EA043163B1 - APPLICATION OF MEANS FOR INDUCING SPECIFIC IMMUNE AGAINST SARS-COV-2 SEVERE ACUTE RESPIRATORY SYNDROME VIRUS FOR POPULATION REVACCINATION (VERSIONS) - Google Patents

Description

Technical field

The invention relates to biotechnology, immunology and virology. The use of agents for revaccination of the population against diseases caused by the SARS-CoV-2 severe acute respiratory syndrome virus has been proposed.

State of the art

Coronaviruses are a large family of viruses that cause a wide range of diseases in humans and animals. At the end of 2019, the world faced a new zoonotic beta-coronavirus SARSCoV-2, which caused an outbreak of coronavirus infection (COVID-19) in Wuhan (People's Republic of China (PRC)). On March 11, 2020, the World Health Organization announced that the spread of the new coronavirus in the world has become a pandemic. As of February 1, 2021, over 100 million cases of COVID-19 have been reported worldwide; over 2 million people have died.

The most common symptoms of COVID-19 include fever, dry cough, shortness of breath, and fatigue. Less common - sore throat, joint pain, runny nose, headache. In this case, the disease can occur in both mild and severe forms. Risk factors are older age and the presence of chronic diseases.

As a result of the transferred disease, both cellular and humoral immune responses are formed. CD8+ and CD4+ T cells specific for SARS-CoV-2 are found in 70% and 100% of patients recovering from COVID-19, respectively. The main target for T-lymphocytes is the S protein of SARS-CoV-2. T-lymphocytes are also found specific to the M and N proteins of the coronavirus and, to a lesser extent, specific to nsp3, nsp4, ORF3a and ORF8 of SARS-CoV-2. The immune response is polarized towards 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).

The humoral immune response is mediated by antibodies that are directed primarily at the surface S protein of the coronavirus. It has been shown that the RBD domain of glycoprotein S, which is responsible for binding to the ACE-2 receptor on human cells, is the main target of virus-neutralizing antibodies. The kinetics of the humoral immune response against SARS-CoV-2 is characterized by sustained seroconversion (IgM and IgG) 7-14 days after symptom onset. IgG titers increase during the first 3 weeks and begin to decline by 8 weeks (Adams ER, Ainsworth M, Anand R. Antibody testing for COVID-19: a report from the National COVID Scientific Advisory Panel. medRxiv. 2020). IgG titers have also been shown 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).

The scientific evidence to date suggests that the natural immunity to COVID-19 that a person acquires as a result of a previous illness is short-lived. (https://www.cdc.gov/coronavirus/2019-ncov/vaccines/facts.html). This is evidenced by observed trends in decreasing antibody levels, as well as cases of re-infection with coronavirus (Akiko Iwasaki. What reinfections mean for COVID-19. Lancet Infect Dis. 2021 Jan; 21(1): 3-5.)

The most effective way to prevent infectious diseases is vaccination. To date, several COVID-19 vaccines have been developed that use different coronavirus antigens.

1) Vaccines containing the whole virion as an antigen.

There are 4 candidate inactivated vaccines developed in China. Currently 3 vaccines (Sinovac with the National Institute of Infectious Disease Control and Prevention; Sinopharm with the Wuhan Institute of Biological Products and the Wuhan Institute of Virology of the Chinese Academy of Sciences; Sinopharm with the Beijing Institute of Biological Products and the Institute of Virus Disease Control and Prevention) are in phase III clinical trials, and one vaccine (Institute of Medical Biology, Chinese Academy of Medical Sciences) is in phase I/II clinical trials.

(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, placebocontrolled, phase 1/2 clinical trial. Lancet Infect Dis. 2021 Feb;21(2): 181-192 Xia S et al.

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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-vaccine, BBIBP-CorV: a randomised, double-blind, placebo-controlled, phase 1/2 trial.

Lancet Infect Dis. 2021 Jan;21(l):39-5L doi: 10.1016/S 1473-3099(20)30831-8. Epub

2020 Oct 15. PMID: 33069281; RMSSH: PMC7561304. CheY 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)

2) Vaccines in which the antigen is a full-length S protein.

There are 3 vaccines based on adenoviruses of various serotypes that express the gene for the full-length S protein of the SARS-CoV-2 virus. CanSino Biological Inc. and the Beijing Institute of Biotechnology have developed a vaccine based on human adenovirus serotype 5; Oxford University, AstraZeneca - vaccine based on chimpanzee adenovirus, N.F. Gamaleya of the Ministry of Health of Russia - a vaccine based on human adenoviruses serotype 26 and serotype 5. In addition, a DNA vaccine containing the full-length S protein gene of the SARS-CoV-2 virus is known, 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-inhuman trial. Lancet. 2020 Jun 13;395 (10240):1845-1854 van Doremalen N et al.

ChAdOxl 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).

3) Vaccines in which the antigen is a full-length S protein with two proline substitutions (K986P and V987P).

Two vaccines are known based on lipid nanoparticles, including mRNA encoding the S protein of SARS-CoV-2, with proline substitutions (Moderna in collaboration with the National Institute of Allergy and Infectious Diseases; BioNTech in collaboration with Fosun Pharma and Pfizer). A protein subunit vaccine developed by Novavax is also known, the antigen in this vaccine is the full-length S protein of SARS-CoV-2 with two proline substitutions (K986P and V987P) and three mutations in the furin cleavage site (R682Q, R683Q and R685Q). In addition, Janssen Pharmaceutical Companies has developed a vaccine based on human adenovirus serotype 26 expressing the full-length S protein of SARS-CoV-2 with two proline substitutions (K986P and V987P) and two mutations in the furin cleavage site (R682S and R685G).

(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(l 1):1694-1700, doi: 10.1038/s41591-0201070-6. Epub 2020 Sep 3).

4) Vaccines in which the antigen is the RBD domain of the S protein.

Known protein subunit vaccine containing RBD-dimer (residues 319-537 in the form of a tandem repeat), developed by Anhui Zhifei Longcom Biopharmaceutical in conjunction with the Institute of Microbiology of the Chinese Academy of Sciences. Also known vaccine based on lipid nanoparticles, including mRNA encoding the RBD-trimer (trimerized by adding the foldon domain of fibrin T4).

- 2 043163 (BioNTech with Fosun Pharma and Pfizer) (Mulligan M.

et al. Phase I/II study of COVID-19 RNA vaccine BNT162bl 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.ell. Dai L et al. Viral targets for vaccines against COVID-19. Nat Rev Immunol. 2021 Feb;21(2):73-82).

Currently, 8 vaccines against COVID-19 are approved for use in the world. The results of clinical studies have shown that immunization with these vaccines forms both a humoral and a cellular immune response to the SARS-CoV-2 virus in humans. However, how long post-vaccination protective immunity will be for each type of vaccine has not yet been established. According to experts, it can vary from person to person and, according to various estimates, range from 1 to 2 years. Such terms determine the need for the development of COVID-specific prophylaxis agents used for human revaccination.

Meanwhile, the choice of means for revaccination is not an easy task.

When developing a means for specific prophylaxis used for revaccination, one should remember the effects that occur during repeated immunization of a person, while having a significant impact on the final structure of anti-infective immunity, including its protective properties.

It is known that when using vaccines containing a large number of antigens in their composition (for example, inactivated vaccines), an immune response is formed for each antigen. However, in this case, the intensity of immunity in relation to the selected antigen will be lower compared to those vaccine preparations that have only this antigen in their structure (the effect of blurring the immune response). In addition, some antigens in the structure of the pathogen may be non-protective, the formation of T and B cell clones for such antigens will not contribute to the overall protectiveness of immunity, interfering with the formation of cell clones important for protection.

Based on the above, we can conclude that the most promising for revaccination are those vaccines that contain one or more proteins with pronounced protective properties. In this case, in the process of revaccination, there will be additional stimulation (boosting) and no less important focusing of the immune response on the antigenic determinants of the pathogen that are most important for human protection, regardless of the primary immunization.

The prior art does not know means for revaccination against coronavirus infection.

