Use of drugs (mutants) for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-COV-2 for revaccination of the population

Abstract

The present invention relates to the fields of biotechnology, immunology and virology. The use of a drug is disclosed, the drug containing component 1, which is a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the ORF6-Ad26 region has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated, and / or component 2, which is a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated. In addition, a drug is used, which contains component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 sites have been deleted and the site ORF6-Ad26 has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, and also contains component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 sites have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, or contains only component 2. Furthermore, a drug is used, which contains component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated, and also contains component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The present invention enables the production of safe and effective drugs that provide extended post-vaccination immunity against the virus SARS-CV-2 and is intended for revaccination of the population against diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the fields of biotechnology, immunology and virology. The use of a drug for the revaccination of a population against the disease caused by the severe acute respiratory syndrome virus SARS-CoV-2. [Background technology]

[0002] Background of the Invention Coronaviruses are a large family of viruses that cause a wide range of diseases in humans and animals. In late 2019, the world was confronted with a zoonotic novel beta-coronavirus, SARS-CoV-2, which caused the coronavirus disease (COVID-19) pandemic in Wuhan, People's Republic of China (PRC). On March 11, 2020, the World Health Organization described the global spread of this disease as a pandemic. As of February 1, 2021, more than 100 million cases of COVID-19 disease had been recorded, and over 2 million people had died.

[0003] The most common symptoms of COVID-19 include fever, dry cough, difficulty breathing, and fatigue. Sore throat, joint pain, runny nose, and headache are rare. The disease can be mild or severe. Older age and the presence of chronic diseases are risk factors.

[0004] Following illness, both cell-mediated and antibody-mediated immune responses are formed. SARS-CoV-2-specific CD8+ and CD4+ T cells are found in 70% and 100% of recovered COVID-19 patients, respectively. The SARS-CoV-2 S protein is the primary target of T cells. In addition, T cells specific for the M and N coronavirus proteins have been found, with fewer T cells specific for SARS-CoV-2 nsp3, nsp4, ORF3a, and ORF8. The immune response is biased toward Th1 (Grifoni et al. Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell. 2020 Jun 25; 181(7): 1489-1501.e15).

[0005] The antibody immune response is primarily mediated by antibodies targeting the coronavirus surface S protein. The RBD of the S glycoprotein, which is involved in binding to the ACE-2 receptor on human cells, has been shown to be the primary target of virus-neutralizing antibodies. The kinetics of the antibody-mediated immune response to SARS-CoV-2 is characterized by sustainable seroconversion (IgM and IgG) within 7–14 days after symptom onset. IgG titers increase during the first 3 weeks and begin to decline by week 8 (Adams ER, Ainsworth M, Anand R. Antibody testing for COVID-19: a report from the National COVID Scientific Advisory Panel. medRxiv. 2020). Furthermore, IgG titers have been demonstrated to correlate with disease severity (Gregory A Poland et al. SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates. Lancet. 2020 14–20 November; 396(10262): 1595–1606).

[0006] Accumulating scientific data to date indicates that natural immunity to COVID-19 developed in subjects who have experienced the disease is somewhat short-lived (https: / / www.cdc.gov / coronavirus / 2019-ncov / vaccines / facts.html), as evidenced by the observed trend of declining antibody levels and cases of coronavirus reinfection (Akiko Iwasaki. What reinfections mean for COVID-19. Lancet Infect Dis. 2021 Jan; 21(1): 3-5).

[0007] Vaccination is the most effective method of preventing infectious diseases. To date, several COVID-19 vaccines have been developed, which are based on various coronavirus antigens.

[0008] 1) Vaccines that contain the whole virus as the antigen. There are four known candidate inactivated vaccines developed in China: three vaccines (Sinovac in collaboration with the National Institute for Prevention and Control of Infectious Diseases; Sinopharm in collaboration with the Wuhan Institute of Biological Products and the Wuhan Institute of Virology of the Chinese Academy of Sciences; and Sinopharm in collaboration with the Beijing Institute of Biological Products and the Institute of Control and Prevention of Viral Diseases) are currently in Phase III of their clinical programs, and one vaccine (Institute of medical biology, Chinese Academy of Medical Sciences) is in Phase I / II of their clinical programs. (Zhang Y et al. Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: a randomised, double-blind, placebo-controlled, phase 1 / 2 clinical trial. Lancet Infect Dis. 2021 Feb;21(2):181-192;Xia S et al. Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety and Immunogenicity Outcomes: Interim Analysis of 2 Randomized Clinical Trials. JAMA. 2020 Sep 8;324(10):951-960.Xia S et al Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV: a randomised, double-blind, placebo-controlled, phase 1 / 2 trial. Lancet Infect Dis. 2021 Jan;21(1):39-51.doi: 10.1016 / S1473-3099(20)30831-8. Epub 2020 Oct 15. PMID: 33069281;PMCID: PMC7561304.Che Y et al. Randomized, double-blinded and placebo-controlled phase II trial of an inactivated SARS-CoV-2 vaccine in healthy adults. Clin Infect Dis. 2020 Nov 9).

[0009] 2) A vaccine containing the full-length S protein as an antigen. Three vaccines based on adenoviruses of various serotypes expressing the full-length S protein gene of SARS-CoV-2 are known: CanSino Biological Inc. and the Beijing Institute of Biotechnology developed a vaccine based on human adenovirus serotype 5; Oxford University and AstraZeneca developed a vaccine based on chimpanzee adenovirus; and FGBU NFGamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of Russia developed vaccines based on human adenovirus serotypes 26 and 5. In addition, a DNA vaccine containing the full-length S protein gene of SARS-CoV-2 is known and was developed by Inovio Pharmaceuticals in collaboration with the International Vaccine Institute. (Zhu F et al. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet. 2020 Jun 13;395(10240):1845-1854.van Doremalen N et al. ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature. 2020 Oct;586(7830):578-582.Logunov DY et al. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1 / 2 studies from Russia. Lancet. 2020 Sep 26;396(10255):887-897).

[0010] 3) A vaccine in which the full-length S protein with two proline substitutions (K986P and V987P) serves as the antigen. Two vaccines based on lipid nanoparticles containing mRNA encoding the SARS-CoV-2 S protein with proline substitutions are known (Moderna in collaboration with the National Institute of Allergy and Infectious Diseases; BioNTech in collaboration with Fosun Pharma and Pfizer). Additionally, a protein subunit vaccine developed by Novavax is known; in that vaccine, the full-length SARS-CoV-2 S protein has two proline substitutions (K986P and V987P) and three mutations in the furin cleavage site (R682Q, R683Q, and R685Q). Another vaccine based on human adenovirus serotype 26 expressing the full-length SARS-CoV-2 S protein with two proline substitutions (K986P and V987P) and two mutations in the furin cleavage site (R682S and R685G) is being developed by Janssen Pharmaceutical Companies. (L. Baden et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2020 Dec 30;L. Jackson An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med. 2020 Nov 12;383(20):1920-1931.Keech C et al. Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine. N Engl J Med. 2020 Dec 10;Tostanoski L et al. Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters. Nat Med. 2020 Nov;26(11):1694-1700.doi: 10.1038 / s41591-020-1070-6. Epub 2020 Sep 3).

