WO2023128813A1

WO2023128813A1 - Antigens for inducing immunity against the sars-cov-2 virus and gene constructs for the expression thereof

Info

Publication number: WO2023128813A1
Application number: PCT/RU2022/000408
Authority: WO
Inventors: Артур Александрович ИСАЕВ; Александр Викторович КУДРЯВЦЕВ; Мария Евгеньевна ФРОЛОВА; Анна Владимировна ВАХРУШЕВА; Александр Викторович ИВАНОВ; Милана ДЖОНОВИЧ; Тарас Владимирович ИВАНИШИН; Игорь Викторович Красильников
Original assignee: Общество с ограниченной ответственностью "Бетувакс"
Priority date: 2021-12-30
Filing date: 2022-12-30
Publication date: 2023-07-06

Abstract

The invention relates to biotechnology, immunology and virology, and more particularly to an antigen composition that allows the induction of a specific humoral and cellular immune response to the requisite epitope of the SARS-CoV-2 viral coat protein. For this purpose, a gene construct is proposed which includes an expression cassette organized in the form of a modular construct that allows the substitution of regions coding for the signal peptide, the target SARS-CoV-2 recombinant protein-antigen RBD-SD1, fused with an Fc fragment of immunoglobulin by a linker. Also proposed is a method for obtaining recombinant proteins based on a region of the SARS-CoV-2 S protein including RBD and SD1 and fused with an Fc fragment of IgG.

Description

ANTIGENS FOR INDUCING IMMUNE AGAINST SARS-COV-2 VIRUS AND GENE CONSTRUCTIONS FOR THEIR EXPRESSION

The field of technology to which the invention belongs.

The invention relates to biotechnology, immunology, virology and concerns the creation of a gene construct that includes variants of expression cassettes encoding recombinant proteins based on the S-protein region of SARS-CoV-2, including RBD and SD1 , fused with an IgG Fc fragment, which, in combination with betulin can be used as an active substance that causes the production of antibodies against the SARS-CoV-2 virus.

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 time-tested technology for obtaining vaccines based on the subunit of the virus has certain advantages, such as safety and the possibility of repeated administration. However, individual viral subunits have low immunogenicity, which necessitates the use of adjuvants.

Vaccines based on whole inactivated viruses are generally widely used in 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 associated with efficiency - undesirable side effects and problems with the targeting of produced antibodies to the desired epitope of viral envelope proteins.

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. At the moment, by the way, none of the DNA or RNA vaccines have been approved for the prevention of human infectious diseases. Of the shortcomings, low immunogenicity should be noted. In case of use for delivery viral vectors, there is a danger of foreign DNA integrating into the cell genome, the possible development of autoimmune reactions against cells expressing the antigenic protein, or a reaction to foreign DNA. In addition, 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. In addition, adenoviral vaccines can elicit an immune response against the adenovirus itself and cannot be reused without modification.

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 suitable adjuvants. Their use makes it possible to reduce the dose of the antigen, reduce the cost of the final preparation, increase the immunogenicity of "weak" or small antigens, which are extremely difficult to develop immunity and obtain a vaccine against this class of antigens. 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 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. 75 (less than 40 kDa), and it is also rapidly excreted from the body, so RBD conjugated with the Fc fragment of IgG became a candidate for antigens.

The addition of an adjuvant will reduce the amount of protein per dose, reduce the cost of the vaccine, and increase the immune response to the antigen. Currently, in experimental and clinical studies, mineral (hydroxide 80 or aluminum 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 ISKOM 85 (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 90 (patent RU2545714). 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.

95 From patent RU2749193, options for using antigens are known, including RBD for induction of specific immunity against the SARS-CoV-2 virus as an antigenic component, together with betulin spherical amorphous nanoparticles (SANP) with a size of 80-160 nm as an adjuvant. Spherical particles based on birch bark triterpenoids - betulin (betulenol, betulinol, lupendiol) 100 are characterized in FIG. 1 and FIG. 2. This adjuvant is produced on an industrial scale in accordance with the requirements of the pilot industrial regulation, and was also tested earlier in preclinical studies of influenza vaccines, where it showed good immunogenic properties and safety of use.

105 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 software of the 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 betulin as an adjuvant and a recombinant protein as an active component. The technical result is achieved by creating a genetic construct that includes an expression cassette organized in the format of a modular construct that allows for the replacement of regions encoding the signal peptide, the target recombinant SARS-CoV-2 RBD-SD1 antigen protein, fused with the immunoglobulin Fc fragment through a linker.

More specifically, the solution of the problem and the achievement of the technical result of the invention is carried out by developing an antigen for inducing immunity against the SARS-CoV-2 virus, which is a recombinant protein that includes a segment of the S-protein of the SARS-CoV-2 virus, consisting of amino acid residues 302-638 domains RBD and SD1, fused with the Fc fragment of IgG through a polyglycine-serine linker.

In some embodiments, the polyglycine-serine linker has the sequence shown in SEQ ID NO: 19.

