EA042672B1 - APPLICATION OF A DRUG FOR INDUCING SPECIFIC IMMUNE AGAINST THE SARS-CoV-2 SEVERE ACUTE RESPIRATORY SYNDROME VIRUS IN PERSONS OVER 60 AND/OR HAVING CHRONIC DISEASES (OPTIONS) - Google Patents

Description

Technical field

The invention relates to biotechnology, immunology and virology. Proposed is the use of an agent for the prevention of diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2-vaccination, including revaccination, in persons aged 60 years and older and/or with chronic diseases.

State of the art

The ongoing COVID-19 pandemic has affected millions of people around the world. Currently, more than 100 million cases and more than 2 million deaths from this disease have been registered.

In the course of the studies, it was found that the cause of the disease is the RNA-containing virus SARS-CoV-2, belonging to the Coronaviridae family, to the Beta-CoV B line. It was shown that the virus spreads by direct, contact transmission, as well as by airborne droplets. The median incubation period is 5.1 days, after which the first symptoms develop (Lauer SA, Grantz KH, Bi Q, et al. The incubation period of coronavirus disease 2019 (COVID19) from publicly reported confirmed cases: estimation and application. Ann Intern Med. 2020. 10.7326/M200504). For COVID-19, the characteristic symptoms are: fever, dry cough, shortness of breath, fatigue. Less common - sore throat, joint pain, runny nose, headache.

COVID-19 can be both mild and severe. The most formidable complications of this disease are pneumonia, acute respiratory distress syndrome, acute respiratory failure, acute heart failure, acute renal failure, septic shock, cardiomyopathy, etc. In this case, a severe form of the disease often develops in older patients and those with chronic diseases. So, according to the Centers for Disease Control and Prevention, the risk of death from COVID-19 at the age of 50-64 years increases 30 times, at 65-74 years old - 90 times, at 75-84 years old - 220 times, and in people older than 85 years - 630 times (compared to younger people (18-29 years old) (https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/older-adults.html).

SARS-CoV-2 enters the human body by binding the viral S protein to the angiotensin-converting enzyme-2 (ACE-2) receptor, which is expressed in the lungs, endothelium, heart, kidneys, and gastrointestinal tract. It is assumed that due to this specific mechanism, the risk of infection in the elderly increases. According to the CDC, 63.1% of adults over 60 have hypertension, 38% of people over 65 have chronic kidney disease (CKD), and 26.8% of adults over 65 have diabetes. Many of these patients use ACE-2 inhibitors and angiotensin receptor blockers, which activate the ACE-2 receptor. Thus, it is hypothesized that older adults with these comorbidities may be at increased risk and have a more severe course of SARS-CoV-2 infection (Z. Shahid et al. COVID-19 and Older Adults: What We Know. J Am Geriatr Soc. 2020 May; 68(5): 926-929).

Thus, older people and/or those with chronic diseases are in particular need of vaccination against COVID-19.

To date, several vaccines against Covid-19 have been developed. Pharmaceutical company Moderna, in collaboration with the National Institutes of Health (USA), has developed the mRNA-1273 vaccine, which consists of mRNA encoding the S protein of SARS-CoV-2 protected by a lipid envelope. At the moment, phase 3 clinical trials of this vaccine have ended. This study included more than 7,000 volunteers over the age of 65, as well as more than 5,000 volunteers under the age of 65, who have chronic conditions that put them at increased risk of severe COVID-19 (diabetes, obesity, and heart disease). According to published data, the effectiveness of the vaccine for people over 65 years of age was 86.4% (L. Baden et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2020 Dec 30).

Pharmaceutical company Pfizer has partnered with biotech company BioNTech to develop the BNT162b2 (tozinameran) vaccine. This vaccine is a lipid nanoparticle encapsulated with a modified mRNA encoding the mutant form S of the SARS-CoV-2 protein. More than 18,000 people aged 16 to 89 years old were vaccinated during the latest phase of clinical trials, with 7,971 people (42.3%) over 55 years of age. It is also reported that some patients had chronic diseases (obesity, etc.), which are risk factors for an unfavorable outcome of COVID-19. The effectiveness of the vaccine for patients over 55 years old was 93.7%, over 65 years old - 94.7%, over 75 years old - 100% (Polack F. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec).