[0011] 4) A vaccine containing the RBD of the S protein as an antigen. A protein subunit vaccine containing an RBD dimer (residues 319-537 as a tandem repeat) is being developed by Anhui Zhifei Longcom Biopharmaceutical in collaboration with the Institute of Microbiology of the Chinese Academy of Sciences. Additionally, a lipid nanoparticle-based vaccine containing mRNA encoding an RBD trimer (trimerization by the addition of the foldon domain from T4 fibritin) is known (collaboration between BioNTech, Fosun Pharma, and Pfizer) (Mulligan M. et al. Phase I / II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature. 2020 Oct;586(7830):589-593. Dai L. A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS. Cell. 2020 Aug 6;182(3):722-733.e11. Dai L et al. Viral targets for vaccines against COVID-19. Nat Rev Immunol. 2021 Feb;21(2):73-82).

[0012] Currently, eight vaccines for the prevention of COVID-19 have been approved worldwide. Clinical studies have shown that immunization with these vaccines results in the generation of both antibody- and cellular-mediated immune responses against SARS-CoV-2. However, the extent to which each type of vaccine provides durable post-vaccination protective immunity has yet to be established. Experts believe that this may show interindividual variability and may last for 1 to 2 years, according to various estimates. This duration dictates the need for the development of drugs specific for COVID prevention to be used in human revaccination.

[0013] However, the selection of a revaccination agent is a difficult task.

[0014] In the process of developing specific prophylactic agents targeting revaccination, one must bear in mind the effects that arise in humans from booster immunizations, which have a considerable impact on the overall structure of anti-infective immunity, including its protective properties.

[0015] It is known that the use of vaccines containing multiple antigens (e.g., inactivated vaccines) results in the formation of an immune response against each antigen. However, in this case, the level of immunity to a given antigen is lower than that of a vaccine containing only this one antigen (dilution effect of the immune response). Furthermore, some antigens within the pathogen structure may not be protective, and the formation of T cell and B cell clones in response to such antigens may not contribute to the overall protectiveness of immunity and may prevent the formation of cell clones that are important for protection.

[0016] Based on the above, it can be concluded that vaccines containing one or more proteins with significant protective properties are more promising for revaccination, which would then involve an additional stimulus (booster) and (equally important) focusing the immune response on antigenic determinants of the pathogen, which are most important for human protection, regardless of initial immunization.

[0017] No drugs for revaccination against coronavirus infection are known from the current state of the art.

[0018] The technical solution disclosed in RF Patent No. 2731342 (published 01.09.2020) was selected as a prototype by the authors of the claimed invention. A variant of an agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 is known from this patent. - containing a component 1, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated, and also containing a component 2, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated. - containing a component 1, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the ORF6-Ad26 region has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated, and also containing a component 2, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated. - A pharmaceutical composition comprising component 1, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:3 has been incorporated, and also comprising component 2, which is a pharmaceutical agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 has been incorporated.

[0019] Additionally, the patent discloses the use of the indicated variants of the drug for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2, which comprises administering effective amounts of component 1 and component 2 consecutively with a time interval of at least one week.

[0020] A disadvantage of this drug is that its use for the extension of post-vaccination immunity has not been described.

[0021] The background to the present invention therefore demonstrates the need for the development of agents that can be used for revaccination against diseases caused by the SARS-CoV-2 virus. Summary of the Invention [Problem to be solved by the invention]

[0022] Disclosure of the Invention The technical problem of the claimed inventions is the development of a drug that provides extended post-vaccination immunity against the SARS-CoV-2 virus. [Means for solving the problem]

[0023] The technical result is the creation of a safe and effective drug that provides extended post-vaccination immunity against the SARS-CoV-2 virus.

[0024] Said technical result is achieved by the disclosure of the use of a drug, component 1, in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been integrated, and / or a drug, component 2, in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been integrated, for revaccination against the disease caused by the severe acute respiratory syndrome virus SARS-CoV-2.

[0025] In addition, the use of a drug for revaccination against a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2 is disclosed, said drug containing component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 sites have been deleted and the site ORF6-Ad26 has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, and also containing component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 sites have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, or only containing component 2.

[0026] In addition, the use of another agent for revaccination against a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2 is disclosed, which agent contains component 1, an agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, and also contains component 2, an agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated.

[0027] The agent is used in liquid or lyophilized form.

[0028] Additionally, the buffer for the liquid form may contain, in weight percent: Tris 0.1831~0.3432 Sodium chloride 0.3313~0.6212 Sucrose 3.7821~7.0915 Magnesium chloride hexahydrate 0.0154~0.0289 EDTA 0.0029~0.0054 Polysorbate-80 0.0378~0.0709 Ethanol 95% 0.0004~0.0007 Water remaining Contains:

[0029] Additionally, the reconstituted lyophilized drug contains, in wt %: Tris 0.0180~0.0338 Sodium chloride 0.1044~0.1957 Sucrose 5.4688~10.2539 Magnesium chloride hexahydrate 0.0015~0.0028 EDTA 0.0003~0.0005 Polysorbate-80 0.0037~0.0070 Water remaining The buffer solution comprises:

[0030] Additionally, during use, component 1 and component 2 are in separate containers. [Brief explanation of the drawings]

[0031] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 illustrates the results of the evaluation of the efficacy of immunization in volunteers with the liquid formulation of the development drug according to variant 1, by assessing the percentage of proliferating CD8+ lymphocytes restimulated with the S antigen of SARS-CoV-2. Y-axis - amount of proliferating cells (%). X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 2] Figure 1 illustrates the results of the evaluation of the efficacy of immunization in volunteers with the liquid formulation of the development drug according to variant 1, by assessing the percentage of proliferating CD4+ lymphocytes restimulated with the S antigen of SARS-CoV-2. Y-axis - amount of proliferating cells (%). X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 3]Figure 1 illustrates the results of the evaluation of the efficacy of immunization in volunteers with a lyophilized form of the development drug according to variant 1, by assessing the percentage of proliferating CD8+ lymphocytes restimulated with the S antigen of SARS-CoV-2. Y-axis - amount of proliferating cells (%). X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 4] Figure 1 illustrates the results of the evaluation of the efficacy of immunization in volunteers with a lyophilized form of the development drug according to variant 1, by assessing the percentage of proliferating CD4+ lymphocytes restimulated with the S antigen of SARS-CoV-2. Y-axis - amount of proliferating cells (%). X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 5]Figure 1 illustrates the fold increase in IFNγ concentrations in the culture medium of peripheral blood mononuclear cells of volunteers immunized with the liquid form of the development drug according to variant 1, after restimulation with the S antigen of SARS-CoV-2, before immunization (day 0) and on days 14 and 28 of the study. Y-axis - fold increase in interferon-gamma concentrations. X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 6] Figure 1 illustrates the fold increase in IFNγ concentrations in the culture medium of peripheral blood mononuclear cells of volunteers immunized with a lyophilized form of the development drug according to variant 1, after restimulation with the S antigen of SARS-CoV-2, before immunization (day 0) and on days 14 and 28 of the study. Y-axis - fold increase in interferon-gamma concentrations. X-axis - number of days. ● - indicates the individual value for each volunteer on day 0. ■ - indicates the individual value for each volunteer on day 14. ▲ - indicates the individual value for each volunteer on day 28. Dots indicate the individual value for each volunteer who participated in the study. The median is indicated by a black line for each data set. Statistical significance between the values ​​obtained on days 0, 14 and 28 is indicated by brackets and the symbols *, p<0.05; **, p<0.01; ****, p<0.001, using the Mann-Whitney test. [Figure 7] Figure 1 illustrates the results of the evaluation of antibody-mediated immune responses against antigens of SARS-CoV-2 in volunteers immunized with the liquid form of the development drug according to variant 1. Y-axis - IgG titers against the RBD of the S glycoprotein of SARS-CoV-2. X-axis - number of days.