In some particular embodiments of the invention, the antigen has the sequence represented by SEQ ID NO: 3 or SEQ ID NO: 8.

Another variant of the invention is also an antigenic composition for inducing immunity against the SARS-CoV-2 virus, containing the specified antigen, a buffer solution and an adjuvant, where a corpuscular adjuvant based on triterpenoids from betulin birch bark is used as an adjuvant.

The solution of the problem is also carried out by using the above antigenic composition to induce an immune response against the SARS-CoV-2 virus.

In some embodiments of the invention, the antigenic composition is administered intramuscularly.

In some embodiments of the invention, the induction of immunity refers to the induction of antibody and cellular immunity.

The solution of the task and the achievement of the technical result of the invention is also carried out by developing a gene construct for the expression of a recombinant protein, including a section of the S-protein of the SARS-CoV-2 virus, consisting of amino acid residues 302-638 of the RBD and SD1 domains, fused to the Fc fragment of IgG through polyglycine -serine linker, including an expression cassette consisting of sections encoding:

- SARS-CoV-2 S-protein signal peptide or anti-TNF immunoglobulin antibody F10 signal peptide, - a section from 302 to 638 amino acid residues of the S-protein RBD-SD1 of the SARS-CoV-2 virus,

- polyglycine-serine linker,

- Fc fragment of IgG.

In some embodiments of the gene construct, the coding sequence for the SARS-CoV-2 S protein signal peptide is represented by SEQ ID NO: 4.

In some embodiments of the gene construct, the coding sequence for the signal peptide of the anti-TNF immunoglobulin antibody F10 is represented by SEQ ID NO: 10.

In some embodiments of the gene construct, the coding sequence for the SARS-CoV-2 S protein region has the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments of the gene construct, the polyglycine-serine linker has the sequence shown in SEQ ID NO: 19.

In some embodiments of the gene construct, the coding sequence for the Fc fragment of IgG is represented by SEQ ID NO: 6.

In some particular embodiments of the invention, the gene construct contains an expression cassette represented by the sequence of SEQ ID NO: 27.

In some particular embodiments of the invention, the gene construct contains an expression cassette represented by the sequence of SEQ ID NO: 29.

In some particular embodiments of the invention, the gene construct contains an expression cassette represented by the sequence of SEQ ID NO: 28.

In some particular embodiments of the invention, the gene construct contains an expression cassette represented by the sequence of SEQ ID NO: 30.

The solution of the task and the achievement of the technical result of the invention is also carried out by developing a method for obtaining a recombinant protein - antigen for inducing immunity against the SARS-CoV-2 virus, including:

1) transfection of Chinese hamster ovary cells with the CHO-S gene construct described above;

2) cultivation of transfected Chinese hamster ovary CHO-S cells;

3) selection of the culture medium;

4) chromatographic purification of the resulting recombinant protein;

5) confirmation of the authenticity of the obtained recombinant protein.

Brief description of the drawings.

Fig. 1. Characterized spherical particles consisting of betulin, a) betulin formula, b) electron photographs of betulin spherical particles, c) the size of betulin corpuscular particles is about 120-160 nm. Fig. 2. Image of adjuvant in the form of betulin in combination with antigen after sonication, obtained by transmission electron microscopy.

Fig. Fig. 3. Recombinant proteins deposited on betulin spherical RBD-SDI-Fc nanoparticles, a) schematic representation of the betulin/RBD-SDI-Fc complex, b) typical image of the SARS-CoV-2 virion, c) chromatographic peak of RBD-SDI-Fc before application on betulin particles, d) chromatograms of the supernatant after applying the protein to the particles, confirming the absorption of the antigen from the solution.

Fig. 4. Scheme of SARS-CoV-2 S-protein and recombinant protein based on RBD-SDI-Fc. a) scheme of the S-protein on the surface of SARS-CoV-2, b) scheme of the structure of the recombinant RBD-SDI-Fc antigen, c) scheme of the S-protein of SARS-CoV-2, with the specified RBD-SD1 part taken as the basis for recombinant proteins .

Fig. 5. Expression cassettes of four gene constructs encoding recombinant proteins based on the S-protein region of SARS-CoV-2, including RBD and SD1 , fused to the Fc fragment of IgG.

Fig. 6. Linearized pcDNA3.4 vector for TOPO® TA cloning of PCR products and for high level expression.

Fig. 7. Scheme for obtaining a gene construct for the expression of recombinant RBD-SDI-Fc proteins.

Fig. 8. Map of the 83/1 gene construct containing the S-protein signal peptide, the RBD region (Wuhan), and the Fc fragment.

Fig. 9. Map of 86/12 gene construct containing F10 antibody signal peptide, RBD-SD1 region (Wuhan), and IgG Fc fragment.

Fig. 10. Map of the 78/9 gene construct containing the S protein signal peptide, RBD-SD1 region (Beta), and IgG Fc fragment.