The University of Oxford, in collaboration with the pharmaceutical company AstraZeneca, has developed a ChAdOx1 nCoV-19 vector vaccine (AZD1222). The active component of this vaccine is the chimpanzee adenovirus ChAdOx1 containing a codon-optimized coding sequence of the full-length S protein of the SARS-CoV-2 virus (GenBank MN908947) with a tissue plasminogen activator leader sequence. Individuals aged 56 years and older were enrolled in clinical trials of the drug, which accounted for 12.2% of the total cohort (1006 (13.3%) in the UK and 412 (10.1%) in Brazil). Of these, patients aged 56-69 years - 974 people, from 70 years and older - 1 042672

444 people. The vaccine had good safety profiles. However, according to the authors of the study, it was not possible to evaluate the effectiveness of the vaccine in age groups over 56 years of age due to insufficient data (Voysey M. et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomized controlled trials in Brazil, South

Africa, and the UK. Lancet. 2021 Jan 9;397(10269):99-111).

The Johnson and Johnson and Janssen Pharmaceutical research teams, in collaboration with the Beth Israel Deaconess Medical Center, have developed the Ad26.COV2.S vaccine. The active component of this vaccine is a recombinant vector based on human adenovirus serotype 26, with a deletion of the E1 and E3 regions, containing the S gene of the SARS-CoV-2 protein, with a furin cleavage site mutation and with two proline-stabilizing mutations. The results of a Phase 1-2b clinical trial have now been published. This study included a cohort of participants aged 18 to 55 years and a cohort of participants over 65 years of age. The results of the study showed that neutralizing antibodies could be detected in 90% of participants, regardless of age group, on day 29 after 1 dose of the vaccine. By day 57, neutralizing antibodies were detected in all vaccinated volunteers. The introduction of the second dose of the vaccine provided a 2.6-2.9-fold increase in antibody titer (Sadoff J. Interim Results of a Phase 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine. N Engl J Med. 2021 Jan 13. doi: 10.1056/NEJMoa2034201. Epub ahead of print. PMID: 33440088.). This solution was chosen by the authors for the prototype. The disadvantage of the prototype is that after the first injection of the vaccine, it is possible to form antibodies to the vector part of the expression vector, which, upon subsequent administration, will reduce the effectiveness of vaccination.

Thus, there is a need in the art for an agent that is safe and capable of inducing an immune response against the SARS-CoV-2 virus in elderly and/or chronically ill patients.

Implementation of the invention

The technical objective of the claimed invention is the creation of agents that provide effective induction of an immune response against the SARS-CoV-2 virus in persons aged 60 and older, as well as those with chronic diseases.

The technical result consists in creating a safe and effective agent that ensures the development of humoral and cellular immune responses against the SARS-CoV2 virus in persons aged 60 and older, as well as those with chronic diseases.

With age, the aging of the immune system occurs, which is characterized by a decrease in the qualitative and quantitative characteristics of the immune response, which can affect the safety and efficacy profile of developed vaccines.

Quantitative changes are characterized by a decrease in the number of naive T-lymphocytes, as well as a change in the ratio of CD4+:CD8+ cells, mainly due to a significant decrease in the number of CD8+ T-lymphocytes. Aging also leads to a decrease in the repertoire of T-cell receptors, and in general reduces the survival of T-lymphocytes. Qualitative changes include the preferential production of short-lived effector T cells over memory cell progenitors, resulting in an impaired immune response. The number of B cells does not change as much with age, however, due to the decrease in the expression of selected proteins, less functional antibodies are produced in old age. In addition to age, various chronic diseases can influence the formation of the immune response to the vaccine, since many of them are often associated with disorders in the immune system.

Thus, the development of an agent for use by elderly people and / or with chronic diseases, which provides effective induction of an immune response against the SARS-CoV-2 virus, including the development of humoral and cellular immune responses against the SARS-CoV-2 virus, is a complex scientific task. .

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 to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in persons over 60 years of age and / or with chronic diseases.

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

- 2 042672 genome of a 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 or containing only component 2 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in persons over 60 years of age and / or with chronic diseases.

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 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 to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in individuals over 60 years of age and/or with chronic diseases.

In this case, when using the agent, it is administered intranasally and / or intramuscularly.

Also, when using the agent, the components are administered sequentially with an interval of more than 1 week.