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[0032] The first step in developing a drug to induce specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 was the selection of a vaccine antigen. As part of this study, a literature review was conducted and demonstrated that the coronavirus S protein was the most promising antigen for generating a candidate vaccine. This type I transmembrane glycoprotein is involved in the binding, fusion, and entry of virus particles into cells. As shown, it induces the production of neutralizing antibodies (Liang M et al., SARS patient-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity. Biomed Environ Sci. 2005 Dec;18(6):363-74).

[0033] The authors developed different variants of the expression cassette to achieve the most effective induction of the immune response against the S protein of SARS-CoV-2.

[0034] Implementing the invention The expression cassette SEQ ID NO:1 contains the CMV promoter, the gene encoding the SARS-CoV-2 S protein, and a polyadenylation signal. The CMV promoter is a promoter for early cytomegalovirus genes and provides constitutive expression in many cell types. However, the intensity of target gene expression controlled by the CMV promoter varies depending on the cell type. In addition, it has been shown that the level of transgene expression controlled by the CMV promoter decreases with increasing cell culture time due to DNA methylation-related inhibition of gene expression [Wang W., Jia YL., Li YC., Jing CQ., Guo X., Shang XF., Zhao CP., Wang TY. Impact of different promoters, promoter mutations, and an enhancer on recombinant protein expression in CHO cells. / / Scientific Reports - 2017. - Vol. 8. - P. 10416].

[0035] The expression cassette SEQ ID NO:2 contains the CAG promoter, the gene encoding the SARS-CoV-2 S protein, and a polyadenylation signal. The CAG promoter is a synthetic promoter that drives the CMV promoter early enhancer, the chicken β-actin promoter, and a chimeric intron (chicken β-actin and rabbit β-globin). Experiments have shown that the transcriptional activity of the CAG promoter is higher than that of the CMV promoter [Yang CQ, Li XY, Li Q., ​​Fu SL, Li H., Guo ZK, Lin JT, Zhao ST. Evaluation of three different promoters driving gene expression in developing chicken embryos by in vivo electroporation. / / Genet. Mol. Res. - 2014. - Vol. 13. - P. 1270-1277].

[0036] The expression cassette SEQ ID NO:3 contains the EF1 promoter, the gene encoding the SARS-CoV-2 S protein, and a polyadenylation signal. The EF1 promoter is the promoter for human eukaryotic translation elongation factor 1β (EF-1α). This promoter is constitutively active in a wide range of cell types [PMID: 28557288. The EF-1α promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells]. The gene EF-1α encodes elongation factor 1α, one of the most common proteins in eukaryotic cells, and is expressed in almost all mammalian cell types. EF-1α is often active in cells where viral promoters are unable to express controlled genes and where viral promoters are gradually lost.

[0037] The expression cassette SEQ ID NO: 4 contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal.

[0038] An adenovirus-based vector system was chosen to effectively deliver the gene encoding the S protein of the SARS-CoV-2 coronavirus into the human body. Adenovirus vectors offer several advantages: they cannot replicate in human cells, can enter both dividing and non-dividing cells, can induce cellular and antibody-mediated immune responses, and provide high levels of target antigen expression.

[0039] The authors developed a variant of the drug containing two components based on different adenovirus serotypes, in which any immune response against the adenovirus vector portion that may occur after administration of the first component of the drug is not boosted in the future and does not affect the generation of antigen-specific immune responses against the vaccine antigen.

[0040] Furthermore, the developed agent expands the armamentarium of agents for inducing an immune response against the SARS-CoV-2 coronavirus, which would offer to overcome the difficulties arising from the existence of pre-existing immune responses to some adenovirus serotypes in parts of the population.

[0041] Thus, efforts have resulted in the development of the following drug variants: 1. A drug for revaccination against a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2, comprising component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been integrated, and / or component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been integrated. 2. A drug for revaccination against a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2, comprising component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, and also comprising component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions have been deleted from the genome and in which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated, or comprising component 2 alone. 3. A drug for revaccination against diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2, comprising component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated, and also comprising component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 has been incorporated. [Example]

[0042] Example 1 Obtaining an expression vector containing the genome of a recombinant strain of human adenovirus serotype 26 In step 1, the authors developed the design of a plasmid construct, pAd26-Ends, which carries two sites of homology with the genome of human adenovirus serotype 26 (two homology arms) and an ampicillin resistance gene. One homology arm is the viral genome sequence containing the start of human adenovirus serotype 26 (from the left inverted terminal repeat to the E1 site) and the pIX protein. The second homology arm contains the nucleotide sequence from ORF3 of the E4 site to the end of the genome. The pAd26-Ends construct was synthesized by ZAO "Eurogene" (Moscow).

[0043] Human adenovirus serotype 26 DNA isolated from viral particles was mixed with the pAd26-Ends construct, and homologous recombination between pAd26-Ends and the viral DNA resulted in the generation of the plasmid pAd26-dlE1, which contains the human adenovirus serotype 26 genome with a deleted E1 region.

[0044] Next, in the resulting plasmid pAd26-dlE1, the sequence containing open reading frame 6 (ORF6-Ad26) was replaced with the analogous sequence from human adenovirus serotype 5 using conventional cloning methods to enable efficient replication of human adenovirus serotype 26 in HEK293 cell culture, resulting in the plasmid pAd26-dlE1-ORF6-Ad5.

[0045] Next, to enhance the vector's packaging capacity, the E3 region of the adenovirus genome (approximately 3321 bp between the pill gene and the U-exon) was deleted from the construct plasmid pAd26-dlE1-ORF6-Ad5 using conventional genetic engineering techniques. This resulted in the recombinant vector pAd26-only-null, which contains the open reading frame ORF6 of human adenovirus serotype 5 and is based on the genome of a recombinant strain of human adenovirus serotype 26 in which the E1 and E3 regions of the genome have been deleted. SEQ ID NO: 5 was used as the maternal sequence of human adenovirus serotype 26.

[0046] In addition, the authors developed several designs of expression cassettes: - the expression cassette SEQ ID NO: 1 contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 2 contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 3 contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2 and a polyadenylation signal.

[0047] Using the plasmid construct pAd26-Ends, we used genetic engineering techniques to obtain constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, and pArms-26-EF1-S-CoV2, each containing the expression cassette SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and also carrying homology arms of the genome of human adenovirus serotype 26. Next, constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, and pArms-26-EF1-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, and each plasmid was mixed with the recombinant vector pAd26-only-null. By homologous recombination, the plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, and pAd26-only-EF1-S-CoV2 were obtained, which carry the genome of a recombinant strain of human adenovirus serotype 26 containing the open reading frame ORF6 of human adenovirus serotype 5 and a deletion of the E1 and E3 regions of the genome, and which contain the expression cassettes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0048] In step 4, the plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, and pAd26-only-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion, and the resulting DNA products were used to transfect HEK293 cell cultures.

[0049] Thus, an expression vector was obtained containing the genome of a recombinant strain of human adenovirus serotype 26 in which the E1 and E3 regions have been deleted from the genome and the site ORF6-Ad26 has been replaced by ORF6-Ad5, and incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

[0050] Example 2 Obtaining an immunobiological agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been integrated. In this study, anion exchange and exclusion chromatography was used to purify the expression vector obtained in Example 1. The resulting suspension contained adenoviral particles in a buffer solution for the liquid form of the drug or in a buffer solution for the lyophilized form of the drug.