Fig. 11. Map of the 85/1 gene construct containing the F10 antibody signal peptide, RBD-SD1 region (Beta), and IgG Fc fragment.

Fig. 12. General scheme of the expression cassette, including the sequence of the signal peptide, the RBD-SD1 region of SARS-CoV-2, the linker sequence, and the Fc fragment of IgG, organized in a modular design format that allows modification or replacement of regions according to restriction sites.

Fig. Fig. 13. Electropherogram of separation of samples of recombinant RBD-SDI-Fc proteins from Wuhan and Beta strains in 10% PAAG under reducing (+BME) and non-reducing conditions (-BME). M - molecular weight marker (15, 20, 25, 30, 40, 50, 60, 70, 85, 100, 120, 150, 200 kDa).

Fig. 14. Design of an experiment for two-time immunization of BALB/C mice with a period of 2 weeks. Fig. Fig. 15. The level of specific antibodies to the mutant RBD-Fc protein in the sera of immunized BALB/c mice after double immunization with vaccine preparations at a dose of 5 µg and 20 µg. Separate symbols show the individual titer value for each animal, the horizontal line shows the geometric mean titer for the group.

Fig. 16. The level of virus-neutralizing antibodies to SARS-CoV-2 coronavirus in the sera of vaccinated BALB/c mice.

Separate symbols show the individual titer value for each animal, the horizontal line shows the geometric mean titer for the group.

Fig. Fig. 17. Results of studying the cellular immune response - specific responses of CD4+ and CD8+ Tern after immunization, (a) the relative presence of virus-specific CD4+ and CD8+ Tern among CD4+/CD8+ CD44+ CD62L- splenocytes, (b) the relative frequency of virus-specific cytokines producing CD4+ and CD8+ Tet.*p <0.05.

Sequence listing.

Nucleotide sequence numbers (SEQ ID NO) in the text of the description and the claims correspond to those in the Sequence Listing according to the ST.26 standard, which is part of the present description of the invention:

SEQ ID NO: 1 - pcDNA3.4 S-RBD(Wuhan)-Fc,

SEQ ID NO: 2 - pcDNA3.4,

SEQ ID NO: 3 - RBD-FcAA (Wuhan),

SEQ ID NO: 4 - Signal peptide, S-protein (NA),

SEQ ID NO: 5 - RBD(Wuhan) NA

SEQ ID NO: 6 - Fc NA,

SEQ ID NO: 7 - pcDNA3.4 S-RBD(Beta)-Fc,

SEQ ID NO: 8 - RBD-FcAA (Beta),

SEQ ID NO: 9 - RBD(Beta) NA,

SEQ ID NO: 10 - Signal peptide, antibody F10 NA,

SEQ ID NO: 11 - pcDNA3.4 F10-RBD(Wuhan)-Fc,

SEQ ID NO: 12 - pcDNA3.4 F10-RBD(Beta)-Fc,

SEQ ID NO: 13 - LSpF1 ,

SEQ ID NO: 14 - LSpR1 ,

SEQ ID NO: 15 - LSpF2,

SEQ ID NO: 16 - LSpR2,

SEQ ID NO: 17 - FclinkerF,

SEQ ID NO: 18 - pcDNA3.4-R, SEQ ID NO 19 - Linker,

SEQ ID NO: 20 pAL2-T, S-protein,

SEQ ID NO 21 pAL2-T, Fc,

SEQ ID NO 22 LidF HF,

SEQ ID NO 23 LidF10HR1 ,

SEQ ID NO 24 LidF10HF2,

SEQ ID NO 25 LSpR2,

SEQ ID NO 26 pAL2-T, F10,

SEQ ID NO 27 S-RBD(Wuhan)-Fc NA,

SEQ ID NO 28 F10-RBD(Wuhan)-Fc NA,

SEQ ID NO 29 S-RBD(Beta)-Fc NA,

SEQ ID NO 30 F10-RBD(Beta)-FcNA,

SEQ ID NO 31 S-RBD(Wuhan)-Fc,

SEQ ID NO 32 F10-RBD(Wuhan)-Fc,

SEQ ID NO 33 S-RBD(Beta)-Fc,

SEQ ID NO 34 F10-RBD(Beta)-Fc.

Implementation of the invention.

Design of RBD-SD1-Fc expression cassettes.

At the first stage of the work, a gene construct was created based on the pcDNA3.4 vector (SEQ ID NO: 2) containing the following regions: CMV promoter/enhancer, Start-End (cloning site for cloning PCR products), WPRE (groundhog post-transcriptional regulatory element), NeoR/KanR (neomycin/kanamycin resistance gene), HSV TK poly(A) signal (herpes simplex virus thymidine kinase (TK) polyadenylation signal), f1 ori (origin of replication), SV40 promoter and SV40 ori, SV40 poly(A) signal (SV40 early polyadenylation signal), AmpR, AmpR promoter (ampicillin resistance gene (Na) (β-lactamase) and its promoter), restriction sites are indicated by their respective names. Expression cassettes are cloned in the Start-End position.