The technical solution according to the patent of the Russian Federation No. 2731342 (published on 09/01/2020) was chosen by the authors of the claimed invention as a prototype. Variants of the agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 are known from this patent:

containing component 1, which is an 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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5, with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with built-in expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

containing component 1, which is an 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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5, with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of the recombinant strain of simian adenovirus of the 25th serotype, in which the E1 and E3 regions are deleted, with built-in expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3;

containing component 1, which is an agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted, with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

In addition, this patent discloses the use of these options for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2, including the use of component 1 and 2 in an effective amount sequentially with an interval of at least 1 week.

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The disadvantage of this tool is that its use to prolong post-vaccination immunity is not described.

Thus, there is a need in the art to develop an agent that can be used for revaccination against diseases caused by the SARS-CoV-2 virus.

Implementation of the invention

The technical task of the claimed group of inventions is the development of tools that ensure the prolongation of post-vaccination immunity against the SARS-CoV-2 virus.

The technical result is to create a safe and effective agent that ensures the extension of post-vaccination immunity against the SARS-CoV-2 virus.

This technical result is achieved by the proposed use of an agent containing component 1, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 26th serotype, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6 -Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or containing component 2, which is an expression vector agent based on the genome of a recombinant strain of human adenovirus 5- serotype, in which the E1 and E3 regions with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 are deleted for revaccination against diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2 .

Also proposed is the use of an agent containing component 1, which is an 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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which E1 are deleted and E3 regions with an inserted expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 or containing only component 2 for revaccination against diseases caused by severe acute respiratory syndrome virus SARS-CoV-2.

In addition, the proposed use of an agent containing component 1, which is an agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted with an integrated expression cassette, selected of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 for revaccination against diseases caused by SARS-CoV-2 severe acute respiratory syndrome virus.

In this case, when used, the agent is in liquid or lyophilized form.

Moreover, the buffer solution for the liquid form of the agent contains, wt.%:

tris from 0.1831 to 0.3432 sodium chloride from 0.3313 to 0.6212 sucrose from 3.7821 to 7.0915 magnesium chloride hexahydrate from 0.0154 to 0.0289 EDTA from 0.0029 to 0.0054 polysorbate-80 from 0.0378 to 0.0709 ethanol 95% from 0.0004 to 0.0007 water rest. Also, the reconstituted lyophilized form of the agent contains a buffer solution, wt.%: tris from 0.0180 to 0.0338 sodium chloride from 0.1044 to 0.1957

sucrose from 5.4688 to 10.2539 magnesium chloride hexahydrate from 0.0015 to 0.0028 EDTA from 0.0003 to 0.0005 polysorbate-80 from 0.0037 to 0.0070

water the rest.

In addition, when using the product, component 1 and component 2 are in different packages.

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Brief description of the figures

In FIG. 1 shows the results of assessing the effectiveness of immunization of volunteers with a liquid form of the developed agent according to option 1 in assessing the proportion of proliferating CD8+ lymphocytes restimulated by SARS-CoV-2 S antigen.

The y-axis is the number of proliferating cells, %. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ - values for each volunteer on day 28 are indicated.

The median of the values is represented as a black line for each data group. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; **, p<0.01; ****, p<0.001 according to the Mann-Whitney test.

In FIG. Figure 2 presents the results of evaluating the effectiveness of immunization of volunteers with a liquid form with the developed agent according to option 1 in assessing the proportion of proliferating CD4+ lymphocytes restimulated by SARS-CoV-2 S antigen.

The y-axis is the number of proliferating cells, %. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ - values for each volunteer on day 28 are indicated.

The median of the values is represented as a black line for each data group. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; **, p<0.01; ****, p<0.001 according to the Mann-Whitney test.

In FIG. Figure 3 presents the results of evaluating the effectiveness of immunization of volunteers with the lyophilized form of the developed agent according to option 1 in assessing the proportion of proliferating CD8+ lymphocytes restimulated by SARS-CoV-2 S antigen.

The y-axis is the number of proliferating cells, %. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ - values for each volunteer on day 28 are indicated.

The median of the values is represented as a black line for each data group. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; **, p<0.01; ****, p<0.001 according to the Mann-Whitney test.

In FIG. Figure 4 shows the results of evaluating the effectiveness of immunization of volunteers with the lyophilized form of the developed agent according to option 1 in assessing the proportion of proliferating CD4+ lymphocytes restimulated by the SARS-CoV-2 S antigen.

The y-axis is the number of proliferating cells, %. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ indicates the values for each volunteer on day 28.

The median of the values is represented as a black line for each data group. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; **, p<0.01; ****, p<0.001 according to the Mann-Whitney test.

In FIG. 5 Shows the increase in the concentration of IFNy (in times) in the culture medium of peripheral blood mononuclear cells of volunteers immunized with the liquid form of the developed agent according to option 1, after their restimulation with the SARS-CoV-2 S antigen before immunization (day 0) and after 14 and 28 days of the study .

Y-axis - increase in the concentration of gamma-interferon, times. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ - values for each volunteer on day 28 are indicated.

The median of the values is represented as a black line for each data group. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; ****, p<0.001 according to the Mann-Whitney test.

In FIG. Figure 6 shows the increase in the concentration of IFNy (in times) in the culture medium of peripheral blood mononuclear cells of volunteers immunized with the lyophilized form of the developed agent according to option 1, after their restimulation with SARS-CoV-2 S antigen before immunization (day 0) and after 14 and 28 days of the study .

Y-axis - increase in the concentration of gamma-interferon, times. The abscissa axis is days.

• - values for each volunteer on day 0 are indicated.

- values are indicated for each volunteer on day 14.

▲ - values for each volunteer on day 28 are indicated.

Dots indicate the values for each volunteer participating in the study. Median

- 5043163 values are represented as a black line for each group of data. Statistically significant difference between the values of 0, 14 and 28 days is indicated by brackets and symbols *, p<0.05; ****, p<0.001 according to the Mann-Whitney test.

In FIG. Figure 7 shows the results of assessing the humoral immune response to the antigen of the SARS-CoV2 virus in volunteers immunized with the liquid form of the developed agent according to option 1.

Y-axis - IgG titer to the RBD domain of the glycoprotein S of the SARS-CoV-2 virus.

The abscissa axis is days.

- data for each volunteer.

In FIG. Figure 8 shows the results of assessing the humoral immune response to the antigen of the SARS-CoV2 virus in volunteers immunized with a lyophilized form of the developed agent according to option 1.

Y-axis - IgG titer to the RBD domain of the glycoprotein S of the SARS-CoV-2 virus.

The abscissa axis is days.

- data for each volunteer.

Implementation of the invention

The first step in the development of a tool for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 was the selection of a vaccine antigen. In the course of the work, a literature search was carried out, which showed that the most promising antigen for creating a candidate vaccine is the coronavirus S protein. It is a type I transmembrane glycoprotein that is responsible for the binding, fusion and penetration of viral particles into the cell. It has been shown to be an inducer of neutralizing antibodies (Liang M et al., SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity. Biomed Environ Sci. 2005 Dec;18(6):363-74 ).

To achieve the most effective induction of immune responses against the SARSCoV-2 S protein, the authors developed various variants of expression cassettes.

The expression cassette of SEQ ID NO: 1 consists of the CMV promoter, the S gene of the SARS-CoV2 virus protein, and a polyadenylation signal. The CMV promoter is a cytomegalovirus early gene promoter that provides for constitutive expression in a variety of cell types. However, the strength of target gene expression driven by the CMV promoter varies across cell types. In addition, it was shown that the level of transgene expression under the control of the CMV promoter decreases with increasing cell culture time due to the suppression of gene expression, which is associated with DNA methylation [Wang W., Jia YL., Li YC, Jing CQ., Guo X., Shang XF., Zhao SR., Wang TY. Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.//Scientific Reports - 2017. - Vol. 8. - P. 10416].