[0051] Thus, the following immunobiological agents were obtained based on the genome of a recombinant strain of human adenovirus serotype 26 in which the E1 and E3 regions had been deleted from the genome and the region ORF6-Ad26 had been substituted for ORF6-Ad5: 1. An immunobiological agent (Ad26-CMV-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and comprising the expression cassette SEQ ID NO: 1, which contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 2. An immunobiological agent (Ad26-CMV-S-CoV2) in a buffer solution for a lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been substituted for ORF6-Ad5, and which comprises the expression cassette SEQ ID NO: 1, which contains the CMV promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 3. An immunobiological agent (Ad26-CAG-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and which contains the expression cassette SEQ ID NO: 2, which contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 4. An immunobiological agent (Ad26-CAG-S-CoV2) in a buffer solution for a lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been substituted for ORF6-Ad5, and which comprises the expression cassette SEQ ID NO: 2, which contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 5. Immunobiological agent (Ad26-EF1-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and which comprises the expression cassette SEQ ID NO: 3, which contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 6. An immunobiological agent (Ad26-EF1-S-CoV2) in a buffer solution for a lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions have been deleted from the genome and the region ORF6-Ad26 has been replaced by ORF6-Ad5, and which comprises the expression cassette SEQ ID NO: 3, which contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal.

[0052] Each of the immunobiological agents provided is a component 1 of variant 1 and variant 2 of the development agent.

[0053] Example 3 Obtaining an expression vector containing the genome of a recombinant strain of simian adenovirus serotype 25 In step 1, we developed the design of a plasmid construct, pSim25-Ends, which contains two homologous regions (two homology arms) to the genome of simian adenovirus serotype 25. One homology arm is the sequence from the beginning of simian adenovirus serotype 25 (from the left inverted terminal repeat to the E1 site) and from the end of the E1 site to the pIVa2 protein. The second homology arm contains the terminal nucleotide sequence of the adenovirus genome, including the right inverted terminal repeat. The pSim25-Ends construct was synthesized by ZAO "Eurogene" (Moscow).

[0054] Simian adenovirus serotype 25 DNA isolated from viral particles was mixed with pSim25-Ends. Homologous recombination between pSim25-Ends and the viral DNA generated the plasmid pSim25-dlE1, which contains the E1-deleted simian adenovirus serotype 25 genome.

[0055] Next, to enhance the vector's packaging capacity, the E3 region of the adenovirus genome (3921 bp, from the start of gene 12.5K to gene 14.7K) was deleted from the construct plasmid pSim25-dlE1 using conventional genetic engineering techniques. This resulted in the plasmid construct pSim25-null, which encodes the full-length genome of simian adenovirus serotype 25, with the E1 and E3 regions of the genome deleted. SEQ ID NO: 6 was used as the maternal sequence of simian adenovirus serotype 25.

[0056] In addition, the authors developed several designs of expression cassettes: - the expression cassette SEQ ID NO: 4 contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 2 contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 3 contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2 and a polyadenylation signal.

[0057] Using the plasmid construct pSim25-Ends, we used genetic engineering techniques to obtain constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, and pArms-Sim25-EF1-S-CoV2, each containing the expression cassette SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:3, and also carrying homology arms from the genome of simian adenovirus serotype 25. Next, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, and pArms-Sim25-EF1-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, and each plasmid was mixed with the recombinant vector pSim25-null. Homologous recombination yielded the recombinant plasmid vectors pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, and pSim25-EF1-S-CoV2, which contain the full-length genome of simian adenovirus serotype 25 with deleted E1 and E3 regions and the expression cassettes SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0058] In step 3, the plasmids pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, and pSim25-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion. The resulting DNA products were used to transfect HEK293 cell cultures. The resulting material was used to accumulate preparative amounts of recombinant adenovirus.

[0059] As a result of the research, 25 human adenovirus serotypes containing the gene encoding the S protein of SARS-CoV-2 were obtained: simAd25-CMV-S-CoV2 (containing expression cassette SEQ ID NO: 4), simAd25-CAG-S-CoV2 (containing expression cassette SEQ ID NO: 2), and simAd25-EF1-S-CoV2 (containing expression cassette SEQ ID NO: 3).

[0060] Thus, an expression vector was obtained containing the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions had been deleted from the genome, and incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3.

[0061] Example 4 Obtaining an immunobiological agent in the form of an expression vector, which is based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions have been deleted from the genome, and which incorporates an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 In this study, anion exchange and exclusion chromatography was used to purify the expression vector obtained in Example 3. The resulting suspension contained adenoviral particles in a buffer solution for the liquid form of the drug or in a buffer solution for the lyophilized form of the drug.

[0062] Thus, the following immunobiological agents were obtained based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome: 1. An immunobiological agent (simAd25-CMV-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome, and comprising the expression cassette SEQ ID NO: 1, which contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 2. An immunobiological agent (simAd25-CMV-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 1 containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 3. An immunobiological agent (simAd25-CAG-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 2 containing the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 4. An immunobiological agent (simAd25-CAG-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 2, which contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 5. An immunobiological agent (simAd25-EF1-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 6. An immunobiological agent (simAd25-EF1-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of simian adenovirus serotype 25 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal.

[0063] Each of the immunobiological agents provided is component 2 of variant 1 and component 1 of variant 3 of the development agent.

[0064] Example 5 Obtaining an expression vector containing the genome of a recombinant strain of human adenovirus serotype 5 In step 1, the design of the plasmid construct pAd5-Ends was developed, which carries two sites of homology (two homology arms) with the genome of human adenovirus serotype 5. One homology arm is the sequence containing the start of human adenovirus serotype 5 (from the left inverted terminal repeat to the E1 site) and the pIX protein of the viral genome. The second homology arm contains the nucleotide sequence from after the E4 site ORF3 to the end of the genome. The pAd5-Ends construct was synthesized by ZAO "Eurogene" (Moscow).

[0065] Human adenovirus serotype 5 DNA isolated from viral particles was mixed with the pAd5-Ends construct. Homologous recombination between pAd5-Ends and the viral DNA resulted in the generation of the plasmid pAd5-dlE1, which contains the human adenovirus serotype 5 genome with a deleted E1 region.

[0066] Next, to enhance the vector's packaging capacity, the E3 region of the adenovirus genome (approximately 2685 bp from the end of gene 12.5K to the start of the U-exon sequence) was deleted from the construct plasmid pAd5-dlE1 using conventional genetic engineering techniques. This resulted in the recombinant plasmid vector pAd5-too-null, based on the genome of human adenovirus serotype 5, in which E1 and E3 were deleted from the genome. SEQ ID NO: 7 was used as the maternal sequence of human adenovirus serotype 5.

[0067] In addition, the authors developed several designs of expression cassettes: - the expression cassette SEQ ID NO: 1 contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 2 contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; - the expression cassette SEQ ID NO: 3 contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2 and a polyadenylation signal.

[0068] Next, based on the plasmid construct pAd5-Ends, genetic engineering techniques were used to obtain constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, and pArms-Ad5-EF1-S-CoV2, which contain the expression cassettes SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and also have homology arms of the genome of human adenovirus serotype 5.

[0069] Next, constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, and pArms-Ad5-EF1-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, and each plasmid was mixed with the recombinant vector pAd5-too-null. Homologous recombination resulted in the plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, and pAd5-too-EF1-S-CoV2, which carried the genome of a recombinant strain of human adenovirus serotype 5 in which the E1 and E3 regions had been deleted from the genome, and the expression cassettes SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0070] In step 4, the plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, and pAd5-too-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion. The resulting DNA products were used to transfect HEK293 cell cultures. The resulting material was used to accumulate preparative amounts of recombinant adenovirus.

