500 μL at 3000g using a VivaSpin6 centrifugal concentrator (molecular weight cutoff of 10 kDa).

[0037] 50 μL of 3M sodium acetate was added to 500 μL of plasmid DNA solution. Ethanol precooled to -20°C was added to the mixture (1400 μL), the mixture was stirred, and incubated at -20 ± 2°C for 20 minutes. At 4 ± 2°C, the resulting solution was centrifuged at 14000g for 20 minutes, the supernatant was completely discarded, and 500 μL of 70% ethanol was carefully added to the plasmid DNA precipitate. The sample was centrifuged at 14000rpm for 10 minutes, and the supernatant was discarded. The precipitate was dried in a laminar flow of sterile air for 10 minutes and dissolved in 20 μL of sterile water without DNase and RNase.

[0038] 1 μL was taken to measure the DNA concentration and determine the RBD / S21 / S14-Fc gene sequence. To sequence the embedded gene using the Sanger method, primers SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 were used.

[0039] Sequencing reaction peak diagram Figure 2-7 The results of the final sequencing using primers covering the entire sequence of the RBD / S21 / S14-Fc gene confirmed the consistency of the RBD / S21 / S14-Fc fusion gene sequence with the reference sequence.

[0040] Transfect and select transfectants. Create production strains.

[0041] To transfect CHO cells, a pure (transfection grade) DNA solution of P-Pharma-Zeo-RBD / S21 / S14-Fc plasmid was obtained at a concentration of 8.6 mg / mL. After transfection with the expression vector P-Pharma-Zeo-RBD / S21 / S14-Fc, a cell pool was obtained with a cell viability of 92% 48 hours after transfection. A control transfection was performed with a vector containing the green fluorescent protein (GFP) gene, with a transfection efficiency of 50±20%. After 72 hours, selection of transfected strains was started by adding 400 μg / mL bleomycin and 500nM MTX for cultivation. Under more than 10 days of selection pressure, cell viability reached a minimum of 55±5%. On the 16th day, the viability of cells in the cell pool recovered to 90±5%, after which some of the cells in the cell pool were frozen and diluted to a maximum density of 0.5 cells per well. A total of 100 plates were used. From the 96-well plates used in the cloning stage, 105 clones were selected using the limiting dilution method and transferred to 24-well plates. All 77 clones grown in 24-well plates were transferred to 6-well plates, and the content of anti-cell agglomeration agent B was increased to 1.4% to destroy cell aggregation. After expansion, 10 subclones with the best single cell yield (PCD) ≥ 20pg / cell, viable cell content ≥ 900,000 / mL, and protein content in the culture medium ≥ 30μg / mL were selected to adapt to culturing in flasks during stirring. The subclones were passaged twice (4 days per passage), and after the second passage, the cells were frozen with a cell content of 10±1.5 million / freezing tube. Table 3 lists the productivity data of the clones in the second passage calculated from the start of 96-well plate inoculation.

[0042] Based on the CHO-HP cell line transfected with the P-Pharma-Zeo-RBD / S21 / S14-Fc vector, 4 primary clones were obtained, which produced RBD / S21 / S14-Fc fusion protein at a rate of at least 17±2pg / (cell*day). The clone with the highest productivity was selected as the production strain (Table 3, clone 10).

[0043]

[0044] Therefore, a CHO-HP cell line obtained from the CHO-K1 cell line (Cricetulus griseus species) was generated by adapting to suspension culture, containing the gene for the fusion protein RBD / S21 / S14 of the SARS-CoV-2 S protein domain coupled to the Fc fragment of human IgG1 as part of the P-Pharma-Zeo vector. Thus, RBD / S21 / S14-Fc was assembled and tested, and a RBD / S21 / S14-Fc protein production strain was created.

[0045] Analysis of secretions and confirmation of protein identity. Figure 8 Shown are the results of estimating RBD / S21 / S14-Fc expression by Western immunoblotting.