The S-RBD-SD1 (Wuhan)-Fc gene construct (SEQ ID NO: 1) included an expression cassette encoding a recombinant protein (SEQ ID NO: 3) based on the S protein region of the Wuhan-Hu-1 SARS-CoV-2 strain (hereinafter: Wuhan), including RBD and SD1 fused with the Fc fragment of IgG (Fig. 46). The S protein region (amino acids 302-638) including RBD and SD1 is shown in FIG. 4c as "RBD-SD1 antigen". Thus, the expression cassette included three constructive sequences:

(a) SARS CoV-2 S protein signal peptide (SEQ ID NO: 4),

(b) Wuhan strain S protein region (SEQ ID NO: 5), (c) Fc fragment of IgG (SEQ ID NO: 6).

Additionally, in this invention, based on the above gene construct S-RBD-SD1 (Wuhan)-Fc, a variant of the expression cassette was created, where the region of the S-protein of the Wuhan strain, including RBD and SD1 , was replaced with a region with mutations (K417N, E484K, N501 Y, D614G) of SARS-CoV-2 strain Beta, B1.351 (hereinafter: Beta). As a result, the gene construct S-RBD-SD1 (Beta)-Fc (SEQ ID NO: 7) was created, expressing the recombinant protein (SEQ ID NO: 8) based on the same region of the S-protein of the Beta strain (amino acids 302-638) , including RBD and SD1, fused with the Fc fragment of IgG. Thus, the gene construct included three constructive sequences:

(a) SARS CoV-2 S protein signal peptide (SEQ ID NO: 4),

(b) S-protein region of strain Beta (SEQ ID NO: 9),

(c) Fc fragment of IgG (SEQ ID NO: 6).

Signal peptides are sequences that play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The secretion of the recombinant protein directly depends on the choice of the signal peptide. In order to allow high levels of protein production in the culture medium, the SARS-CoV-2 S protein signal peptide sequence is used in this invention due to the presence of the SARS-CoV-2 S protein region in the recombinant protein. Also, due to the presence of an IgG Fc fragment in the recombinant protein, the S protein signal peptide sequence in the two previously described genetic constructs S-RBD-SD1(Wuhan)-Fc and S-RBD-SD1(Beta)-Fc expressing the S-region protein of strains Wuhan and Beta, replaced by the signal peptide of the antibody F10 (SEQ ID NO: 10). Thus, genetic constructs F10-RBD-SD1(Wuhan)-Fc (SEQ ID NO: 11) and F10-RBD-SD1-Fc (SEQ ID NO: 12) expressing recombinant proteins based on the S-protein region of Wuhan strains were created. and Beta SARS-CoV-2, including RBD and SD1 fused to an IgG Fc fragment.

Fc fragment - a crystallizing fragment of an immunoglobulin, a part of an immunoglobulin molecule that interacts with Fc receptors on the cell surface and with some proteins of the complement system. The Fc fragment dimerizes with each other via disulfide bonds, forming a dimer of identical halves, forming a Y-shaped structure. Association with the Fc fragment of antigenic fragments thus mimics the antigen-antibody complex, which increases the immune response, correcting it towards a specific immune response and preventing IgE-related allergenicity. Also, the connection with the Fc fragment allows for affinity purification by chromatography through the sorbent Sepharose with protein A as a ligand, which ensures high purity of the final product after purification.

Thus, taking into account the used signal peptide sequence variants, the SARS-CoV-2 S protein, and the IgG Fc fragment sequence, four expression cassettes were ultimately constructed (Table 1) expressing two recombinant proteins based on the S protein region. strains Wuhan and Beta, which are schematically represented in Fig. 5.

Method for obtaining recombinant proteins RBD-SD1-Fc.

The developed method for obtaining recombinant RBD-SDI-Fc proteins includes the following steps:

1) Generation of signal peptide sequences and Fc fragment of IgG,

2) Obtaining pAL2-T gene constructs, including the sequences of the signal peptide and the Fc fragment of IgG,

3) Ligation of fragments (sequences) of the signal peptide, the Fc fragment of IgG, and the S-protein region of SARS-CoV-2 into the pcDNA3.4 gene construct,

4) Cultivation of cells to obtain a recombinant protein based on RBD-SDI-Fc,

5) Selection of culture medium,

6) Chromatographic purification of the recombinant protein based on RBD-SDI-Fc,

7) Authentication of the obtained recombinant protein based on RBD-SDI-Fc.

At the first stage of the work, sequences were synthesized by PCR the signal peptide of the S-protein and the Fc fragment of IgG. The signal peptide sequence of the S protein was synthesized using primers LSpF1-LSpR1 (SEQ ID NO: 13, SEQ ID NO: 14) and LSpF2-LSpR2 (SEQ ID NO: 15, SEQ ID NO: 16). The sequence of the Fc fragment was synthesized using primers FclinkerF (SEQ NO ID: 17) and pcDNA3.4-R (SEQ NO ID: 18) based on a plasmid containing the Hc gene of the monoclonal antibody trastuzumab as a template.