[0071] As a result of the research, human adenovirus serotype 5 containing the gene encoding the S protein of SARS-CoV-2 was obtained: Ad5-CMV-S-CoV2 (containing expression cassette SEQ ID NO: 1), Ad5-CAG-S-CoV2 (containing expression cassette SEQ ID NO: 2), Ad5-EF1-S-CoV2 (containing expression cassette SEQ ID NO: 3).

[0072] Thus, an expression vector was obtained containing the genome of a recombinant strain of human adenovirus serotype 5 in which the E1 and E3 regions had been deleted from the genome, and incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0073] Example 6 Obtaining an immunobiological agent in the form of an expression vector, based on the genome of a recombinant strain of human adenovirus serotype 5, in which the E1 and E3 regions have been deleted from the genome, and incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 In this study, anion exchange and exclusion chromatography was used to purify the expression vector obtained in Example 5. The resulting suspension contained adenoviral particles in a buffer solution for the liquid form of the drug or in a buffer solution for the lyophilized form of the drug.

[0074] Thus, the following immunobiological agents are based on the genome of a recombinant strain of human adenovirus serotype 5 in which the E1 and E3 regions have been deleted from the genome: 1. An immunobiological agent (Ad5-CMV-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted from the genome, and comprising the expression cassette SEQ ID NO: 1, which contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 2. An immunobiological agent (Ad5-CMV-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted, and comprising the expression cassette SEQ ID NO: 1, which contains a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 3. An immunobiological agent (Ad5-CAG-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 2 containing the CAG promoter, the gene encoding the S protein of SARS-CoV-2 and a polyadenylation signal. 4. An immunobiological agent (Ad5-CAG-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted, and comprising the expression cassette SEQ ID NO: 2, which contains the CAG promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal. 5. An immunobiological agent (Ad5-EF1-S-CoV2) in a buffer solution for the liquid form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5 in which the E1 and E3 regions have been deleted from the genome and comprising the expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene encoding the S protein of SARS-CoV-2 and a polyadenylation signal. 6. An immunobiological agent (Ad5-EF1-S-CoV2) in a buffer solution for the lyophilized form of the agent, based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted, and comprising the expression cassette SEQ ID NO: 3, which contains the EF1 promoter, the gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal.

[0075] Each of the immunobiological agents provided is a component 1 of variant 1 and variant 2 of the development agent.

[0076] Each of the immunobiological agents provided is a component 2 of the development agents Variant 1 and Variant 3.

[0077] Example 7 Preparation of buffer solutions The agent developed in accordance with the claimed invention comprises two components, packaged in separate vials, each of which is an immunobiological agent based on a recombinant adenovirus containing an expression cassette in a buffer solution.

[0078] The authors of the present invention have designed the composition of a buffer solution to ensure the stability of recombinant adenoviral particles. The solution comprises: 1. Tris(hydroxymethyl)aminomethane (Tris) needed to maintain the pH of the solution 2. Sodium chloride is added to achieve the appropriate ionic strength and osmolality 3. Sucrose used as a cryoprotectant 4. Magnesium chloride hexahydrate is required as a source of divalent cations 5. EDTA used as an inhibitor of free radical oxidation 6. Polysorbate-80 used as a surfactant 7. Ethanol 95% used as an inhibitor of free radical oxidation 8. Water used as a solvent

[0079] The authors of the present invention have developed two variants of buffer solutions, one for the liquid form of the drug and one for the lyophilized form of the drug.

[0080] To determine the concentration of compounds in the composition of the buffer solution for the liquid form of the drug, several variants of the experimental group were prepared (Table 1). One of the components of the drug was added to each of the prepared buffer solutions: 1. An immunobiological agent based on recombinant human adenovirus serotype 26, which contains an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; * 10 11Virus particles. 2. Immunobiological agent based on recombinant human adenovirus serotype 5 containing an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal, 1 * 10 11 Virus particles. 3. Immunobiological agent based on recombinant simian adenovirus serotype 25 containing an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal, 1 * 10 11 Virus particles.

[0081] Therefore, the stability of each adenovirus serotype in the pharmaceutical composition was tested: the prepared pharmaceuticals were stored at temperatures of -18°C and -70°C for 3 months, then thawed, and the change in recombinant adenovirus titer was evaluated.

[0082] [Table 1]

[0083] The results of the experiment showed that the titer of the recombinant adenovirus did not change after storage in a buffer solution for the liquid form of the drug at temperatures of -18°C and -70°C for 3 months.

[0084] Therefore, the buffer solution developed for the liquid form of the drug provides stability of all components of the developed drug in the following ranges of active ingredient (wt %): Tris: 0.1831 wt% to 0.3432 wt%; Sodium chloride: 0.3313 wt%~0.6212 wt%; Sucrose: 3,7821% to 7,0915% by weight; Magnesium chloride hexahydrate: 0.0154 wt% to 0.0289 wt%; EDTA:0.0029wt%~0.0054wt%; Polysorbate-80: 0.0378% by weight to 0.0709% by weight; Ethanol 95%: 0.0004 wt%~0.0007 wt%; Solvent: Remainder.

[0085] To determine the concentration of compounds in the composition of the buffer solution for the lyophilized form of the drug, several variants of the experimental group were prepared (Table 2). One of the components of the drug was added to each of the prepared buffer solutions: 1. An immunobiological agent based on recombinant human adenovirus serotype 26, which contains an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal; * 10 11 Virus particles. 2. Immunobiological agent based on recombinant human adenovirus serotype 5 containing an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal, 1 * 10 11 Virus particles. 3. Immunobiological agent based on recombinant simian adenovirus serotype 25 containing an expression cassette containing a CMV promoter, a gene encoding the S protein of SARS-CoV-2, and a polyadenylation signal, 1 * 10 11 Virus particles.

[0086] Therefore, the stability of each adenovirus serotype in the pharmaceutical composition was tested: the prepared pharmaceutical products were stored at temperatures of +2 and +8°C for 3 months, then thawed and the change in recombinant adenovirus titer was evaluated.

[0087] [Table 2]

[0088] The results of the experiment showed that the titer of the recombinant adenovirus did not change after storage in a buffer solution for the lyophilized form of the drug at temperatures of +2°C and +8°C for 3 months.

[0089] Therefore, the buffer solution developed for the lyophilized form of the drug provides stability of all components of the developed drug in the following ranges of active ingredient (wt %): Tris: 0.0180 wt% to 0.0338 wt%; Sodium chloride: 0.1044 wt%~0.1957 wt%; Sucrose: 5,4688% by weight to 10.2539% by weight; Magnesium chloride hexahydrate: 0.0015% by weight to 0.0028% by weight; EDTA:0.0003wt%~0.0005wt%; Polysorbate-80: 0.0037% by weight to 0.0070% by weight; Solvent: Remainder.

[0090] Example 8 Studying the immunogenicity of investigational drugs by assessing the cellular immune response to SARS-CoV-2 viral antigens in the blood of volunteers at different time points after vaccination Clinical studies of the development drug, Mutant 1, included investigating the strength of cell-mediated immunity.