The expression cassette of SEQ ID NO: 2 consists of the CAG promoter, the S gene of the SARS-CoV2 virus protein, and a polyadenylation signal. The CAG promoter is a synthetic promoter that includes an early enhancer of the CMV promoter, a chicken P-actin promoter, and a chimeric intron (chicken P-actin and rabbit P-globin). It has been experimentally shown that the transcriptional activity of the CAG promoter is higher than that of the CMV promoter [Yang C.Q., Li X.Y., Li Q., Fu S.L., Li H., Guo Z.K., Lin J.T., Zhao S.T. Eval-uation of three different promoters driving gene expression in developing chicken embryo by using in vivo electroporation.//Genet. Mol. Res. - 2014. - Vol. 13. - P. 1270-1277].

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

The expression cassette of SEQ ID NO: 4 consists of the CMV promoter, the S gene of the SARS-CoV2 virus protein, and a polyadenylation signal.

For efficient delivery of the SARS-CoV-2 coronavirus S gene into the human body, a vector system based on adenoviruses was chosen. Adenovirus vectors have a number of advantages: they are unable to multiply in human cells, penetrate both dividing and non-dividing cells, are able to induce cellular and humoral immune responses, and provide a high level of target antigen expression.

The authors have developed variants of the agent containing two components based on adenoviruses of different serotypes. Thus, the immune response to the adenovirus vector part, which can

- 6 043163 occur after the introduction of the first component of the agent, is not further boosted and does not affect the generation of antigen-specific immune responses against the vaccine antigen.

In addition, the developed tools expand the arsenal of tools for inducing an immune response against the SARS-CoV-2 coronavirus, which will overcome the difficulties associated with the problem of a pre-existing immune response in a part of the population to certain adenovirus serotypes.

Thus, as a result of the work carried out, the following options were developed.

1. An agent for revaccination against diseases caused by the SARS-CoV-2 severe acute respiratory syndrome virus, containing component 1, which is an 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 are deleted , and the ORF6-Ad26 region is replaced by an ORF6-Ad5 with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, in which E1 and E3 regions are delegated with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

2. An agent for revaccination against diseases caused by the SARS-CoV-2 severe acute respiratory syndrome virus, containing component 1, which is an agent in the form of an expression vector based on the genome of the recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted , and the ORF6-Ad26 region was replaced with an ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector on based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 or containing only component 2.

3. An agent for revaccination against diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2, containing component 1, which is an agent in the form of an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

Example 1

Obtaining an expression vector containing the genome of a recombinant human adenovirus strain of the 26th serotype.

At the first stage of the work, the design of the pAd26-Ends plasmid construct carrying two regions homologous to the human adenovirus serotype 26 genome (two arms of homology) and the ampicillin resistance gene was developed. One arm of homology is the beginning of the human adenovirus serotype 26 genome (from the left inverted terminal repeat to the E1 region) and the sequence of the viral genome, including the pIX protein. The second arm of the homology contains the nucleotide sequence after the E4 ORF3 region to the end of the genome. The pAd26-Ends construct was synthesized by ZAO Evrogen (Moscow).

Virion-isolated DNA of human adenovirus serotype 26 was mixed with pAd26-Ends. As a result of homologous recombination between pAd26-Ends and viral DNA, plasmid pAd26-d1E1 was obtained, carrying the genome of human adenovirus serotype 26 with the deleted E1 region.

Then, in the resulting plasmid pAd26-d1E1, using standard cloning methods, the sequence containing open reading frame 6 (ORF6-Ad26) was replaced with a similar sequence from the genome of human adenovirus serotype 5 in order for human adenovirus serotype 26 to be able to multiply efficiently in HEK293 cell culture. As a result, plasmid pAd26-d1E1-ORF6-Ad5 was obtained.

Further, using standard genetic engineering methods, the E3 region of the adenovirus genome (approximately 3321 bp between the pVIII and U-exon genes) was removed from the constructed plasmid pAd26-d1E1-ORF6-Ad5 to increase the packaging capacity of the vector. As a result, a pAd26-only-null recombinant vector based on the human adenovirus serotype 26 genome with an open reading frame ORF6 of human adenovirus serotype 5 and with a deletion of the E1 and E3 regions was obtained. SEQ ID NO: 5 was used as the maternal sequence for human adenovirus serotype 26.

In addition, the authors developed several designs of the expression cassette:

the expression cassette of SEQ ID NO: 1 consists of the CMV promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 2 consists of the CAG promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 3 consists of the EF1 promoter, the S protein gene of the SARS-CoV-2 virus

- 7 043163 and polyadenylation signal.

Based on the pAd26-Ends plasmid construct, pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EF1-S-CoV2 constructs containing expression cassettes SEQ ID NO : 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, as well as bearing arms of the homology of the genome of adenovirus serotype 26. After that, constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EF1-S-CoV2 were linearized according to the unique hydrolysis site between the homology arms, each plasmid was mixed with the recombinant vector pAd26-only -null. As a result of homologous recombination, plasmids pAd26-only-CMV-S-CoV2, pAd26-onlyCAG-S-CoV2, pAd26-only-EF1-S-CoV2 were obtained, carrying the genome of recombinant human adenovirus 26 serotype with an open reading frame ORF6 human adenovirus 5 -th serotype and with a deletion of E1 and E3 regions, with an expression cassette of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

At the fourth stage, plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-onlyEF1-S-CoV2 were digested with specific restriction endonucleases to remove the vector part. The resulting DNA preparations were transfected into HEK293 culture cells.

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 were deleted, and the ORF6-Ad26 region was replaced with an ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

At this stage of the work, the expression vectors obtained in example 1 were purified by anion exchange and size exclusion chromatography. The finished suspension contained adenoviral particles in a buffer solution for the liquid form of the agent or in a buffer solution for the lyophilized form of the agent.

Thus, the following immunobiological agents were obtained based on the genome of the recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions were deleted, and the ORF6-Ad26 region was replaced by ORF6-Ad5:

1. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 1 (Ad26-CMV-S-CoV2) in a buffer solution for the liquid form of the agent.

2. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 1 (Ad26-CMV-S-CoV2) in lyophilized buffer.

3. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 2 (Ad26-CAG-S-CoV2) in a buffer solution for the liquid form of the agent.

4. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 2 (Ad26-CAG-S-CoV2) in lyophilized buffer.

5. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 3 (Ad26-EF1-S-CoV2) in a buffer solution for the liquid form of the agent.

6. An immunobiological agent based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and polyadenylation signal, SEQ ID NO: 3 (Ad26-EF1-S-CoV2) in lyophilized buffer.

Each of the presented immunobiological agents is component 1 in option 1 and in option 2 of the developed agent.

Example 3

Obtaining an expression vector containing the genome of a recombinant strain of simian adenovirus serotype 25.

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At the first stage of the work, the design of the pSim25-Ends plasmid construct was developed, carrying two regions homologous to the genome of simian adenovirus serotype 25 (two arms of homology). One arm of homology is the beginning of the simian adenovirus serotype 25 genome (from the left inverted terminal repeat to the E1 region) and the sequence from the end of the E1 region to the pIVa2 protein. The second arm of the homology contains the sequence of the end of the adenovirus genome, including the right inverted terminal repeat. Synthesis of the pSim25-Ends construct was carried out by ZAO Evrogen (Moscow).

Virion-isolated simian adenovirus serotype 25 DNA was mixed with pSim25-Ends. As a result of homologous recombination between pSim25-Ends and viral DNA, the plasmid pSim25-d1E1 was obtained, carrying the genome of simian adenovirus serotype 25 with the deleted E1 region.

Then, using standard genetic engineering methods, the E3 region of the adenovirus genome (3921 bp from the beginning of the 12.5K gene to the 14.7K gene) was removed from the constructed plasmid pSim25-d1E1 to increase the packaging capacity of the vector. As a result, the pSim25-null plasmid construct was obtained, encoding the complete genome of simian adenovirus serotype 25 with the deletion of the E1 and E3 regions. SEQ ID NO: 6 was used as the maternal sequence of simian adenovirus serotype 25.