The primers were annealed at 58°C, the number of amplification cycles was set equal to 15. The fragments were separated by DNA electrophoresis in 1% agarose gel, fragments of 78 and 850 bp were isolated, and cloned separately into the T-vector pAL2-T. As a result of the work, two pAL2-T gene constructs were obtained, including the sequences of the S-protein signal protein and the polyglycine-serine linker (SEQ ID NO: 19) fused to the IgG Fc fragment, respectively. The resulting sequences were confirmed by Sanger sequencing.

Selected pAL2-T gene constructs (SEQ ID NO: 20, SEQ ID NO: 21) carrying the S protein signal peptide sequence and the polyglycine serine linker sequence fused to the IgG Fc fragment, respectively, were treated with restriction endonucleases Nhel, Xhol, HinDIII, were isolated fragments, and in the course of three-component ligation cloned into pcDNA3.4 (Fig. 6) at the recognition sites of restriction endonucleases Nhel, Xhol.

The gene construct containing the nucleotide sequence of the S-protein region of the Wuhan strain (Wuhan-1, GenBanklD: NC_045512 302:638) was treated with restriction endonucleases BamHI and HinDIII, the reaction products were separated in a 1% agarose gel during electrophoresis, and a 1080 bp fragment was isolated. O. The isolated fragments were cloned at the BamHI and HinDIII sites into a preliminarily prepared pcDNA3.4 gene construct carrying the nucleotide sequences encoding the signal peptide of the S protein and the Fc fragment of IgG. Thus, a pcDNA3.4 S-RBD-SD1 (Wuhan)-Fc gene construct (SEQ ID NO: 1) was obtained, containing an expression cassette encoding an S protein signal peptide, a Wuhan strain S protein region, and an IgG Fc fragment, which can then be used to produce RBD-SD1 (Wuhan)-Fc recombinant (SEQ ID NO: 3).

To obtain the RBD-SDI-Fc recombinant protein containing the S-protein region of the Beta strain (SEQ ID NO: 8), an expression cassette was obtained, where the region encoding the S-protein, including RBD and SD1, was replaced with a region with mutations (K417N, E484K, N501 Y, D614G). The gene construct containing the nucleotide sequence of the S-protein region of the Beta strain (SEQ ID NO: 9) was treated with restriction endonucleases BamHI and HinDIII, the reaction products were separated in a 1% agarose gel during electrophoresis, and a 1080 bp fragment was isolated. The isolated fragments were cloned BamHI and HinDIII sites into a pre-prepared pcDNA3.4 gene construct carrying nucleotide sequences encoding the S-protein signal peptide and an IgG Fc fragment. Thus, a pcDNA3.4 S-RBD-SD1 (Beta)-Fc gene construct (SEQ ID NO: 7) was obtained, comprising an S protein signal peptide, a Beta strain S protein region, and an IgG Fc fragment, which can then be used for the production of RBD-SD1(Beta)-Fc recombinant protein (SEQ ID NO: 8).

To test the operation of the expression cassette when replacing the signal sequence, on the basis of previously obtained constructs, expression cassettes encoding recombinant proteins RBD-SD1(Wuhan)-Fc (SEQ ID NO: 3) and RBD-SD1(Beta)-Fc (SEQ ID NO : 8), where the sequence of the signal peptide of the S-protein was replaced with the sequence of the signal peptide F10 of anti-TNF immunoglobulin (SEQ ID NO: 10).

The F10 signal peptide sequence was synthesized using primers LidF10HF1-LidF10HR1 (SEQ ID NO: 22, SEQ ID NO: 23) and LidF10HF2-LSpR2 (SEQ ID NO: 24, SEQ ID NO: 25). The primers were annealed at 58°С, the number of amplification cycles was set equal to 15. The fragments were separated by DNA electrophoresis in 1% agarose gel, 84 bp fragments were isolated, and cloned into the T-vector pAL2-T. The selected pAL2-T gene construct (SEQ ID NO: 26) carrying the F10 antibody signal peptide sequence and the previously obtained pAL2-T construct carrying the IgG Fc fragment sequence (SEQ ID NO: 21) were treated with restriction endonucleases Nhel, Xhol, HinDIII, the fragments were isolated and cloned into pcDNA3.4 in the course of three-component ligation at the sites of recognition of restriction endonucleases Nhel, Xhol.