[0046] For mass spectrometric identification of proteins, Coomassie-stained bands were cut after SDS-PAGE and trypsin digested in gel. Tryptic peptides were separated by nano-electrospray (Thermo Fisher Scientific) using an Ultimate 3000Nano LC chromatography system (Thermo Fisher Scientific) and a QExactive Plus mass spectrometer (Thermo Fisher Scientific). MS / MS data analysis was performed using PEAKS Studio 8 software. Primary peptide structures generated by PEAKS Studio were analyzed using a database of relevant protein sequences. The following settings were used for analysis: Met oxidation, Asn / Gln deamidation, and Cys carbamidomethylation. Fig. 9 Coverage of the RBD / S21 / S14-Fc sequence by tryptic peptides is shown. Fig.10 Shown are the results of gel filtration chromatography using a Superdex 200 Increase column in sodium phosphate buffer at pH 7.4.

[0047] A method for obtaining a recombinant antigen from a production strain cell. The established method for obtaining a recombinant antigen comprises the following steps: 1) culturing a strain producing a recombinant SARS-CoV-2 RBD protein; 2) collecting and clarifying the filtered culture fluid; 3) chromatographically purifying the recombinant SARS-CoV-2 RBD protein; 4) confirming the identity of the obtained recombinant SARS-CoV-2 RBD protein.

[0048] Culture. 7A master cell bank (MasterCell Bank) tube of cells of the strain producing the recombinant RBD / S21 / S14-Fc antigen was thawed in a 37°C water bath for 10 minutes, and then 4 mL of ActiPro medium (Hyclone, USA) containing 4 mM L-alanine-L-glutamine (Gibco, USA) and 0.4% anti-cell aggregation agent was added, stirred, and centrifuged at 200g for 5 minutes, the supernatant was discarded, and 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% anti-cell aggregation agent, and cultured in an incubator at 37°C and at least 70% humidity for one day. When cultured in a bioreactor, the reactor was inoculated with the amount required for initial inoculation, that is, the cell density was 0.5×10 6 cells / mL, that is, at least 400×10 6 Cells were re-inoculated into the increased volume flask. The cell culture was transferred to a 125 mL flask containing 20 mL of medium and cultured for 4 days in a CO2 incubator shaker at a platform speed of 120 ± 5 rpm, a temperature of 37 ± 1 °C, a CO2 level of 5 ± 1%, and a relative humidity of 70 ± 5%. The entire culture was transferred to a 500 mL flask containing 125 mL of medium and cultured under the same conditions for 3 days. The required volume of cells was transferred to a BioFlo 320 bioreactor (Eppendorf, Germany) with a working volume of 1 liter to a cell density of 0.5 × 10 6 Cells / mL, the final culture volume is 800mL. In a BioFlo 320 bioreactor (Eppendorf, Germany) with a working volume of 1 liter, the production strain cells were cultured with ActiPro medium (Hyclone, USA) containing 4mM L-alanine-L-glutamine (Gibco, USA) and 0.4% anti-cell aggregation agent. From the third day of culture, a composite supplement was added every day: Cell Boost 7A (Cytiva, USA), accounting for 1.5-3.5% of the culture volume, and a mixture of L-cysteine ​​(152mM), L-tryptophan (90mM) and L-tyrosine (250mM) (AppliChem, USA).

[0049] The amount of amino acid mixture used was 10 times less than that of Cell Boost 7A. Throughout the process, a certain concentration (200 g / L) of glucose solution (Panreac, USA) was added daily to maintain the glucose concentration in the culture medium at about 5 g / L. All of these supplements were added daily through the pump of the bioreactor controller, so that the calculated daily volume would not cause significant changes in pH and osmotic pressure. On the first day of culture, the prepared biomass was transferred to the bioreactor so that the initial concentration of viable cells was 0.5 ± 0.1 × 10 6 cells / mL. Cultivation was carried out at a pH of 7.0±0.2 and a dissolved oxygen concentration of 40±5%. The pH was adjusted by adding CO2 and 0.5M NaHCO3 (Sigma-Aldrich, USA). At the beginning of the process, the dissolved oxygen concentration was adjusted by nitrogen and air, and as the concentration of living cells and oxygen consumption increased, the dissolved oxygen concentration was adjusted by air and oxygen. The gas mixture was provided by microbubbles at a rate of 0.2 to 1.5 L / min. During the cultivation process, the speed of the marine impeller agitator was 60 to 100 rpm per minute (cascade control), the temperature was 37°C, then dropped to 35°C during the exponential growth phase, and to 33°C on the second day of cultivation.