[0091] The 40 volunteers taking part in the study were: 1) Liquid form of the development drug, variant 1: 1x10 11 dose of viral particles, followed by immunization with component 2 within 21 days after immunization with component 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2) (20 volunteers). 2) Lyophilized form of the development drug, Mutant 1: 1x10 11 dose of viral particles, followed by immunization with component 2 within 21 days after immunization with component 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2) (20 volunteers).

[0092] Blood samples were collected from volunteers on days 0 (before drug administration), 14, and 28 and centrifuged on a Ficcol density gradient to isolate mononuclear cells. Isolated cells were stained with CFSE fluorescent dye (Invivogen, USA) and added to plate wells. Lymphocytes were then restimulated in vitro by adding coronavirus S protein to the culture medium (to a final protein concentration of 1 μg / mL). Intact cells without added antigen served as a negative control. 72 hours after antigen addition, the percentage of proliferating cells was measured, and medium was collected to measure interferon-gamma levels.

[0093] The percentage of proliferating cells was assessed by staining cells with antibodies against the T cell marker molecules CD3, CD4, and CD8 (anti-CD3 Pe-Cy7 (BD Biosciences, clone SK7), anti-CD4 APC (BD Biosciences, clone SK3), and anti-CD8 PerCP-Cy5.5 (BD Biosciences, clone SK1)). A flow cytofluorometer (BD FACS AriaIII, BD Biosciences, USA) was used to identify proliferating (lower CFSE expression) CD4 and CD8 T cells in the cell mixture. To determine the percentage of proliferating cells obtained in each sample, the results obtained from the analysis of intact cells were subtracted from those obtained from the analysis of cells restimulated with coronavirus antigen S. The final results are shown in Figures 1 and 2 (for the liquid form of the vaccine) and Figures 3 and 4 (for the lyophilized form of the vaccine).

[0094] Interferon gamma (IFNγ) concentrations in the culture medium of human blood mononuclear cells were measured within 72 hours of restimulation with coronavirus S protein using an interferon gamma EIA-BEST kit (VECTOR BEST, Russia) according to the manufacturer's instructions. The results are shown in Figure 5 (for the liquid form of the vaccine) and Figure 6 (for the lyophilized form of the vaccine).

[0095] The study results showed that the strength of cellular immunity induced by sequential immunization of volunteers with both forms of the drug, variant 1, increased with time since immunization, as evidenced by the median percentages of proliferating CD4+ and CD8+ T cells. In both groups, the maximum number of proliferating CD4+ and CD8+ T cells was observed on day 28 after immunization. The maximum statistically significant difference (p<0.001) in the percentages of proliferating CD4+ and CD8+ T cells was observed between days 0 and 28.

[0096] From the results shown in Figures 5 and 6, it can be concluded that, based on the median increment in IFNγ concentrations, the increasing strength of cellular immunity resulting from sequential immunization of volunteers with both forms of the drug, variant 1, became more pronounced with increasing days since immunization. The statistically significant difference in the increment in IFNγ concentrations between pre-immunization levels (day 0) and levels 14 days after vaccination was p<0.001. The maximum increment in IFNγ concentrations is observed on day 28 after immunization. The maximum statistically significant difference (p<0.001) in the increment in IFNγ concentrations was observed between days 0 and 28 of the study.

[0097] Therefore, based on the above data, it can be concluded that immunization with the developmental drug induces a potent antigen-specific cellular anti-infective immune component, supported by high statistical significance of parameters measured before and after immunization.

[0098] Example 9 Studying the immunogenicity of investigational drugs by assessing antibody titers against SARS-CoV-2 antigens in the blood of volunteers at different time points after vaccination The 40 volunteers taking part in the study were: 1) Liquid form of the development drug, variant 1: 1x10 11 dose of viral particles, followed by immunization with component 2 within 21 days after immunization with component 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2) (20 volunteers). 2) Lyophilized form of the development drug, Mutant 1: 1x10 11 dose of viral particles, followed by immunization with component 2 within 21 days after immunization with component 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2) (20 volunteers).

[0099] Blood samples were collected on days 14, 21 and 28, after which serum was separated.

[0100] The titers of antibodies against the RBD of the SARS-CoV-2 S protein were measured using the kit SARS-CoV-2-RBD-EIA-Gamaleya according to the manufacturer's instructions.

[0101] The results of the assay of antibody titers against SARS-CoV-2 antigens in the serum of volunteers after administration of the liquid form of the drug are shown in Figure 7.

[0102] The results of the assay of antibody titers against SARS-CoV-2 antigens in the serum of volunteers after administration of the lyophilized form of the drug are shown in Figure 8.

[0103] As is evident from the data presented, immunization of volunteers with the developmental drug in both liquid and lyophilized form allows the development of strong antibody-mediated immunity (statistically significantly different from the values ​​obtained in non-immunized control animals), characterized by elevated levels of antibodies against the S protein of SARS-CoV-2. An increase in the strength of the antibody-mediated immune response was observed over time after immunization.

[0104] Example 10 Use of a developmental agent to extend post-vaccination immunity against SARS-CoV-2 following immunization with a model subunit vaccine The objective of this study was to evaluate the potential use of the developmental agent for revaccination of animals immunized with a model subunit vaccine.

[0105] In this experiment, female Balb / c mice weighing 18 g were used. In phase 1, animals were immunized with a model vaccine containing the S protein of SARS-CoV-2 (10 μg / mouse) in phosphate-buffered saline containing aluminum hydroxide (100 μg / mouse). Two doses of the vaccine were administered with an interval of 21 days. On day 180, animals were re-immunized with different variants of the developed drug. In the case of two-component drugs, the first component (10 10 viral particles / mouse) was administered on day 180 of the experiment, and the second component (10 10 vp / mouse) was administered on day 201. In the case of the single component drugs, immunization took place on day 201 of the experiment. Thus, the following experimental and control groups of animals were studied: 1) Model vaccines: Ad26-CMV-S-CoV2 and Ad5-CMV-S-CoV2 2) Model vaccines: Ad26-CAG-S-CoV2 and Ad5-CAG-S-CoV2 3) Model vaccines / Ad26-EF1-S-CoV2 / Ad5-EF1-S-CoV2 4) Model vaccine / Ad26-CMV-S-CoV2 / simAd25-CMV-S-CoV2 5) Model vaccine / Ad26-CAG-S-CoV2 / simAd25-CAG-S-CoV2 6) Model vaccine / Ad26-EF1-S-CoV2 / simAd25-EF1-S-CoV2 7) Model vaccine / simAd25-CMV-S-CoV2 / Ad5-CMV-S-CoV2 8) Model vaccine / simAd25-CAG-S-CoV2 / Ad5-CAG-S-CoV2 9) Model vaccine / simAd25-EF1-S-CoV2 / Ad5-EF1-S-CoV2 10) Model vaccine / Ad26-CMV-S-CoV2 11) Model vaccine / Ad26-CAG-S-CoV2 12) Model vaccine / Ad26-EF1-S-CoV2 13) Model vaccine / Ad5-CMV-S-CoV2 14) Model vaccine / Ad5-CAG-S-CoV2 15) Model vaccine / Ad5-EF1-S-CoV2 16) Model vaccine / simAd25-CMV-S-CoV2 17) Model vaccine / simAd25-CAG-S-CoV2 18) Model vaccine / simAd25-EF1-S-CoV2

[0106] Blood was collected from the tail vein and serum was separated on days 21, 180, and 222 of the experiment. Anti-SARS-CoV-2 antibody titers were determined by enzyme immunoassay (EIA) according to the following protocol: 1) The antigen was adsorbed onto the wells of a 96-well microtitration plate at +4°C for 16 hours. 2) To eliminate non-specific binding, the plate was "locked" with blocking buffer (100 μL / well added to each well). The plate was incubated on a shaker at +37°C for 1 hour. 3) The serum from the immunized mice was diluted 100-fold, and then a series of 2-fold dilutions were prepared. 4) 50 μL of each diluted serum sample was added to the plate wells. 5) The plate was then incubated at +37°C for 1 hour. 6) After the incubation was completed, the wells were washed three times with phosphate buffer. 7) Next, a horseradish peroxidase-conjugated secondary anti-mouse IgG antibody was added. 8) The plate was then incubated at +37°C for 1 hour. 9) After the incubation was completed, the wells were washed three times with phosphate buffer. 10) Next, a solution of tetramethylbenzidine (TMB), a horseradish substrate that converts to a colored compound during the reaction, was added. Within 15 minutes, sulfuric acid was added to stop the reaction. The optical density (OD) of the solution was then measured in each well at a wavelength of 450 nm using a spectrophotometer.