The pcDNA3.4 gene constructs, including the nucleotide sequences of the S protein region of the Wuhan and Beta strains, were treated with BamHI and Nhel restriction endonucleases, the reaction products were separated in a 1% agarose gel during electrophoresis, and 1080 bp fragments were isolated. The isolated fragments were cloned at the BamHI and HinDIII sites into pre-prepared pcDNA3.4 gene constructs carrying sequences encoding the F10 signal peptide and the IgG Fc fragment. Thus, the pcDNA3.4 F10-RBD-SD1(Wuhan)-Fc (SEQ ID NO: 11) and F10-RBD-SD1(Beta)-Fc (SEQ ID NO: 12) gene constructs were obtained, which can then be used to production of RBD-SD1(Wuhan)-Fc or RBD-SD1(Beta)-Fc recombinant proteins. The scheme for obtaining a gene construct with variants of expression cassettes encoding recombinant proteins based on RBD-SDI-Fc is shown in Fig. 7. As a result of the work, gene constructs 83/1 (Fig. 8), 86/12 (Fig. 9), 78/9 (Fig. 10), and 85/1 (Fig. 11) were obtained. These gene constructs included the following expression cassettes: S-RBD-SD1(Wuhan)-Fc (SEQ ID NO: 27), F10-RBD- SD1(Wuhan)-Fc (SEQ ID NO: 28), S-RBD-SD1 (Beta)-Fc (SEQ ID NO: 29), F10-RBD-SD1(Beta)-Fc (SEQ ID NO: 30). The expression cassettes encoded the following recombinant proteins (with signal sequences): SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. A general scheme of expression cassettes for the production of RBD-SD1-Fc recombinant proteins is shown in fig. 12.

This modular design allows, according to the LEGO Genetics principle, to easily obtain new variants of recombinant proteins - antigens against SARS-CoV-2 and its mutant strains, as well as increase the productivity of the expression system and replace in the expression cassette, in addition to the above modifications, in general, also the following:

1) Replace the region encoding the S-protein of SARS-CoV-2, including RBD and SD1, with alternative ones, incl. mutant and/or homologous, for example against other viruses by treatment with BamHI and HinDIII endonucleases and subsequent ligation.

2) Change the signal sequences for the expression of the recombinant protein to alternative ones, incl. in order to increase the productivity of the expression system or introduce additional post-translational modifications, by treating the initial expression cassette with Nhel and Hindi II endonucleases and subsequent ligation.

3) Change the Fc fragment and the linker to alternative ones, for example, to Fc fragments of other immunoglobulins, incl. to change the pharmacokinetic characteristics of the resulting antigen, by treatment with BamHI and Xhol endonucleases and subsequent ligation.

4) Cloning and excision of the entire signal peptide-RBD-SD1-Fc genetic construct, incl. to replace the promoter by processing the original expression cassette with Xhol and Nhel endonucleases and subsequent cloning into another vector.

At the same time, replacements in the expression cassette should not be less than 85%.

Cell culture for production of recombinant proteins RBD-SDI-Fc.

The resulting gene constructs (83/1, 86/12, 78/9, 85/1) were then generated in preparative amounts and purified using the commercial Plasmid Midiprep 2.0 kit (Evrogen, cat. no. BC124). Recombinant RBD-SDI-Fc proteins were obtained based on the CHO-S cell line and the gene constructs developed above. This is a commercial cell line based on Chinese Hamster Ovary CHO cells, adapted for cultivation in dense suspension in a special culture medium for increased protein expression. CHO-S cells were cultured in a shaker incubator at 5% CO2, 80% humidity, and a shaking speed of 120 rpm in HyClone HyCell TransFx-C transfection media (GE Healthcare, USA) supplemented with 8 mM glutamine (PanEco , Russia), until a density of 2 * 106 cells / ml is reached in 1 liter of suspension. Next, the resulting suspension was transfected with plasmid vectors 83/1 , 86/12, 78/9, and 85/1 using 1 mg/ml Polyethylenimine reagent (Polyscience, USA) according to the manufacturer's instructions. Biosynthesis of recombinant RBD-SDI-Fc proteins was performed in CHO cells in transient expression mode. The transfected cells were then selected for 2 weeks on the antibiotic hygromycin B. This antibiotic was chosen because the plasmid vectors contain the antibiotic resistance gene Hygromycin B. The production of this protein ranged from 5 mg/l to 10 mg/l.

Isolation of recombinant RBD-SDI-Fc proteins was carried out from the culture liquid at the end of cultivation of CHO cells. The cells were pelleted by centrifugation for 5 min at 1000 rpm, the supernatant was transferred into clean tubes and pelleted by centrifugation for 30 min at 4000 rpm. The supernatant was again transferred to clean tubes, 1/10 volume of 10x Tris-HCI buffer (20 mM Tris-HCI, 150 mM NaCI pH 7.2 1x buffer) was added and applied to a pre-equilibrated 1 ml column with HiTrap MabSelect SuRe ( Cytiva, USA). The isolation of protein preparations was carried out on an Acta Pure chromatograph (GE Healthcare, USA) at a liquid flow rate of 2–4 ml/min and a pressure of no more than 0.5 MPa. After loading, the column was washed with 10 column volumes of Tris-HCI buffer. The recombinant RBD-SD1-Fc protein was eluted with citrate buffer (20 mM citric acid-NaOH, 200 mM NaCI, pH 3.0), and protein detection was performed in real time by optical absorption at 280 nm. The fraction containing the desired product was neutralized by adding 1/10 of the volume fraction of neutralization buffer (1 M Tris-HCI pH 8.0). The eluate was dialyzed against two changes of phosphate buffer (10 mM NaH2PO4, 1.7 mM K2HPO4, 137 mM NaCl, KCl 2.7 mM pH=7.4), sterilized by filtration and stored at +4°C for short-term storage (no more than a month) and -20°C for long-term storage.