[0050] Sampling and clarification and filtration of the culture fluid. Starting from the third day of culture, samples were taken from the culture fluid every day to determine the number of viable cells and the concentrations of glucose and lactate. The culture was stopped when the cell survival rate dropped below 90%. The culture fluid was clarified by two stages of centrifugation: 10000g for 30 minutes and 3270g for 15 minutes. The culture fluid was sterilized using a 0.22μm filter. The volume of the sterile culture fluid obtained was 1.05L. From the culture fluid, 375mg of recombinant RBD / S21 / S14-Fc antigen was obtained by purification according to the following scheme.

[0051] Chromatographic protein purification. The protein in the clarified culture broth was purified chromatographically using a preparative chromatograph. The first stage of purification used Mab Select Shure LX affinity adsorbent (Cytiva), as well as MabSelect Prisma (Cytiva) and Mab Select (Cytiva) in a volume of 4.7 mL. Equilibrate with 20 mM sodium phosphate and 150 mM sodium chloride buffer, pH 7.0, 10 column volumes, at a rate of 2.5 mL / min. The culture broth was applied to the column with a contact time of 1 minute. After application, the column was rinsed with 20 mM sodium phosphate and 150 mM sodium chloride buffer (pH 7.0), 5 column volumes, at a rate of 5 mL / min. After the rinse was completed, elution was performed with 20 Mm sodium citrate buffer, pH 3.0, 4 column volumes, at a rate of 1 mL / min. The pH of the intermediate product was adjusted to 7.0 with 1 M Tris solution. The second stage of purification used HA Ultrogel mixed reaction resin (Sartorius), as well as CHT ceramic hydroxyapatite (Bio-Rad) and Ca++Pure-HA (Tosoh Biosciences) in a volume of 10 mL. Equilibrated with 20 mM sodium phosphate and 150 mM sodium chloride buffer (PH 7.0) for 8 column volumes at a rate of 2 mL / min. The intermediate obtained from the first stage of purification was applied to the column with a 50% gradient of 20 mM sodium phosphate, 150 mM sodium chloride buffer (PH 7.0) for a contact time of 5 min. After application, the column was rinsed with 20 mM sodium phosphate, 150 mM sodium chloride buffer (PH 7.0) for 4 column volumes at a rate of 2 mL / min. After the rinse was completed, eluted with 20 mM sodium phosphate, 150 mM sodium chloride buffer (PH 7.0) for 4 column volumes at a rate of 1 mL / min. NaCl was added to the intermediate until the final concentration was 2 M. Use 1M phosphoric acid solution that the pH value of intermediate product is adjusted to 6.0.The purification in the third stage uses Butyl Sepharose HP hydrophobic resin (Cytiva), and Capto Butyl ImpRes (Cytiva), Phenyl Sepharose HP (Cytiva) and Capto Phenyl ImpRes (Cytiva), and volume is 5mL.Use 20mM sodium phosphate and 2M sodium chloride buffer (pH 6.0), 10 column volumes, balance at a speed of 2.5mL / min.The intermediate product that the second stage purification obtains is applied to post, and contact time is 5 minutes.After applying, use 20mM sodium phosphate, 2M sodium chloride buffer (pH6.0), 5 column volumes, rinse post at a speed of 2.5mL / min.After the flushing is completed, 100% gradient of 20mM sodium phosphate buffer (pH 6.0) is used for 3 column volumes at a rate of 2mL / min for elution. The fourth stage of purification is to transfer 53mL of Sephadex G25 resin to the finished dosage form buffer. Before use, the column is balanced with 20mM sodium phosphate buffer (pH 7.0) at a rate of 1mL / min. After use, the column is rinsed with 3 columns of 20mM sodium phosphate buffer (pH 7.0) at a rate of 10mL / min. The concentration is adjusted to 2mg / mL with 20mM sodium phosphate buffer (pH 5.0-7.0), and the solution is sterile filtered with a Steritop 0.22μm filter (Merck Millipore, Germany). As a result, 180mL of sterile purified recombinant antigen RBD / S21 / S14-Fc is obtained. The recombinant antigen is stored in a refrigerator at a temperature of +4°C.