In addition, the authors have developed several designs for the expression cassette:

the expression cassette of SEQ ID NO: 4 consists of the CMV promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 2 consists of the CAG promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 3 consists of the EF1 promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal.

Then, based on the pSim25-Ends plasmid construct, pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EF1-S-CoV2 constructs containing SEQ ID expression cassettes were obtained by genetic engineering. NO: 4, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, as well as shoulder-bearing homologies from the simian adenovirus serotype 25 genome. After that, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EF1-S-CoV2 were linearized according to a unique hydrolysis site between the homology arms, each plasmid was mixed with the recombinant vector pSim25- null. As a result of homologous recombination, recombinant plasmid vectors pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EF1-S-CoV2 were obtained, containing the complete genome of monkey adenovirus serotype 25 with deletion of E1 and E3 regions and an expression cassette of SEQ ID NO: 4, SEQ ID NO: 2, or SEQ ID NO: 3, respectively.

At the third stage, plasmids pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EF1-S-CoV2 were digested with a specific restriction endonuclease to remove the vector part. The resulting DNA preparations were transfected into HEK293 culture cells. The resulting material was used to accumulate preparative amounts of recombinant adenoviruses.

As a result, recombinant human adenoviruses of serotype 25 containing the S gene of the SARS-CoV-2 virus protein were obtained: simAd25-CMV-S-CoV2 (containing the expression cassette SEQ ID NO: 4), simAd25-CAG-S-CoV2 (containing the SEQ ID NO: 2), simAd25-EF1-S-CoV2 (containing the expression cassette of SEQ ID NO: 3).

Thus, an expression vector was obtained containing the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are delegated with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 .

Example 4

Obtaining an immunobiological agent in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 are deleted .

At this stage of the work, the expression vectors obtained in example 3 were purified by anion exchange and size exclusion chromatography. The finished suspension contained adenoviral particles in a buffer solution for the liquid form of the agent or in a buffer solution for the lyophilized form of the agent.

Thus, the following immunobiological agents were obtained based on the genome of the recombinant strain of simian adenovirus of the 25th serotype of the serotype, in which the E1 and E3 regions were deleted:

1. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 1 (simAd25- CMVS-CoV2) in a buffer solution for the liquid form of the agent.

2. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 1 (simAd25- CMV-

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S-CoV2) in a buffer solution for the lyophilized form of the agent.

3. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 2 (simAd25- CAGS-CoV2) in a buffer solution for the liquid form of the agent.

4. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 2 (simAd25- CAGS-CoV2) in the buffer solution for the lyophilized form of the agent.

5. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 3 (simAd25- EF1-SCoV2) in a buffer solution for the liquid form of the agent.

6. An immunobiological agent based on the genome of a recombinant strain of simian adenovirus serotype 25, in which E1 and E3 regions are deleted with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 3 (simAd25- EF1-SCoV2) in the buffer solution for the lyophilized form of the agent.

Each of the presented immunobiological agents is component 2 in option 1 of the developed agent and component 1 in option 3 of the developed agent.

Example 5

Obtaining an expression vector containing the genome of a recombinant human adenovirus strain of the 5th serotype.

At the first stage of the work, the design of the pAd5-Ends plasmid construct carrying two regions homologous to the genome of human adenovirus serotype 5 (two arms of homology) was developed. One arm of homology is the beginning of the human adenovirus serotype 5 genome (from the left inverted terminal repeat to the E1 region) and the sequence of the viral genome, including the pIX protein. The second arm of homology contains the nucleotide sequence after the ORF3 of the E4 region until the end of the genome. The pAd5-Ends construct was synthesized by ZAO Evrogen (Moscow).

Virion-isolated DNA of human adenovirus serotype 5 was mixed with pAd5-Ends. As a result of homologous recombination between pAd5-Ends and viral DNA, plasmid pAd5d1E1 was obtained, carrying the genome of human adenovirus serotype 5 with a deleted E1 region.

Then, using standard genetic engineering methods, the E3 region of the adenovirus genome (2685 bp from the end of the 12.5K gene to the beginning of the U-exon sequence) was removed from the constructed plasmid pAd5-d1E1 to increase the packaging capacity of the vector. As a result, a recombinant plasmid vector pAd5-too-null was obtained based on the genome of human adenovirus serotype 5 with a deletion of the E1 and E3 regions of the genome. SEQ ID NO: 7 was used as the maternal sequence for human adenovirus serotype 5.

In addition, the authors have developed several designs for the expression cassette:

the expression cassette of SEQ ID NO: 1 consists of the CMV promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 2 consists of the CAG promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal;

the expression cassette of SEQ ID NO: 3 consists of the EF1 promoter, the S gene of the SARS-CoV-2 virus protein, and a polyadenylation signal.

Then, based on the pAd5-Ends plasmid construct, pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EF1-S-CoV2 constructs containing SEQ ID expression cassettes were obtained by genetic engineering. NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, as well as carrying arms homology from the genome of adenovirus serotype 5.

After that, constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EF1-SCoV2 were linearized at a unique site of hydrolysis between homologous arms, each plasmid was mixed with the recombinant vector pAd5-too- null. As a result of homologous recombination, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too-EF1-S-CoV2 were obtained, carrying the genome of recombinant human adenovirus serotype 5 with an E1 deletion and E3 regions and expression cassettes of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively.

At the fourth stage, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too-EF1-SCoV2 were digested with a specific restriction endonuclease to remove the vector part. The resulting DNA preparation was transfected into HEK293 culture cells. The resulting material was used to accumulate preparative amounts of recombinant adenovirus.

As a result, recombinant human adenoviruses of the 5th serotype were obtained, containing the S gene of the SARS-CoV-2 virus protein: Ad5-CMV-S-CoV2 (containing the expression cassette SEQ ID NO: 1), Ad5-CAG-S-CoV2 (containing expression cassette of SEQ ID NO: 2), Ad5-EF1-S-CoV2 (containing expression cassette of SEQ ID NO: 3).

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Thus, an expression vector was obtained containing the genome of a recombinant strain of human adenovirus serotype 5, in which E1 and E3 regions were deleted with an integrated expression cassette selected from SeQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 .

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 E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 .

At this stage of the work, the expression vectors obtained in example 5 were purified by anion exchange and size exclusion chromatography. The finished suspension contained adenoviral particles in a buffer solution for the liquid form of the agent or in a buffer solution for the lyophilized form of the agent.

Thus, the following immunobiological agents were obtained based on the genome of the recombinant strain of human adenovirus serotype 5, in which the E1 and E3 regions are deleted:

1. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 1 (Ad5 -CMV-S-CoV2) in a buffer solution for the liquid form of the agent.

2. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 1 (Ad5 -CMV-S-CoV2) in the buffer solution for the lyophilized form of the agent.

3. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 2 (Ad5 -CAG-S-CoV2) in a buffer solution for the liquid form of the agent.

4. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the CAG promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 2 (Ad5 -CAG-S-CoV2) in a buffer solution for the lyophilized form of the agent.

5. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 3 (Ad5 -EF1-S-CoV2) in a buffer solution for the liquid form of the agent.

6. An immunobiological agent based on the genome of recombinant human adenovirus serotype 5, in which the E1 and E3 regions are deleted with an expression cassette containing the EF1 promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, SEQ ID NO: 3 (Ad5 -EF1-S-CoV2) in the buffer solution for the lyophilized form of the agent.

Each of the presented immunobiological agents is component 1 in option 1 and in option 2 of the developed agent.

Each of the presented immunobiological agents is component 2 in option 1 and in option 3 of the developed agent.

Example 7

Obtaining a buffer solution.

The developed tool according to this invention consists of two components located in different vials. Each component is an immunobiological agent based on a recombinant adenovirus with an expression cassette in a buffer solution.