Authentication.

According to the results of the electropherogram, the mobility of the recombinant RBD-SDI-Fc protein under reducing conditions (+BME) corresponded to =60 kDa, which is due to its glycosylation (the mass of the amino acid sequence without glycosylation is about 64.5 kDa). Under non-reducing conditions (-BME), the molecular weight of the resulting protein was =120-130 kDa, which corresponds to the dimeric state of RBD-SDI-Fc. Thus, during the initial analysis of the drug, its expected molecular weight was confirmed as in both monomeric and dimeric states. The electrophoregram results are shown in Fig. 13.

Preparation of betulin as an adjuvant and buffer solutions.

The adjuvant was prepared using a solution of betulin in tetrahydrofuran (TGF) (OOO Berezovy Mir, Russia). At the first stage, sterilizing filtration was carried out through a nylon membrane (NRG Pall N66 +) with a pore diameter of 0.22 μm. To obtain a homogeneous dispersion of the adjuvant and reduce the concentration of TGF, a 1% solution of betulin was mixed with a 25-fold volume of a sterile 0.05 M Tris buffer solution (pH 7, 0-9.0). The suspension was treated with ultrasound (35–40 kHz) for 10 min.

Preparation of antigenic compositions based on recombinant protein RBD-SD1-Fc and betulin in buffer solution.

The recombinant RBD-SD1-FC protein and a pre-prepared adjuvant in 0.05 M Tris-HCI buffer solution with the addition of 0.15 M NaCI (pH 7.5) were mixed so that the final antigen concentration was 40 μg/ml or 10 μg/ml, and adjuvant - 400 mcg/ml. The components were thoroughly mixed and placed in a shaker in the refrigerator for 3 hours. The vials with RBD-SD1-FC and adjuvant were then stored in a refrigerator at 2-8°C.

Study of the immunogenic properties (antibody titer) of the developed antigenic compositions based on the recombinant RBD-SDI-Fc protein at various times after immunization.

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. The number of animals used in the study was sufficient to assess the immunogenicity of the studied objects, and also made it possible to subject the results of the study to statistical processing. At the same time, the number of animals was minimal in terms of ethical principles. The animals were kept under standard conditions (Table 2), 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 3 shows preparations based on recombinant RBD-SDI-Fc proteins for animal immunization and study design. Physiological saline (0.9% NaCl) (Medpolimer, Russia) was used as a 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 (Fig. 14). 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. Serum was stored at -20°C until use. 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 a posteriori pairwise comparisons of groups with each other. The group significance level was taken equal to a = 0.05. Differences were considered significant at p < a.

Linked immunosorbent assay.

The presence of specific class G immunoglobulins (IgG) was determined in mouse sera samples after double immunization. The protein (SARS-CoV-2; mutant RBD-Fc Lot:2452-139-066 2.95mg/ml (0.22mkm Filt) Gly/TRIS/HCI pH 7.4) provided for the study was used as an antigen. Sorption of the antigen diluted in sterile DPBS to a concentration of 1.0 µg/ml was performed overnight at +4°C in Maxisorp 96-well plates (Nunc) (100 µl/well). The plates were then washed twice with a solution of 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 dilution of 1:400 for the groups of animals that received vaccine preparations and from a dilution of 1:50 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 (Abeam) at a dilution of 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 1 N H2SO4 (Vekton, Russia). Optical density (OD) at a wavelength of 450 nm was measured on a multiplate reader with LVF monochromators CLARIOstar (BMG LABTECH).

For four vaccine preparations, after double immunization at a dose of 5 μg and 20 μg, the level of specific antibodies to the mutant RBD-Fc IgG protein in the sera of immunized animals was determined using an indirect ELISA using an experimental test system. The results of determining the level of specific antibodies are shown in Fig. 15. 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 mutant RBD-Fc protein after a double immunization with both vaccine preparations in both doses used.

Microneutralization reaction.

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 the 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 was 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. Petersburg No. 77. PCh.01.000. M.000053.08.20 dated 08.05.2020 for work with PBA II-IV pathogenicity groups).

The results of determining the level of neutralizing antibodies against the SARS-CoV-2 coronavirus in the blood sera of mice after double immunization showed that virus-neutralizing antibodies in an amount significantly different from the placebo group are formed only in the group of animals that received an antigenic composition containing the recombinant protein RBD- SDI-Fc strain Wuhan at a dose of 20mcg. The level of virus-neutralizing antibodies formed in other experimental groups did not differ significantly from that in the placebo group. The results are shown in FIG. 16. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by pairwise comparison of groups using Tukey's test.