[0052] Confirmation of protein identity. Confirm the identity of the obtained recombinant RBD / S21 / S14-Fc antigen using polyacrylamide gel electrophoresis followed by immunoblotting. Electrophoresis was performed using a 4-20% gradient polyacrylamide gel (Bio-Rad, catalog number 4561093). Electrophoresis analysis of the samples was performed under reducing conditions. 100 ng of protein solution was added to the gel wells. Separation was performed at a voltage of 200 V for 50 minutes. Transfer to a PVDF membrane using the Trans Blot Turbo (Bio-Rad) system according to the manufacturer's instructions. After blocking the membrane with a 3% bovine serum albumin solution, it was bound to an alkaline phosphatase-conjugated anti-IgG1 antibody (Abcam, catalog number ab6760). After rinsing the membrane, the conjugate was detected using a BCIP / NBT solution.

[0053] Obtaining a homogeneous dispersion of a betulin-based particulate adjuvant (PA). To obtain a homogeneous dispersion of a betulin-based particulate adjuvant, 25 volumes of 0.01 M sterile Tris buffer (pH 9.0 ± 0.1) were added to a sterile mixture of tetrahydrofuran (TFH) containing a 1% betulin solution, filtered with a 0.22 μm nylon membrane according to the technical protocol, and stirred continuously for 15 minutes using a peristaltic pump and a propeller magnetic stirrer. After adding the buffer, the THF concentration in the solution was 3.85%. After that, the formed suspension was treated with ultrasound at a frequency of 35 kHz for 10 minutes. To this end, a glass container containing the particulate adjuvant suspension was placed in an ultrasonic bath and treated as described above. As a result of the treatment, a white homogeneous suspension without agglomerates and inclusions was obtained. The homogeneity of the suspension was checked by light scattering for 30 seconds until two identical particle distribution spectra in the range of 100-150 nm were obtained. In the next stage, the PA dispersion was purified from tetrahydrofuran. In filtration mode, ultrafiltration was performed using hollow fibers with a molecular weight cutoff of 100 kDa. To prevent adsorption, a 2-3-fold diluted initial PA dispersion was used. The ultrafiltration rate was 1.0-1.2 liters / min and the pressure was set at 0.6-0.8 atmospheres. For diafiltration, 10 volumes (25 liters) of 0.01 M Tris buffer were used. The dispersion was then concentrated to a betulin concentration of 1.0-2.0 mg / mL (4.5 L buffer). The concentration of betulin was determined using HPLC. The containers with the particulate adjuvant were stored in a refrigerator at a temperature of approximately +4°C.

[0054] The candidate vaccine was obtained for immunization of animals and volunteers. 100 mL of sterile RBD / S21 / S14-Fc recombinant antigen dissolved in Tris-HCl buffer was added to a container with 1 liter of sterile adjuvant prepared in 0.05M Tris-HCl buffer in advance, and 0.15M NaCl at pH 7.5 was added and mixed thoroughly at +4°C for 2 hours. The protein concentration of the semi-finished product containing RBD / S21 / S14-Fc (which passed all controls) formed was adjusted to 40 μg / mL and the adjuvant concentration was adjusted to 400 μg / mL in phosphate buffer at pH 7.5. The components were mixed thoroughly and placed in a shaker in a refrigerator at 2 to 8°C for 3 hours. The batch of candidate vaccine was then evenly divided into vials (0.5 mL) and stored in a refrigerator at 2-8°C. The antigen and adjuvant content of each dose (0.5 mL) of the vaccine were 20 μg and 200 μg, respectively. Candidate vaccine samples containing antigen and adjuvant (0.5 mL per dose, 5 μg of recombinant RBD / S21 / S14-Fc antigen and 200 μg of particulate adjuvant) were obtained according to the same protocol.

[0055] The obtained vaccine is used as a candidate vaccine for nonclinical and clinical studies.