The authors of the invention selected the composition of the buffer solution, which ensures the stability of recombinant adenovirus particles. This solution includes:

1. Tris (hydroxymethyl) aminomethane (Tris), which is necessary to maintain the pH of the solution.

2. Sodium chloride, which is added to achieve the required ionic strength and osmolarity.

3. Sucrose, which is used as a cryoprotectant.

4. Magnesium chloride hexahydrate, which is needed as a source of divalent cations.

5. EDTA, which is used as a free radical oxidation inhibitor.

6. Polysorbate-80, which is used as a surfactant.

7. Ethanol 95%, which is used as a free radical oxidation inhibitor.

8. Water, which is used as a solvent.

The authors of the invention developed 2 variants of the buffer solution: for the liquid form of the agent and for the lyophilized form of the agent.

To determine the concentration of the substances that make up the buffer solution for the liquid form of the agent, several variants of the experimental groups were obtained (Table 1). One of the components of the agent was added to each of the resulting buffer solutions:

1. An immunobiological agent based on a recombinant human adenovirus of the 26th serotype

- 11 043163 pa with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and the polyadenylation signal, 1x1011 viral particles.

2. An immunobiological agent based on recombinant human adenovirus serotype 5 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and a polyadenylation signal, 1x1011 viral particles.

3. An immunobiological agent based on recombinant simian adenovirus serotype 25 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and a polyadenylation signal, 1x1011 viral particles.

Thus, the stability of each of the adenovirus serotypes included in the product was tested. The resulting pharmaceuticals were stored at -18°C and -70°C for 3 months, then thawed and the change in titrarecombinant adenoviruses was assessed.

Table 1

Composition e experimental b buffer solutions for the liquid form of the product Buffer composition group number Tris (mg) Sodium chloride (mg) Sucrose (mg) Magnesium chloride hexahydrate (mg) EDTA (mg) Polysorbate 80 (mg) Ethanol 95% (mg) Water 1 0.968 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 2 1.815 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 3 1.21 1.752 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 4 1.21 3.285 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 5 1.21 2.19 20 0.102 0.019 0.25 0.0025 up to 0.5 ml 6 1.21 2.19 37.5 0.102 0.019 0.25 0.0025 up to 0.5 ml 7 1.21 2.19 25 0.0816 0.019 0.25 0.0025 up to 0.5 ml 8 1.21 2.19 25 0.153 0.019 0.25 0.0025 up to 0.5 ml 9 1.21 2.19 25 0.102 0.0152 0.25 0.0025 up to 0.5 ml 10 1.21 2.19 25 0.102 0.0285 0.25 0.0025 up to 0.5 ml AND 1.21 2.19 25 0.102 0.019 0.2 0.0025 up to 0.5 ml 12 1.21 2.19 25 0.102 0.019 0.375 0.0025 up to 0.5 ml 13 1.21 2.19 25 0.102 0.019 0.25 0.002 up to 0.5 ml 14 1.21 2.19 25 0.102 0.019 0.25 0.00375 up to 0.5 ml 15 1.21 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml

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

Thus, the developed buffer solution for the liquid form of the agent ensures the stability of all components of the developed agent in the following range of active ingredients, wt.%:

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

- 12 043163 ethanol 95% 0.0004-0.0007 solvent the rest.

To determine the concentration of the substances that make up the buffer solution for the lyophilized form of the agent, several variants of the experimental groups were obtained (Table 2). One of the components of the agent was added to each of the resulting buffer solutions:

1. An immunobiological agent based on recombinant human adenovirus serotype 26 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and a polyadenylation signal, 1x1011 viral particles.

2. An immunobiological agent based on recombinant human adenovirus serotype 5 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and a polyadenylation signal, 1x1011 viral particles.

3. An immunobiological agent based on recombinant simian adenovirus serotype 25 with an expression cassette containing the CMV promoter, the S gene of the SARS-CoV-2 virus protein and a polyadenylation signal, 1x1011 viral particles.

Thus, the stability of each of the adenovirus serotypes included in the product was tested. The resulting pharmaceuticals were stored at +2 and +8°C for 3 months, then thawed and the change in the titer of recombinant adenoviruses was assessed.

Table 2 __________ Composition of experimental buffer solutions __________

group number Buffer composition Tris (mg) Sodium chloride (mg) Sucrose (mg) Magnesium chloride hexahydrate (mg) EDTA (mg) Polysorbate 80 (mg) Water 1 0.1936 1.403 73.5 0.0204 0.0038 0.05 up to 1 ml 2 0.363 1.403 73.5 0.0204 0.0038 0.05 up to 1 ml 3 0.242 1.1224 73.5 0.0204 0.0038 0.05 up to 1 ml 4 0.242 2.1045 73.5 0.0204 0.0038 0.05 up to 1 ml 5 0.242 1.403 58.8 0.0204 0.0038 0.05 up to 1 ml 6 0.242 1.403 110.25 0.0204 0.0038 0.05 up to 1 ml 7 0.242 1.403 73.5 0.01632 0.0038 0.05 up to 1 ml 8 0.242 1.403 73.5 0.0306 0.0038 0.05 up to 1 ml 9 0.242 1.403 73.5 0.0204 0.00304 0.05 up to 1 ml 10 0.242 1.403 73.5 0.0204 0.0057 0.05 up to 1 ml AND 0.242 1.403 73.5 0.0204 0.0038 0.04 up to 1 ml 12 0.242 1.403 73.5 0.0204 0.0038 0.075 up to 1 ml 13 0.242 1.403 73.5 0.0204 0.0038 0.05 up to 1 ml

The results of the experiment showed that the titer of recombinant adenoviruses after their storage in a buffer solution for the lyophilized form of the agent at a temperature of +2°C and +8°C did not change for 3 months.

Thus, the developed buffer solution for the lyophilized form of the vaccine ensures the stability of all components of the developed agent in the following range of active ingredients, 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 solvent the rest.

Example 8

Study of the immunogenicity of the developed agent by assessing the cellular immune response to the antigen of the SARS-CoV-2 virus in the blood of volunteers at various times after vaccination.

Within the framework of clinical trials of the developed agent according to option 1, a study was made of the intensity of cellular immunity.

The study involved 40 volunteers who were immunized with:

1) Component 1, and then after 21 days with component 2 of the liquid form of the developed agent according to option 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2), dose of 1x1011 viral particles (20 people ).

2) Component 1, and then 21 days later, component 2 of the lyophilized form of the developed agent according to option 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2), dose of 1x1011 viral particles (20 people ).

On day 0 (before drug administration), day 14 and day 28, blood samples were taken from volunteers, from which mononuclear cells were isolated by centrifugation in a ficoll solution density gradient. Next, the isolated cells were stained with CFSE fluorescent dye (Invivogen, USA) and seeded onto the wells of the plate. Then, lymphocytes were re-stimulated under in vitro conditions by adding coronavirus S protein to the culture medium (final protein concentration 1 μg/ml). Intact cells, to which no antigen was added, were used as a negative control. 72 hours after the addition of the antigen, the % proliferating cells were measured, and the culture medium was taken to measure the amount of interferon gamma.

To determine the percentage of proliferating cells, they were stained with antibodies against marker molecules of T lymphocytes CD3, CD4, CD8 (anti-CD3 Pe-Cy7 (BD Biosciences, clone SK7), anti-CD4 APC (BD Biosciences, clone SK3), anti-CD8 PerCP -Cy5.5 (BD Biosciences, clone SKI)). Proliferating (with less CFSE cell dye) CD4+ and CD8+ T lymphocytes were determined in the cell mixture using a BD FACS AriaIII flow cytometer (BD Biosciences, USA). The resulting percentage of proliferating cells in each sample was determined by subtracting the result obtained from the analysis of intact cells from the result obtained from the analysis of cells restimulated with coronavirus S antigen. The results obtained are presented in Fig. 1, 2 (for the liquid form of the vaccine), in Fig. 3, 4 (for the lyophilized form of the vaccine).

Quantitative determination of the concentration of gamma-interferon (IFNy) in the culture medium of human mononuclear blood cells 72 hours after their restimulation by the S protein of the coronavirus was carried out using the gamma-Interferon-ELISA-BEST kit (VECTOR-BEST, Russia) according to the manufacturer's instructions. The data obtained is shown in Fig. 5 (for the liquid form of the vaccine), FIG. 6 (for the lyophilized form of the vaccine).