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 that received an antigenic composition containing the recombinant RBD-SD1-Fc protein of the Wuhan strain at a dose of 20 μg.

Study of the immunogenic properties (assessment of the cellular immune response) of the developed antigenic compositions based on the recombinant RBD-SDI-Fc protein at various times after immunization.

To assess the T-cell immune response, C57/black SPF mice (n=5 per group) were injected with RBD1 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 control. The study design is presented in Table. 4.

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 SARS-CoV-2 S protein peptide pool (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, IFNy-BV780, TNFa-BV421, IL2-PE (BioLegend). Dead cells were detected using Zombie Red (BioLegend). True Stain containing anti-CD16/CD32 (Bio-Legend) 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+ tag cells was measured after two immunizations of 20 µg, while immunization with 5 µg of antigen resulted in a statistically significant increase in CD4+ Tern only (p<0.05). There was a trend towards dose-dependent growth of cells producing TM-cytokine (IFNy, IL-2, TNFa) among both Tern populations. The effect reached significance for Tern producing IL-2 (IFNy-IL-2 + TNFa-) and for multifunctional CD8+ T cells (IFNy + IL-2 + TNFa+) in both dosing groups. In the high dose group, the increase in IL-2 produced by CD8+ Tern was also significant (FIG. 17).

Claims

Claim

1. An antigen for inducing immunity against the SARS-CoV-2 virus, which is a recombinant protein that includes a region of the S-protein of the SARS-CoV-2 virus, consisting of amino acid residues 302-638 of the RBD and SD1 domains, fused to the Fc fragment of IgG through polyglycine -serine linker.

2. The antigen of claim 1, wherein the polyglycine-serine linker has the sequence shown in SEQ ID NO: 19.

3. Antigen according to claim 1, having the sequence represented by SEQ ID NO: 3.

4. The antigen of claim 1 having the sequence shown in SEQ ID NO: 8.

5. An antigenic composition for inducing immunity against the SARS-CoV-2 virus, containing an antigen according to any of paragraphs 1-4, a buffer solution and an adjuvant, where a corpuscular adjuvant based on triterpenoids from betulin birch bark is used as an adjuvant.

6. Use of an antigenic composition according to claim 5 to induce an immune response against the SARS-CoV-2 virus.

7. Use according to claim 6, wherein the antigenic composition is administered intramuscularly.

8. Use according to claim 6, wherein the induction of immunity is the induction of antibody and cellular immunity.

9. A gene construct for the expression of a recombinant protein comprising a portion of the S protein of the SARS-CoV-2 virus, consisting of amino acid residues 302-638 of the RBD and SD1 domains, fused to an IgG Fc fragment via a polyglycine-serine linker, including an expression cassette consisting of sections encoding:

- SARS-CoV-2 S-protein signal peptide or immunoglobulin anti-TNF antibody F10 signal peptide,

- a section from 302 to 638 amino acid residues of the S-protein RBD-SD1 of the SARS-CoV-2 virus,

- polyglycine-serine linker,

- Fc fragment of IgG.

10. The gene construct according to claim 9, wherein the SARS-CoV-2 S protein signal peptide coding sequence is shown in SEQ ID NO: 4.

11. The gene construct of claim 9, wherein the coding sequence for the signal peptide of the anti-TNF immunoglobulin antibody F10 is shown in SEQ ID NO: 10.

12. The gene construct according to claim 9, wherein the coding sequence of the SARS-CoV-2 S protein region has the sequence shown in SEQ ID NO: 5.

13. The gene construct according to claim 9, wherein the coding sequence of the SARS-CoV-2 S protein region has the sequence shown in SEQ ID NO: 9.

14. Gene construct according to claim 9, in which the polyglycine-serine linker has the sequence shown by SEQ ID NO: 19.

15. The gene construct according to claim 9, wherein the Fc coding sequence of the IgG fragment is represented by SEQ ID NO: 6.

16. A gene construct according to claim 9, containing an expression cassette represented by the sequence SEQ ID NO: 27.

17. A gene construct according to claim 9, containing an expression cassette represented by the sequence of SEQ ID NO: 29.

18. A gene construct according to claim 9, containing an expression cassette represented by the sequence of SEQ ID NO: 28.

19. A gene construct according to claim 9, containing an expression cassette represented by the sequence SEQ ID NO: 30.

20. A method for producing a recombinant protein - antigen for inducing immunity against the SARS-CoV-2 virus according to any one of paragraphs 1-4, including:

- transfection of Chinese hamster ovary cells CHO-S gene construct according to any one of paragraphs 9-19;

- cultivation of transfected Chinese hamster ovary cells CHO-S;

- selection of culture medium;

- chromatographic purification of the resulting recombinant protein;

- confirmation of the authenticity of the obtained recombinant protein.

21. The method of production according to claim 20, in which the recombinant protein has the sequence represented by SEQ ID NO: 3.

22. The production method according to claim 20, wherein the recombinant protein has the sequence represented by SEQ ID NO: 8.