[0056] Determination of the content of particulate adjuvant (PA): The content of PA was determined by reverse phase HPLC. A liquid chromatograph of Shimadzu LC-2010 (Japan) and an analytical column Kromasil 300-5C8 250×4.6 mm with a particle size of 5 μm, Akzo Nobel (Netherlands), was used, the isocratic elution mode was used, and the detection wavelength was 210 nm.

[0057] The results are shown in Table 4.

[0058]

[0059] Evaluation of vaccine efficacy. The aim of this study was to compare the immunogenicity of vaccines in animal experiments. In this study, antibody production in male and female BALB / c mice weighing 12-14 g immunized with experimental vaccines was compared. The study drug was injected intraperitoneally twice (according to the 0-14 schedule) with a 1 mL syringe, 0.5 mL each time. Similarly, 0.9% sodium chloride solution was administered to the control group animals. Blood samples were collected on the 14th day after the first immunization and on the 14th day after the second immunization. Antibodies in the serum were detected using ELISA. The efficacy of vaccines containing only RBD (two doses, each dose (0.5 mL) containing 5 μg and 20 μg of recombinant protein) and vaccines without adjuvant were studied. Table 5 lists information such as the number of animals used for immunization, vaccines, and doses.

[0060]

[0061] The ELISA TS was used to detect the presence of antibodies to SARS-CoV-2 antigens in serum obtained from mouse blood (taken from each animal separately). The following commercial reagents were used for the development of ELISA TS: a / mouse IgG (H+L) strong enzyme HRP conjugate (SDT, Germany), chromogen-3,3,5,5-tetramethylbenzidine (Hema, Russia), blocking solution was protein salt solution, and 5% sulfuric acid was used as a stop reagent. The ELISA reaction was performed in polystyrene plates produced by Greiner Bio-One, Germany. 1 μg of SARS-CoV-2 antigen was added to each well and incubated for 1 day. The results of the ELISA were recorded at two wavelengths of 450 / 620 nm using a PR 2100 spectrophotometer produced by Sanofi Pasteur Diagnostics, France, and the results are shown in Table 6.

[0062]

[0063] Study of the immunogenicity (antibody titer) of the candidate vaccine based on the RBD / S21 / S14-Fc recombinant protein and natural betulin granule adjuvant. Female mice of the BALB / c strain (obtained from the Stolpovoya branch of the Scientific Center for Biomedical Technologies of the Federal State Budgetary Agency for Science and Technology of the Russian Federation) were used in this study. The animals were kept under standard conditions (18-22 ° C, relative humidity 50-70%, NH3 concentration 0.001 mg / m 3 Animals were given free access to pelleted complete extruded diet for mice, rats and hamsters (Laboratorkorm LLC, Moscow) in accordance with Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and in accordance with the sanitary and epidemiological rules SR 2.2.1.3218-14 “Sanitary and epidemiological requirements for the design, equipment and maintenance of experimental biological clinics (Vivaria)” (approved by Order of the Chief State Sanitary Physician of the Russian Federation No. 51 of 29 August 2014).

[0064] Table 7 lists the candidate vaccine formulations containing particulate adjuvants based on RBD / S21 / S14-Fc recombinant proteins for animal immunization and shows the study design. Physiological saline (0.9% NaCl) was used as a placebo.

[0065]

[0066] According to the experimental design, BALB / c mice weighing 18-20g were injected intraperitoneally with 0.5mL of the preparation twice with a 1mL syringe on the 1st and 14th days. The preparation was mixed 5-6 times before filling the syringe each time. A 0.9% sodium chloride solution was administered to the control group mice of the same batch. Blood was obtained from the mandibular vein 28 days after the first immunization. The blood samples were allowed to stand at room temperature for 2 hours, centrifuged at 5000rpm for 10 minutes, and whole serum was collected. The serum samples were stored at -20°C before use. The presence of antibodies in the serum obtained from the blood of the mice (taken from each animal separately) was detected by enzyme-linked immunosorbent assay (ELISA) and microneutralization test (MNT). The raw data were statistically analyzed using GraphPad Prism v9 software. The data were presented using the following statistical parameters: geometric mean, standard deviation, arithmetic mean, and mean error. To determine the significance of the differences between the groups, univariate analysis of variance (ANOVA) was used, followed by post hoc pairwise comparisons of the differences between the groups using the Tukey test. The significance level of each group was α = 0.05. When p < α, it was considered to be significantly different.