The results of the study showed that an increase in the intensity of cellular immunity caused by sequential immunization of volunteers with both forms of the agent according to option 1, according to the median values of proliferating CD4+ and CD8+ T-lymphocytes, is observed with an increase in days after immunization. In both groups, the maximum values of proliferating both CD4+ and CD8+ T-lymphocytes are observed on the 28th day after immunization. Between 0 and 28 days of the study, there is a maximum statistically significant difference in the values of proliferating CD4+ and CD8+ T-lymphocytes - p<0.001.

From the results shown in FIG. 5, 6, it can be concluded that the increase in the intensity of cellular immunity caused by the sequential immunization of volunteers with both forms of the agent according to option 1, according to the median increase in the concentration of IFNy, is observed with an increase in days after immunization. A statistically significant difference in the values of the increase in the concentration of IFNy before immunization (day 0) and 14 days after vaccination was p<0.001. The maximum increase in the concentration of IFNy is observed on the 28th day after immunization. Between days 0 and 28 of the study, there is a maximum statistically significant difference in the values of the increase in the concentration of IFNy - p<0.001.

Thus, based on the above data, it can be concluded that immunization with the developed agent can cause the formation of a tense antigen-specific cellular link of anti-infective immunity, which is confirmed by a high degree of statistical significance in the measured parameters before and after immunization.

Example 9

Study of the immunogenicity of the developed agent by assessing the titer of antibodies to the antigen of the SARS-CoV-2 virus in the blood of volunteers at various times after vaccination.

The study involved 40 volunteers who were immunized with:

3) Component 1, and then after 21 days with component 2 of the liquid form of the developed agent according to option 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2), dose of 1x1011 viral particles (20 people ).

4) Component 1, and then 21 days later, component 2 of the lyophilized form of the developed agent according to option 1 (component 1: Ad26-CMV-S-CoV2, component 2: Ad5-CMV-S-CoV2), dose of 1x1011 viral particles (20 people ).

On day 14, day 21, day 28, blood samples were taken from volunteers, from which serum was isolated.

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The titer of antibodies to the RBD S domain of the SARS-CoV-2 virus protein was determined by enzyme immunoassay using the SARS-CoV-2-RBD-ELISA-Gamaleya kit. The analysis was carried out according to the manufacturer's instructions.

The results of the analysis of the antibody titer to the antigen of the SARS-CoV-2 virus in the blood serum of volunteers after the administration of the liquid form of the drug are shown in Fig. 7.

The results of the analysis of the antibody titer to the antigen of the SARS-CoV-2 virus in the blood serum of volunteers after the administration of the lyophilized form of the drug are shown in Fig. 8.

As can be seen from the data presented, immunization of volunteers with the developed agent both in liquid form and in lyophilized form allows the formation of a pronounced (statistically significantly different from the indicators of the control non-immunized group of animals) humoral immunity, characterized by an increase in the titer of antibodies to the S protein of the SARS-CoV- virus. 2. At the same time, an increase in the intensity of the humoral immune response is noted with an increase in days after immunization.

Example 10

The use of the developed agent for prolonging post-vaccination immunity to the SARS-CoV-2 virus after immunization with a model subunit vaccine.

The purpose of this work was to determine the possibility of using the developed agent for revaccination of animals immunized with a model subunit vaccine.

In this experiment, Balb/c mice, females, weighing 18 g were used. At the first stage, the animals were immunized with a model vaccine consisting of the S protein of the SARS-CoV-2 virus (10 µg/mouse) in phosphate-buffered saline and aluminum hydroxide (100 µg/mouse). Immunization was carried out twice with an interval of 21 days. On the 180th day of the experiment, repeated immunization was carried out with various variants of the developed agent. In the case of a two-component agent: 1 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 a one-component agent, immunization was carried out on day 201 of the experiment. As a result, the following experimental and control groups of animals were obtained:

1) Model vaccine^26-CMV-S-CoV2/ Ad5-CMV-S-CoV2

2) Model vaccine/Ad26-CAG-S-CoV2/ Ad5-CAG-S-CoV2

3) Model vaccine/ ^A d26- EFl-S-CoV2/ Ad5- EFl-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- EFl-S-CoV2/ simAd25- EFl-S-CoV2

7) Model vaccine/sirnAd25-CMV-S-CoV2/ Ad5-CMV-S-CoV2

8) Model vaccine/ simAd25- CAG-S-CoV2/ Ad5- CAG-S-CoV2

9) Model vaccine/simAd25- EFl-S-CoV2/ Ad5- EFl-S-CoV2

10) Model vaccine/Ad26-CMV-S-CoV2

11) Model vaccine/Ad26-CAG-S-CoV2

12) Model vaccine/Ad26-EFl-S-CoV2

13) Model vaccine/Ad5-CMV-S-CoV2

14) Model vaccine/Ad5-CAG-S-CoV2

15) Model vaccine/Ad5-EFl-S-CoV2

16) Model vaccine/siplAd25-CMV-S-CoV2

17) Model vaccine/ ^s i ^m Ad25-CAG-S-CoV2

18) Model vaccine/simAd25-EFl-S-CoV2

On days 21, 180 and 222 of the experiment, blood was taken from the tail vein of the animals and blood serum was isolated. The titer of antibodies to SARS-CoV-2 was determined by enzyme immunoassay (ELISA) according to the following protocol:

1) The antigen was adsorbed on the wells of a 96-well plate for 16 hours at a temperature of +4°C.

2) Next, to get rid of non-specific binding, the plate was plugged with blocking buffer in a volume of 100 μl per well. Incubated on a shaker at 37°C for an hour.

3) Sera from immunized mice were diluted 100-fold and then a series of 2-fold dilutions was made.

4) Add 50 µl of each diluted serum sample to the wells of the plate.

5) Further incubation was carried out for 1 hour at 37°C.

6) After incubation, the wells were washed three times with phosphate buffer.

7) Secondary antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were then added.

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8) Further incubation was carried out for 1 hour at 37°C.

9) After incubation, the wells were washed three times with phosphate buffer.

10) Then a solution of tetramethylbenzidine (TMB) was added, which is a substrate for horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 min by adding sulfuric acid. Next, using a spectrophotometer measured the optical density of the solution (OD) in each well at a wavelength of 450 nm.

The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results obtained (geometric mean) are presented in table. 3.

Table 3

Titer of antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

Days from the start of the experiment 21 day 180 day Day 222 1 Model vaccine/ Ad26-CMV-S-CoV2/ Ad5-CMV-S-CoV2 1393 606 117627 2 Model vaccine/Ad26-CAG-S-CoV2/ Ad5-CAG-S-CoV2 1600 528 89144 3 Model vaccine/ Ad26-EFl-S-CoV2/ Ad5-EFl-S-CoV2 1213 696 102400 4 Model vaccine/ Ad26-CMV-S-CoV2/ simAd25-CMV-S-CoV2 1600 606 58813 5 Model vaccine/ Ad26- CAG-S-CoV2/ simAd25- CAG-S-CoV2 1393 528 67559 6 Model vaccine/ Ad26-EFl-S-CoV2/ simAd25-EFl-S-CoV2 1393 528 58813 7 Model vaccine/simAd25-CMV-SCoV2/ Ad5-CMV-S-CoV2 1600 606 89144 8 Model vaccine/ simAd25- CAG-SCoV2/ Ad5- CAG-S-CoV2 1213 606 89144 9 Model vaccine/ simAd25- EF1-SCoV2/ Ad5-EFl-S-CoV2 1393 696 102400 10 Model vaccine/ Ad26-CMV-S-CoV2 1600 528 16890 AND Model vaccine/ Ad26-CAG-S-CoV2 1600 528 19401 12 Model vaccine/ Ad26-EFl-S-CoV2 1600 696 19401 13 Model vaccine/ Ad5-CMV-S-CoV2 1600 606 51200 14 Model vaccine/Ad5-CAG-S-CoV2 1600 606 44572 15 Model vaccine/Ad5-EFl-S-CoV2 1600 528 51200 16 Model vaccine/simAd25-CMV-SCoV2 1600 606 33779 17 Model vaccine/ simAd25-CAG-SCoV2 1393 528 38802 18 Model vaccine/simAd25-EF1-SCoV2 1600 528 33779

As can be seen from the presented data, after immunization of mice with a model subunit vaccine, all animals develop antibodies, after 180 days the antibody titer decreases, however, revaccination of animals with various variants of the developed agent leads to a multiple increase in the antibody titer in the blood. Thus, the experimental data confirm the possibility of using the developed agent to prolong post-vaccination immunity against the SARS-CoV-2 virus.