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

Moreover, when used, the liquid form of the agent contains a buffer solution, wt.%:

tris 0.1831 to 0.3432 sodium chloride 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 the rest.

At the same time, when used, 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.

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

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 CD4+ and CD8+ T lymphocytes restimulated by SARS-CoV-2 S antigen.

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

• - denoted by % of proliferating CD4+ T-lymphocytes for each volunteer on day 0.

• - denoted % of proliferating CD4+ T-lymphocytes for each volunteer on day 28.

• - denoted % of proliferating CD8+ lymphocytes for each volunteer on day 0.

• - denoted % of proliferating CD8+ lymphocytes for each volunteer on day 28.

The median values are represented as a black line for each data group, the deviations indicate the 95% confidence interval. Symbol ****, indicates a statistically significant difference between the values of 0 and 28 days (p<0.001, according to the Mann-Whitney test).

- 3 042672

In FIG. Figure 2 shows the increase in the concentration of IFNy (in times) in the culture medium of peripheral blood mononuclear cells after their restimulation with cononavirus S antigen before immunization (day 0) and after 28 days of the study, after sequential immunization of volunteers in the age group over 60 years old with the developed agent according to option 1.

Y-axis - IFNu concentration (times)

The abscissa axis is days.

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

- values are indicated for each volunteer on day 28.

The median of the values is represented as a black line for each data group. Deviations denote 95% confidence interval. Symbol ****, indicates a statistically significant difference between the values of 0 and 28 days (p<0.001, according to the Mann-Whitney test).

In FIG. Figure 3 shows the results of assessing the humoral immune response to the antigen of the SARSCoV2 virus in volunteers immunized with the liquid form of the developed agent according to option 1.

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

The abscissa axis is days.

• - values for each volunteer are indicated.

The geometric mean of the antibody titer is shown as a black bar for each data group. A statistically significant difference between the values on days 21, 28 and 42 is indicated by a bracket above which is the p value according to the Wilcoxon T-test.

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 β-actin promoter, and a chimeric intron (chicken β-actin and rabbit β-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 nondividing cells, are able to induce cellular and humoral immune responses, and provide a high level of expression of the target antigen.

The authors have developed drug variants containing two components, as well as drug variants containing one component based on adenoviruses of different serotypes. Thus, the immune response to the vector part of the adenovirus, which may occur after the introduction of the first component of the agent or a single agent, is not further boosted and does not affect the generation of antigen-specific immune responses against the vaccine antigen in the case of using a two-component agent or, if necessary, repeated administration of a single-component agent. means, since in the latter case a means based on a different adenovirus may be introduced.

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 inducing specific immunity against 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 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 on based on the genome of a recombinant strain of human adenovirus serotype 5, 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.

2. An agent for the induction of specific immunity against 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 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 inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an expression vector agent based on genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 or containing only component 2 are deleted.

3. An agent for the induction of specific immunity against 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 simian adenovirus serotype 25, in which the E1 and E3 regions of co built-in 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 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.

At the same time, the components of the product are in individual packages.

Also, the authors of the invention developed liquid and lyophilized forms of the agent. In addition, the authors of the invention have selected variants of the buffer solution that allow you to store the developed agent both in frozen form at a temperature below -18°C, and in the form of a lyophilisate at a temperature of +2 to +8°C.

A method has also been developed for using an agent for inducing specific immunity against the SARS-CoV-2 severe acute respiratory syndrome virus in people over 60 years of age and/or with chronic diseases by introducing it into the body in an effective amount.

In addition, the tool, including consisting of a single component, can be used once.

The two-component product can be used sequentially at least 1 week apart.

The tool can be used intranasally and / or intramuscularly.

For revaccination, any of the proposed means can be used, regardless of which means the vaccination was carried out.

The implementation of the invention is confirmed by the following examples.

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 was developed, carrying two regions homologous to the genome of human adenovirus serotype 26 (two arms of homology), and the ampicillin resistance gene. 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).

- 5 042672

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-dlE1 was obtained, carrying the genome of human adenovirus serotype 26 with a deleted E1 region.

Then, in the resulting plasmid pAd26-dlE1, 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-dlE1-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-dlE1-ORF6-Ad5 to increase the packaging capacity of the vector. As a result, a pAd26-only-null recombinant vector was obtained based on the genome of human adenovirus serotype 26 with an open reading frame ORF6 of human adenovirus serotype 5 and with a deletion of E1 and E3 regions. 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 gene of the SARS-CoV-2 virus protein, and a 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 a 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 adenovirus26 serotype, 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 adenovirus26 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.