[0067] Enzyme-linked immunosorbent assay. The content of specific G-class immunoglobulins (IgG) in mouse serum samples after two immunizations was determined. Antigens diluted to a concentration of 1.0 μg / mL in sterile DPBS were adsorbed overnight at +4°C in 96-well Maxisorp plates (100 μL / well, Nunc). The plates were then rinsed twice in PBS using an ELx50 (BioTek) automatic washer with 500 μL / well of a 0.1% Tween-20 (Serva) solution. After rinsing, they were incubated for 2 hours at room temperature with blocking buffer (PBS-T containing 5% milk powder, TF Ditol) and rinsed again. After that, a series of double dilutions of serum were added to the plates, starting with a dilution of 1:400 for the group of animals receiving the vaccine preparation and a dilution of 1:50 for the placebo group. The plates containing mouse serum were incubated for 1 hour at room temperature and then rinsed three times. Horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) HRP (Abcam) was used as the detection antibody at a dilution of 1:7500, 100 μL / well. After incubation with the detection antibody, the plate was washed four times. 100 μL / well of TMB solution (Imtek LLC, Russia) was used as the chromogenic substrate, reacted at room temperature for 10 minutes, and then 1N H2SO4 (Vekton, Russia) was added to stop the reaction. The absorbance (OD value) was measured at a wavelength of 450 nm using a CLARIOstar multi-well plate reader and a LVF monochromator (BMGLABTECH). For the vaccine preparation, the level of specific IgG antibodies to the RBD / S21 / S14-Fc protein in the serum of the immunized animals was determined using an indirect ELISA experimental detection system after two immunizations at 5 μg and 20 μg doses. The measured levels of specific antibodies are shown in the table. Statistical analysis was performed using univariate analysis of variance (ANOVA), followed by paired comparisons between groups using the Tukey test. The study showed that after two immunizations with two doses of the vaccine preparation, significant RBD / S21 / S14-Fc protein-specific serum antibodies were produced. The results are shown in Table 8.

[0068] Microneutralization test (MNT). For the microneutralization test, mouse serum was diluted 1:4 with sterile DPBS and heated at 56°C for 30 minutes. Then, serum dilutions were prepared starting from 1:10 (with whole serum as reference) in MEM+2% FBS medium in a volume of 50 μL. An equal volume of SARS-CoV-2 (infectious dose of 25 TCD 50) was added to the serum dilution and incubated at 37°C for 1 hour. The dilution was then transferred to Vero cells in 96-well plates. The plates were incubated at 37°C for 4 days, after which the presence of CPE (cytopathic effect) was detected. The neutralization titer was considered the maximum dilution of serum at which no CPE was observed. All experiments with infectious coronaviruses were performed at the Laboratory of Immunology and Viral Infection Prevention of the Institute of Experimental Medicine, a Federal State Budgetary Scientific Agency (IEM, Sanitary and Epidemiological License published by the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor) of the Russian Federation in St. Petersburg, No. 77.ПЧ.01.000.Μ.000053.08.20 on the Use of Pathogenic Class II-IV Biological Agents on August 5, 2020). The results of the determination of the level of neutralizing antibodies against the SARS-CoV-2 coronavirus in the serum of mice after two immunizations showed that the virus neutralizing antibodies produced by the groups of animals vaccinated with 5μg and 20μg doses of the candidate vaccine containing the recombinant protein were significantly different from those in the placebo group. There was no significant difference in the level of virus neutralizing antibodies between the two groups. Statistical analysis was performed using univariate analysis of variance (ANOVA), followed by a Tukey test for paired comparisons between groups, and the results are shown in Table 8. This study shows that virus neutralizing antibodies were produced only in the animal groups vaccinated with a dose of 5 μg or 20 μg of the candidate vaccine containing the recombinant protein, and the amount was significantly different from that in the placebo group. This proves that the vaccine containing the natural betulin-based particulate adjuvant has strong antigenicity.