Example 11.

Application of the developed agent for prolonging post-vaccination immunity to the virus

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SARS-CoV-2 after immunization with a model inactivated vaccine.

The purpose of this work was to determine the possibility of using the developed agent for revaccination of animals immunized with a model inactivated vaccine.

In this experiment, Balb/c mice, females, weighing 18 g were used. At the first stage, the animals were immunized with a model vaccine consisting of the formalin-inactivated SARS-CoV-2 virus. Immunization was carried out twice with an interval of 21 days. On the 180th day of the experiment, repeated immunization was carried out with various variants of the developed agent. In the case of a two-component agent: 1 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 a one-component agent, immunization was carried out on day 201 of the experiment. As a result, the following experimental and control groups of animals were obtained:

1. Model vaccine/Ad26-CMV-S-CoV2/ Ad5-CMV-S-CoV2

2. Model vaccine/Ad26-CAG-S-CoV2/ Ad5-CAG-S-CoV2

3. Model vaccine/Ad26- EFl-S-CoV2/ Ad5- EFl-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- EFl-S-CoV2/ simAd25- EFl-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/ sirnAd2 5- EFl-S-CoV2/ Ad5- EFl-S-CoV2

10. Model vaccine/Ad26-CMV-S-CoV2

11. Model vaccine/Ad26-CAG-S-CoV2

12. Model vaccine/Ad26-EFl-S-CoV2

13. Model vaccine/Ad5-CMV-S-CoV2

14. Model vaccine/Ad5-CAG-S-CoV2

15. Model vaccine/Ad5-EFl-S-CoV2

16. Model vaccine/ sirnAd25-CMV-S-CoV2

17. Model vaccine/simAd25-CAG-S-CoV2

18. Model vaccine/simAd25-EFl-S-CoV2

On days 21, 180 and 222 of the experiment, blood was taken from the tail vein of the animals and blood serum was isolated. The titer of antibodies to SARS-CoV-2 was determined by enzyme immunoassay (ELISA) according to the following protocol:

1) The antigen was adsorbed on the wells of a 96-well plate for 16 hours at a temperature of +4°C.

2) Next, to get rid of non-specific binding, the plate was plugged with blocking buffer in a volume of 100 μl per well. Incubated on a shaker at 37°C for an hour.

3) Sera from immunized mice were diluted 100-fold and then a series of 2-fold dilutions was made.

4) Add 50 µl of each diluted serum sample to the wells of the plate.

5) Further incubation was carried out for 1 hour at 37°C.

6) After incubation, the wells were washed three times with phosphate buffer.

7) Secondary antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were then added.

8) Further incubation was carried out for 1 hour at 37°C.

9) After incubation, the wells were washed three times with phosphate buffer.

10) Then a solution of tetramethylbenzidine (TMB) was added, which is a substrate for horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 min by adding sulfuric acid. Next, using a spectrophotometer measured the optical density of the solution (OD) in each well at a wavelength of 450 nm.

The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results obtained (geometric mean) are presented in table. 4.

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Table 4

Titer of antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

Days from the start of the experiment 21 day 180 day Day 222 1 Model vaccine/ Ad26-CMV-S-CoV2/ Ad5-CMV-S-CoV2 303 200 135118 2 Model vaccine/Ad26-CAG-S-CoV2/ Ad5-CAG-S-CoV2 348 152 117627 3 Model vaccine/ Ad26- EFl-S-CoV2/ Ad5EFl-S-CoV2 264 174 155209 4 Model vaccine/ Ad26-CMV-S-CoV2/ simAd25-CMV-S-CoV2 303 115 58813 5 Model vaccine/ Ad26- CAG-S-CoV2/ simAd25- CAG-S-CoV2 264 174 77605 6 Model vaccine/ Ad26-EFl-S-CoV2/ simAd25-EFl-S-CoV2 303 174 67559 7 Model vaccine/simAd25-CMV-S-CoV2/ Ad5-CMV-S-CoV2 264 115 135118 8 Model vaccine/simAd25-CAG-S-CoV2/ Ad5-CAG-S-CoV2 264 174 117627 9 Model vaccine/ simAd25- EFl-S-CoV2/ Ad5-EFl-S-CoV2 303 115 117627 10 Model vaccine/ Ad26-CMV-S-CoV2 264 174 19401 AND Model vaccine/ Ad26-CAG-S-CoV2 348 132 19401 12 Model vaccine/ Ad26-EFl-S-CoV2 303 174 16890 13 Model vaccine/ Ad5-CMV-S-CoV2 303 132 58813 14 Model vaccine/Ad5-CAG-S-CoV2 264 152 58813

As can be seen from the data presented, after immunization of mice with a model inactivated vaccine, all animals develop antibodies, after 180 days the antibody titer decreases, however, revaccination of animals with various variants of the developed agent leads to a multiple increase in the antibody titer in the blood. Thus, the experimental data confirm the possibility of using the developed agent to prolong post-vaccination immunity against the SARS-CoV-2 virus.

Example 12.

The use of the developed agent for prolonging post-vaccination immunity to the SARS-CoV-2 virus after immunization with various variants of the developed agent.

The purpose of this work was to determine the possibility of using the developed agent for revaccination of animals immunized with various variants of the developed agent.

In this experiment, Balb/c mice, females, weighing 18 g were used. At the first stage, the animals were immunized with various one-component variants of the developed agent (10 ¹⁰ vp/mouse). After 180 days of the experiment, repeated immunization was carried out with various two-component variants of the developed agent. 1 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 a one-component agent, immunization was carried out on day 201 of the experiment. As a result, the following experimental and control groups of animals were obtained:

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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

On days 21, 180 and 222 of the experiment, blood was taken from the tail vein of the animals and blood serum was isolated. The titer of antibodies to SARS-CoV-2 was determined by enzyme immunoassay (ELISA) according to the following protocol:

1) The antigen was adsorbed on the wells of a 96-well plate for 16 hours at a temperature of +4°C.

2) Next, to get rid of non-specific binding, the plate was plugged with blocking buffer in a volume of 100 μl per well. Incubated on a shaker at 37°C for an hour.

3) Sera from immunized mice were diluted 100-fold and then a series of 2-fold dilutions was made.

4) Add 50 µl of each diluted serum sample to the wells of the plate.

5) Further incubation was carried out for 1 hour at 37°C.

6) After incubation, the wells were washed three times with phosphate buffer.

7) Secondary antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were then added.

8) Further incubation was carried out for 1 hour at 37°C.

9) After incubation, the wells were washed three times with phosphate buffer.

10) Then a solution of tetramethylbenzidine (TMB) was added, which is a substrate of horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 m by adding sulfuric acid. Next, using a spectrophotometer measured the optical density of the solution (OD) in each well at a wavelength of 450 nm.

The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results obtained (geometric mean) are presented in table. 5.