[0107] The antibody titer was determined as the highest dilution that showed a solution optical density significantly higher than the negative control group. The results (geometric means) are shown in Table 3.

[0108] [Table 3]

[0109] [Table 4]

[0110] The data presented demonstrate the development of antibodies in all animals after immunization with the model inactivated vaccine; by day 180 after immunization, antibody titers decline, but revaccination of animals with different variants of the developmental agent results in variable increases in circulating antibody titers. Thus, the experimental data support the use of the developmental agent for the extension of post-vaccination immunity against SARS-CoV-2.

[0111] Example 11 Use of a developmental agent to extend post-vaccination immunity against SARS-CoV-2 after immunization with a model inactivated vaccine The objective of this study was to evaluate the potential use of the developmental agent for revaccination of animals immunized with a model inactivated vaccine.

[0112] In this experiment, female Balb / c mice weighing 18 g were used. In phase 1, animals were immunized with a model vaccine containing formalin-inactivated SARS-CoV-2 virus. Two doses of the vaccine were administered with an interval of 21 days. On day 180, animals were re-immunized with different variants of the developed drug. In the case of two-component drugs, the first component (10 10 vp / mouse) was administered on day 180 of the experiment, and the second component (10 10 vp / mouse) was administered on day 201. In the case of the single component drugs, immunization took place on day 201 of the experiment. Thus, the following experimental and control groups of animals were studied: 1. Model vaccines / Ad26-CMV-S-CoV2 / Ad5-CMV-S-CoV2 2. Model vaccines: Ad26-CAG-S-CoV2 and Ad5-CAG-S-CoV2 3. Model vaccines / Ad26-EF1-S-CoV2 / Ad5-EF1-S-CoV2 4. Model Vaccine / Ad26-CMV-S-CoV2 / simAd25-CMV-S-CoV2 5. Model Vaccine / Ad26-CAG-S-CoV2 / simAd25-CAG-S-CoV2 6. Model Vaccine / Ad26-EF1-S-CoV2 / simAd25-EF1-S-CoV2 7. Model Vaccine / simAd25-CMV-S-CoV2 / Ad5-CMV-S-CoV2 8. Model Vaccine / simAd25-CAG-S-CoV2 / Ad5-CAG-S-CoV2 9. Model Vaccine / simAd25-EF1-S-CoV2 / Ad5-EF1-S-CoV2 10. Model Vaccine / Ad26-CMV-S-CoV2 11. Model Vaccine / Ad26-CAG-S-CoV2 12. Model Vaccine / Ad26-EF1-S-CoV2 13. Model Vaccine / Ad5-CMV-S-CoV2 14. Model Vaccine / Ad5-CAG-S-CoV2 15. Model Vaccine / Ad5-EF1-S-CoV2 16. Model Vaccine / simAd25-CMV-S-CoV2 17. Model Vaccine / simAd25-CAG-S-CoV2 18. Model Vaccine / simAd25-EF1-S-CoV2

[0113] Blood was collected from the tail vein and serum was separated on days 21, 180, and 222 of the experiment. Anti-SARS-CoV-2 antibody titers were determined by enzyme immunoassay (EIA) according to the following protocol: 1) The antigen was adsorbed onto the wells of a 96-well microtitration plate at +4°C for 16 hours. 2) To eliminate non-specific binding, the plate was "locked" with blocking buffer (100 μL / well added to each well). The plate was incubated on a shaker at +37°C for 1 hour. 3) The serum from the immunized mice was diluted 100-fold, and then a series of 2-fold dilutions were prepared. 4) 50 μL of each diluted serum sample was added to the plate wells. 5) The plate was then incubated at +37°C for 1 hour. 6) After the incubation was completed, the wells were washed three times with phosphate buffer. 7) Next, a horseradish peroxidase-conjugated secondary anti-mouse IgG antibody was added. 8) The plate was then incubated at +37°C for 1 hour. 9) After the incubation was completed, the wells were washed three times with phosphate buffer. 10) Next, a solution of tetramethylbenzidine (TMB), a horseradish substrate that converts to a colored compound during the reaction, was added. Within 15 minutes, sulfuric acid was added to stop the reaction. The optical density (OD) of the solution was then measured in each well at a wavelength of 450 nm using a spectrophotometer.

[0114] The antibody titer was determined as the highest dilution that showed a solution optical density significantly higher than the negative control group. The results (geometric means) are shown in Table 4.

[0115] [Table 5]

[0116] [Table 6]

[0117] The data presented demonstrate the development of antibodies in all animals after immunization with the model inactivated vaccine; by day 180 after immunization, antibody titers decline, but revaccination of animals with different variants of the developmental agent results in variable increases in circulating antibody titers. Thus, the experimental data support the use of the developmental agent for the extension of post-vaccination immunity against SARS-CoV-2.

[0118] Example 12 Use of the investigational drug to extend post-vaccination immunity against SARS-CoV-2 following immunization with different variants of the investigational drug The objective of this study was to evaluate the potential use of the developmental agent for revaccination of animals immunized with different variants of the developmental agent.

[0119] In this experiment, female Balb / c mice weighing 18 g were used. In Phase 1, various single-component variants of the development drug (10 10 On day 180, animals were immunized with various two-component variants of the development drug (10 10 The animals were re-immunized with the first component (10 10 vp / mouse) was administered on day 180 of the experiment, and the second component (10 10 vp / mouse) was administered on day 201. In the case of the single component drugs, immunization took place on day 201 of the experiment. Thus, the following experimental and control groups of animals were studied: 1)Ad26-CMV-S-CoV2 / Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 2)Ad26-CMV-S-CoV2 / Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 3)Ad26-CMV-S-CoV2 / simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 4)Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 5)Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 6)Ad5-CMV-S-CoV2 / simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 7)simAd25-CMV-S-CoV2 / Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 8) simAd25-CMV-S-CoV2 / Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 9) simAd25-CMV-S-CoV2 / simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 10)Ad26-CMV-S-CoV2 / Ad26-CMV-S-CoV2 11)Ad26-CMV-S-CoV2 / Ad5-CMV-S-CoV2 12)Ad26-CMV-S-CoV2 / simAd25-CMV-S-CoV2 13)Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2 14)Ad5-CMV-S-CoV2 / Ad5-CMV-S-CoV2 15)Ad5-CMV-S-CoV2 / simAd25-CMV-S-CoV2 16)simAd25-CMV-S-CoV2 / Ad26-CMV-S-CoV2 17)simAd25-CMV-S-CoV2 / Ad5-CMV-S-CoV2 18)simAd25-CMV-S-CoV2 / simAd25-CMV-S-CoV2