[0069]

[0070] Study on the immunogenicity properties (cellular immune response evaluation) of candidate vaccines based on RBD / S21 / S14-Fc recombinant protein and natural betulin granule adjuvant. To evaluate T cell immune response, C57 / black SPF mice (5 mice per group) were intraperitoneally injected with 5 μg or 20 μg of drug twice on day 1 and day 22. Spleens were collected 28 days after the second immunization. The unvaccinated group (n=3) served as the control group. The study design is shown in Table 9.

[0071]

[0072] The spleen homogenate was filtered (pore size 70 μm), washed with DPBS (Gibco) containing 2.5% FCS, red blood cells were removed with RBC lysis buffer (BioLegend), and then washed with DPBS (2.5% FCS). 6Cells were seeded at a density of 100 μg / mL in 96-well flat-bottom plates and stained with SARS-CoV-2 S protein peptide ( SARS-CoV-2Prot S) pool was stimulated for 6 hours. 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). Nonspecific binding was blocked using True Stain containing anti-CD16 / CD32. Cells were stained using Cytofix / Cytoperm (BD Biosciences) as directed. The relative proportions of CD8+ and CD4+ T lymphocytes and cells with a memory effector phenotype (CD44+, CD62L-) were determined. After two immunizations with 20 μg of antigen, it was found that the number of S protein-specific CD4+ and CD8+Tem cells increased significantly, while 5 μg of immunization only led to a significant increase in the number of CD4+Tem cells (p<0.05). In the two Tem cell populations, the growth of cells producing Th1-cytokines (IFNγ, IL-2, TNFα) showed a dose-dependent trend. In both dose groups, the effects on Tem cells producing IL-2 (IFNγ-IL-2+TNFα-) and multifunctional CD8+T cells (IFNγ+IL-2+TNFα+) reached significant levels. In the high-dose group, the level of IL-2 produced by CD8+Tem cells was also greatly increased.

[0073] Study on the immunogenicity characteristics (cellular immune response assessment) of candidate vaccines based on RBD / S21 / S14-Fc recombinant protein and natural betulin granule adjuvant in volunteers.

[0074] To strictly control adverse reactions or any complications after vaccination, volunteers need to be hospitalized for 3 days (4 nights) during both vaccinations / placebo. Volunteers were admitted to the hospital one day before vaccination. Volunteers were discharged two days after the first and second vaccine / placebo injections if no adverse reactions (AEs) and serious adverse reactions (SAEs) occurred. Subsequent follow-up was conducted in outpatient clinics. The study was conducted in two phases.

[0075] Phase I of a Phase I study. Phase I of the clinical trial was an open-label, randomized trial that observed the safety, tolerability (reactogenicity), and immunogenicity of the vaccine given twice intramuscularly at doses of 20μg+5μg and 20μg+20μg. Considering that some ineligible subjects were expected at screening, the study divided 20 healthy volunteers aged 18 to 60 into parallel groups. Group 1 (10 subjects) were vaccinated with the study vaccine product according to the following regimen: the first injection was 20μg-0.5mL intramuscular suspension (40μg / mL), and the second injection was 5μg-0.5mL intramuscular suspension (10μg / mL), with an interval of 28 days between them (hereinafter referred to as "two-dose immunization according to the 20μg+5μg regimen"). Group 2 (10 subjects) was vaccinated with the test product according to the following regimen: the first and second injections of 20 μg-0.5 mL intramuscular suspension (40 μg / mL) were separated by 28 days (hereinafter referred to as "two-dose immunization according to the 20 μg + 20 μg regimen"). Volunteers were admitted to the hospital one day before the two vaccinations. The hospitalization period was two times, each of 4 days (4 visits).