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Table 5

Titer of antibodies to protein S in the blood serum of mice (geometric mean, antibody titer)

Days from the start of the experiment 21 day 180 day Day 222 1 Ad26-CMV-S-CoV2/ Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 2786 2111 204800 2 Ad26-CMV-S-CoV2/ Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 2425 2425 155209 3 Ad26-CMV-S-CoV2/simAd25-CMV-S- CoV2, Ad5-CMV-S-CoV2 2111 1838 178289 4 Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 29407 25600 155209 5 Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 33779 29407 178289 6 Ad5-CMV-S-CoV2/simAd25-CMV-S-CoV2, Ad5-CMV-S-CoV2 38802 33779 155209 7 simAd25-CMV-S-CoV2/ Ad26-CMV-S- CoV2, Ad5-CMV-S-CoV2 12800 9701 155209 8 simAd25-CMV-S-CoV2/ Ad26-CMV-S- CoV2, simAd25-CMV-S-CoV2 14703 11143 178289 9 simAd25-CMV-S-CoV2/ simAd25-CMV-S- CoV2, Ad5-CMV-S-CoV2 16890 11143 155209 10 Ad26-CMV-S-CoV2/ Ad26-CMV-S-CoV2 3200 2111 117627 AND Ad26-CMV-S-CoV2/ Ad5-CMV-S-CoV2 2425 2111 102400 12 Ad26-CMV-S-CoV2/simAd25-CMV-SCoV2 2425 2111 117627 13 Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2 25600 22286 102400 14 Ad5-CMV-S-CoV2/ Ad5-CMV-S-CoV2 29407 25600 117627 15 Ad5-CMV-S-CoV2/simAd25-CMV-S-CoV2 38802 33779 102400 16 simAd25-CMV-S-CoV2/ Ad26-CMV-S- CoV2 16890 12800 117627 17 simAd25-CMV-S-CoV2/ Ad5-CMV-S-CoV2 16890 14703 117627 18 simAd25-CMV-S-CoV2/ simAd25-CMV-S- CoV2 19401 16890 102400

As can be seen from the presented data, after immunization of mice with one-component variants of the developed agent, all animals develop antibodies, after 180 days the antibody titer decreases, however, revaccination of animals with various variants of the developed agent leads to a multiple increase in the antibody titer in the blood. Thus, the experimental data confirm the possibility of using the developed agent to prolong post-vaccination immunity against the SARS-CoV-2 virus.

Example 13

The use of the developed agent for prolonging post-vaccination immunity to the SARS-CoV-2 virus after immunization with various variants of the developed agent.

The purpose of this work was to determine the possibility of using the developed agent for revaccination of animals immunized with various variants of the developed agent.

In this experiment, Balb/c mice, females, weighing 18 g were used. At the first stage, the animals were immunized with variants of the developed two-component agent (10 ¹⁰ vp/mouse) with an interval of 21 days. On the 180th day of the experiment, repeated immunization was carried out with various variants of the developed one-component agent. As a result, the following experimental and control groups of animals were obtained:

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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

On days 42, 180 and 201 days of the experiment, blood was taken from the tail vein of the animals and the blood serum was isolated. The titer of antibodies to SARS-CoV-2 was determined by enzyme immunoassay (ELISA) according to the following protocol:

1) The antigen was adsorbed on the wells of a 96-well plate for 16 hours at a temperature of +4°C.

2) Next, to get rid of non-specific binding, the plate was plugged with blocking buffer in a volume of 100 μl per well. Incubated on a shaker at 37°C for an hour.

3) Sera from immunized mice were diluted 100-fold and then a series of 2-fold dilutions was made.

4) Add 50 µl of each diluted serum sample to the wells of the plate.

5) Further incubation was carried out for 1 hour at 37°C.

6) After incubation, the wells were washed three times with phosphate buffer.

7) Secondary antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were then added.

8) Further incubation was carried out for 1 hour at 37°C.

9) After incubation, the wells were washed three times with phosphate buffer.

10) Then a solution of tetramethylbenzidine (TMB) was added, which is a substrate for horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 min by adding sulfuric acid. Next, using a spectrophotometer measured the optical density of the solution (OD) in each well at a wavelength of 450 nm.

The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results obtained (geometric mean) are presented in table. 6.

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Table 6

Titer of antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

Days from the start of the experiment 21 day 180 day Day 222 1 Ad26-CMV-S-CoV2/ Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 44572 38802 204800 2 Ad26-CMV-S-CoV2/ Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 38802 33779 178289 3 Ad26-CMV-S-CoV2/simAd25-CMV-S- CoV2, Ad5-CMV-S-CoV2 51200 38802 204800 4 Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2 33779 29407 178289 5 Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2, simAd25-CMV-S-CoV2 29407 25600 178289 6 Ad5-CMV-S-CoV2/simAd25-CMV-S- CoV2, Ad5-CMV-S-CoV2 29407 25600 178289

7 simAd25-CMV-S-CoV2/ Ad26-CMV-S- CoV2, Ad5-CMV-S-CoV2 38802 33779 178289 8 simAd25-CMV-S-CoV2/ Ad26-CMV-S- CoV2, simAd25-CMV-S-CoV2 38802 33779 204800 9 simAd25-CMV-S-CoV2/ simAd25-CMV- S-CoV2, Ad5-CMV-S-CoV2 33779 33779 155209 10 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2/ Ad26-CMV-S-CoV2 44572 38802 204800 AND Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2/ Ad5-CMV-S-CoV2 33779 29407 204800 12 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2/simAd25-CMV-S-CoV2 38802 33779 204800 13 Ad26-CMV-S-CoV2, simAd25-CMV-S- CoV2/ Ad26-CMV-S-CoV2 38802 33779 178289 14 Ad26-CMV-S-CoV2, simAd25-CMV-S- CoV2/ Ad5-CMV-S-CoV2 44572 38802 204800 15 Ad26-CMV-S-CoV2, simAd25-CMV-S- CoV2/simAd25-CMV-S-CoV2 38802 33779 178289 16 simAd25-CMV-S-CoV2, Ad5-CMV-S- CoV2/ Ad26-CMV-S-CoV2 51200 38802 204800 17 simAd25-CMV-S-CoV2, Ad5-CMV-S- CoV2/ Ad5-CMV-S-CoV2 33779 29407 178289 18 simAd25-CMV-S-CoV2, Ad5-CMV-S- CoV2/simAd25-CMV-S-CoV2 29407 25600 178289

As can be seen from the presented data, after immunization of mice with two-component variants of the developed agent, all animals develop antibodies, after 180 days the antibody titer decreases, however, revaccination of animals with various variants of the developed agent leads to a multiple increase in the antibody titer in the blood. Thus, experimental data confirm the possibility of using the developed agent to prolong post-vaccination immunity against the SARS-CoV-2 virus.

Thus, the set technical task, namely the creation of agents, agents that ensure the prolongation of post-vaccination immunity against the SARS-CoV-2 virus, has been achieved, which is confirmed by the examples given.

Industrial Applicability

All the above examples confirm the effectiveness of agents that provide effective induction of the immune response, as well as the prolongation of post-vaccination immunity against the SARS-CoV-2 virus and industrial applicability.

Claims (7)

1. The use of an agent containing component 1, which is an 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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an integrated expression cassette , selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which E1 and E3 regions with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 for revaccination against diseases caused by severe acute respiratory syndrome virus SARSCoV-2. 2. The use of an agent containing component 1, which is an 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 are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which E1 are deleted and E3 regions with an inserted expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, or containing only component 2 for revaccination against diseases caused by severe acute respiratory syndrome virus SARS-CoV-2. 3. The use of an agent containing component 1, which is an agent in the form of an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, for revaccination against diseases caused by severe acute respiratory syndrome virus SARS-CoV-2. 4. Use according to claims 1 to 3, characterized in that the agent is in liquid or lyophilized form. 5. The use according to claim 4, characterized in that the buffer solution for the liquid form of the agent contains, wt.%: 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 the rest. 6. Use according to claim 4, characterized in that the reconstituted lyophilized form of the agent contains a buffer solution, wt.%: tris sodium chloride sucrose magnesium chloride hexahydrate EDTA polysorbate-80 water 0.0180-0.0338 0.1044-0.1957 5.4688-10.2539 0.0015-0.0028 0.0003-0.0005 0.0037-0.0070 rest. 7. Use according to claims 1 to 3, characterized in that component 1 and component 2 of the product are in different packages.