[0120] Blood was collected from the tail vein and serum was separated on days 21, 180, and 222 of the experiment. Anti-SARS-CoV-2 antibody titers were determined by enzyme immunoassay (EIA) according to the following protocol: 1) The antigen was adsorbed onto the wells of a 96-well microtitration plate at +4°C for 16 hours. 2) To eliminate non-specific binding, the plate was "locked" with blocking buffer (100 μL / well added to each well). The plate was incubated on a shaker at +37°C for 1 hour. 3) The serum from the immunized mice was diluted 100-fold, and then a series of 2-fold dilutions were prepared. 4) 50 μL of each diluted serum sample was added to the plate wells. 5) The plate was then incubated at +37°C for 1 hour. 6) After the incubation was completed, the wells were washed three times with phosphate buffer. 7) Next, a horseradish peroxidase-conjugated secondary anti-mouse IgG antibody was added. 8) The plate was then incubated at +37°C for 1 hour. 9) After the incubation was completed, the wells were washed three times with phosphate buffer. 10) Next, a solution of tetramethylbenzidine (TMB), a horseradish substrate that converts to a colored compound during the reaction, was added. Within 15 minutes, sulfuric acid was added to stop the reaction. The optical density (OD) of the solution was then measured in each well at a wavelength of 450 nm using a spectrophotometer.

[0121] The antibody titer was determined as the highest dilution that showed a solution optical density significantly higher than the negative control group. The results (geometric means) are shown in Table 5.

[0122] [Table 7]

[0123] [Table 8]

[0124] The data presented demonstrate the development of antibodies in all animals after immunization of mice with a single-component variant of the investigational drug; by day 180 after immunization, antibody titers decline, but revaccination of animals with different variants of the investigational drug results in a variable increase in circulating antibody titers. Thus, the experimental data support the use of the investigational drug for the extension of post-vaccination immunity against SARS-CoV-2.

[0125] Example 13 Use of the investigational drug to extend post-vaccination immunity against SARS-CoV-2 following immunization with different variants of the investigational drug The objective of this study was to evaluate the potential use of the developmental agent for revaccination of animals immunized with different variants of the developmental agent.

[0126] In this experiment, female Balb / c mice weighing 18 g were used. In step 1, the developed two-component drug variant (10 10 vp / mouse) at 21-day intervals. On day 180, animals were re-immunized with different mono-component variants of the developed drug. Thus, the following experimental and control groups of animals were studied: 1)Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2 2)Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad5-CMV-S-CoV2 3)Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 / simAd25-CMV-S-CoV2 4)Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 / Ad26-CMV-S-CoV2 5)Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 / Ad5-CMV-S-CoV2 6)Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 / simAd25-CMV-S-CoV2 7)simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2 8)simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad5-CMV-S-CoV2 9)simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 / simAd25-CMV-S-CoV2 10)Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 11)Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 / Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 12) simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 / simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 13)Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 / Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2

[0127] Blood was collected from the tail vein and serum was separated on days 42, 180, and 222 of the experiment. Anti-SARS-CoV-2 antibody titers were determined by enzyme immunoassay (EIA) according to the following protocol: 1) The antigen was adsorbed onto the wells of a 96-well microtitration plate at +4°C for 16 hours. 2) To eliminate non-specific binding, the plate was "locked" with blocking buffer (100 μL / well added to each well). The plate was incubated on a shaker at +37°C for 1 hour. 3) The serum from the immunized mice was diluted 100-fold, and then a series of 2-fold dilutions were prepared. 4) 50 μL of each diluted serum sample was added to the plate wells. 5) The plate was then incubated at +37°C for 1 hour. 6) After the incubation was completed, the wells were washed three times with phosphate buffer. 7) Next, a horseradish peroxidase-conjugated secondary anti-mouse IgG antibody was added. 8) The plate was then incubated at +37°C for 1 hour. 9) After the incubation was completed, the wells were washed three times with phosphate buffer. 10) Next, a solution of tetramethylbenzidine (TMB), a horseradish substrate that converts to a colored compound during the reaction, was added. Within 15 minutes, sulfuric acid was added to stop the reaction. The optical density (OD) of the solution was then measured in each well at a wavelength of 450 nm using a spectrophotometer.

[0128] The antibody titer was determined as the highest dilution that showed a solution optical density significantly higher than the negative control group. The results (geometric means) are shown in Table 6.

[0129] [Table 9]

[0130] [Table 10]

[0131] The data presented demonstrate the development of antibodies in all animals after immunization of mice with a two-component variant of the investigational agent; by day 180 after immunization, antibody titers decline, but revaccination of animals with different variants of the investigational agent results in a variable increase in circulating antibody titers. Thus, the experimental data support the use of the investigational agent for the extension of post-vaccination immunity against SARS-CoV-2.

[0132] Thus, the specified technical problem, in particular the creation of an agent that provides extended post-vaccination immunity against the SARS-CoV-2 virus, is solved as supported by the examples presented.

[0133] Industrial Use All the examples presented support the efficacy and industrial use of the drug to effectively induce an immune response against the SARS-CoV-2 virus and further provide prolonged post-vaccination immunity.

Claims

1. A drug for treating a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2, comprising a component 1, which is a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the ORF6-Ad26 region has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 has been incorporated, and a component 2, which is a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 has been incorporated, A drug used for revaccinating a patient who has been vaccinated with only component 1, only component 2, or only a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 sites have been deleted and which incorporates an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3, at least 180 days after the vaccine was administered.

2. A drug for treating a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2, comprising a component 1, which is a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 regions have been deleted and the ORF6-Ad26 region has been replaced by ORF6-Ad5, and into which an expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 has been incorporated, and a component 2, which is a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 has been incorporated, A drug used for revaccinating a patient who has been vaccinated with only component 1, only component 2, or only a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 sites have been deleted and which incorporates an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, at least 180 days after the vaccine was administered.

3. A drug for treating a disease caused by severe acute respiratory syndrome virus SARS-CoV-2, comprising: component 1, a drug in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:3 has been incorporated; and component 2, a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, from which the E1 and E3 regions have been deleted and into which an expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 has been incorporated, A drug used for revaccinating, at least 180 days after vaccination, a patient who has been vaccinated with only component 1, only component 2, or only a drug in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, from which the E1 and E3 sites have been deleted and the site ORF6-Ad26 has been replaced by ORF6-Ad5, and in which an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 has been incorporated.

4. The agent according to any one of claims 1 to 3, which is in liquid or lyophilized form.

5. The buffer for the liquid form comprises, in weight percent: Tris 0.1831 to 0.3432 Sodium chloride 0.3313 to 0.6212 Sucrose 3.7821-7.0915 Magnesium chloride hexahydrate 0.0154-0.0289 EDTA 0.0029-0.0054 Polysorbate 80 0.0378-0.0709 Ethanol 95% 0.0004-0.0007 Water remaining The drug according to claim 4, comprising:

6. The reconstituted lyophilized drug contained, in weight percent: Tris 0.0180 to 0.0338 Sodium chloride 0.1044-0.1957 Sucrose 5.4688-10.2539 Magnesium chloride hexahydrate 0.0015-0.0028 EDTA 0.0003-0.0005 Polysorbate 80 0.0037-0.0070 Water remaining The pharmaceutical composition of claim 4, which contains a buffer solution consisting of:

7. The agent according to any one of claims 1 to 3, wherein component 1 and component 2 are in separate containers.

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