[0076] Phase II of Phase I study. Phase II of the clinical trial is a double-blind, randomized, parallel-group clinical trial in which the vaccine is administered intramuscularly twice at a dose of 20μg+5μg and 20μg+20μg to observe its safety, tolerability and immunogenicity. Considering that some ineligible subjects are expected at screening, this study was conducted in 96 healthy volunteers aged 18 to 60 years. Group 3 (32 subjects) was vaccinated with the test product according to the following regimen: the first injection of 20μg-0.5mL intramuscular suspension (40μg / mL) and the second injection of 5μg-0.5mL intramuscular suspension (10μg / mL), with an interval of 28 days between them. Group 4 (32 subjects) received study drug according to the following schedule: the first and second injections were 20 μg-0.5 mL of intramuscular suspension (40 μg / mL), with an interval of 28 days between them; Group 5 (32 subjects) received placebo according to the following schedule: the first and second injections were 0.5 mL of 0.9% sodium chloride solution injected intramuscularly, with an interval of 28 days between them.

[0077] 96 volunteers (Groups 3, 4, and 5) were admitted to the hospital 1 day before the first injection of the test product / placebo. They were observed in the hospital for 4 days (3 nights). All volunteers in Groups 3, 4, and 5 were followed up by the research physician for 6 months (180±5 days) after vaccination with the test product / placebo to identify possible late adverse reactions and evaluate immunogenicity. The final safety and tolerability assessment was performed at the 20th follow-up visit (180±5 days after the first vaccination with the test product / placebo).

[0078] Data analysis showed that there were no significant differences in AEs between the analysis groups in all systems studied. At the fourth follow-up, a volunteer with random number R004 (from group 1) was diagnosed with COVID-19, based on the presence of SARS-CoV-2 RNA confirmed by polymerase chain reaction (PCR) and clinical manifestations (fever, cough), and met the exit criteria. This mild AE phenomenon still existed at the time of the interim data analysis. The differences between group 1 and group 2 were evaluated by Fisher's exact test, and the results showed that there was no statistically significant difference in the proportion of volunteers with COVID-19 symptoms and PCR-confirmed SARS-CoV-2 infection (p=1,000). Comparison of the primary and secondary criteria for safety and tolerability showed no statistically significant differences between the study groups.

[0079] In addition to the safety evaluation of the candidate vaccine, specific antibodies to SARS-CoV-2 were also tested in the blood samples of volunteers from groups 1 to 4. The results of the determination of specific antibodies in the vaccinated volunteers are shown in Table 10.

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[0081]

[0082]

[0083] The results of specific antibody testing after volunteers received two doses of Betuvax-CoV-2 vaccine showed that high titers of specific antibodies were observed in most volunteers when the vaccine was injected intramuscularly at an interval of 28 days according to the dosing schedule (20μg+5μg and 20μg+20μg).

Claims

1. A fusion gene, characterized in that: The fusion gene encodes a recombinant antigen consisting of the RBD receptor, S14P5 and S21P2 epitopes, and the Fc fragment of immunoglobulin IgG1 of the SARS-CoV-2 coronavirus S surface protein, and the amino acid sequence of the recombinant antigen is shown in SEQ ID NO:

1.

2. An expression construct embedded in a P-Pharma-Zeo vector having an amino acid sequence as shown in SEQ ID NO: 4, characterized in that: The expression construct comprises the nucleotide sequence SEQ ID NO: 2 consisting of the fusion gene of claim 1 and the nucleotide sequence SEQ ID NO: 3 encoding the IL-2 leader peptide.

3. A CHO-HP cell line transfected with the expression construct as claimed in claim 2, capable of producing recombinant RBD / S21 / S14-Fc antigen.

4. A method for obtaining a recombinant RBD / S21 / S14-Fc antigen, characterized in that: The CHO-HP cell line of claim 3 is cultured under the condition of adding 4 mM L-alanine-L-glutamine and an anti-cell aggregation agent, and then the culture fluid is collected, clarified, filtered, and chromatographically purified by affinity chromatography, HA resin chromatography, hydrophobic chromatography, and gel permeation chromatography, and then sterile filtered.

5. A recombinant RBD / S21 / S14-Fc antigen obtained according to claim 4, characterized in that: The antigens stimulate an immune response to coronavirus infection.

6. A vaccine composition for preventing coronavirus infection, characterized in that: The buffer contains the recombinant antigen according to claim 5 and a particle adjuvant based on natural betulin.

7. A method for preventing coronavirus infection, characterized in that: Providing the vaccine composition of claim 6 for immunization to a subject who needs to be prevented from coronavirus infection.

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