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 26 serotype, 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 the signal polyadenylation, 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 26 serotype, 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 the signal polyadenylation, SEQ ID NO: 1 (Ad26-CMV-S-CoV2) in buffer solution for lyophilized form of the agent.

3. An immunobiological agent based on the genome of a recombinant strain of human adenovirus 26 serotype, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5 with

- 6 042672 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 (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 26 serotype, 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 the signal polyadenylation, SEQ ID NO: 2 (Ad26-CAG-S-CoV2) in a buffer solution for a lyophilized form of the agent.

5. An immunobiological agent based on the genome of a recombinant strain of human adenovirus 26 serotype, 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 the signal polyadenylation, 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 26 serotype, 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 the signal polyadenylation, SEQ ID NO: 3 (Ad26-EF1-S-CoV2) in buffer solution for 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.

Example 3

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

At the first stage of the work, the design of the plasmid construct pSim25-Ends was developed, carrying two regions homologous to the genome of monkey 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-dlE1 was obtained, carrying the genome of simian adenovirus serotype 25 with a 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 in the constructed plasmid pSim25-dlE1 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 the 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).

- 7 042672

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 are 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 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 the 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 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 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 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 (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 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 (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 pAd5dlE1 was obtained, carrying the genome of human adenovirus serotype 5 with a deleted E1 region.

Further, 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 in the constructed plasmid pAd5-dlE1 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 of human adenovirus 5th serotype.

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 protein gene of the SARS-CoV-2 virus

- 8 042672 and 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 homology bearing arms 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).

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 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 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 delegated 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 delegated 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 delegated 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.

- 9 042672

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 the following.

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 versions of the buffer solution: for the liquid form of the agent and for the lyophilized form of the pharmaceutical 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 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, 1*1011 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, 1*1011 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, 1*1011 viral particles.

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

- 10 042672

Table 1

The composition of experimental buffer solutions for the liquid form of the agent

group number Buffer composition 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 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.%):

- 11 042672

Tris: from 0.1831 wt. % to 0.3432 wt. %;

Sodium chloride: from 0.3313 wt. % to 0.6212 wt. %;

Sucrose: from 3.7821 wt. % to 7.0915 wt. %;

Magnesium chloride hexahydrate: from 0.0154 wt. % to 0.0289 wt. %;

EDTA: from 0.0029 wt. % to 0.0054 wt. %;

Polysorbate-80: from 0.0378 wt. % to 0.0709 wt. %;

Ethanol 95%: from 0.0004 wt. % to 0.0007 wt. %;

Solvent: 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, 1*1011 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, 1*1011 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, 1*1011 viral particles.

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

- 12 042672

table 2

Composition of experimental buffer solutions

No. groups 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 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.

Tris: from 0.0180 wt. % to 0.0338 wt. %;

Sodium chloride: from 0.1044 wt. % to 0.1957 wt. %;

Sucrose: from 5.4688 wt. % to 10.2539 wt. %;

Magnesium chloride hexahydrate: from 0.0015 wt. % to 0.0028 wt. %;

EDTA: from 0.0003 wt. % to 0.0005 wt. %;

Polysorbate-80: from 0.0037 wt. % to 0.0070 wt. %;

Solvent: rest.

- 13 042672

Example 8

Determination of the effectiveness of immunization of older animals with the developed tool for assessing the humoral immune response.

One of the main characteristics of the effectiveness of immunization is the antibody titer. The example presents data regarding the change in antibody titer against the SARS-CoV-2 glycoprotein 21 days after administration of the agent to laboratory animals.

Mammals were used in the experiment - C57/B16 mice, females, 20 months old. All animals were divided into 43 groups of 3 animals, which were injected intramuscularly with either component 1 of the agent at a dose of 10 ¹¹ viral particles/100 μl, and then with an interval of 2 weeks component 2 at a dose of 10 ¹¹ viral particles/100 μl, or only component 1 at a dose of 10 ¹¹ virus particles/100 µl, or only component 2 at a dose of 10 ¹¹ virus particles/100 µl. Thus, the following groups of animals were obtained:

1) Ad26-CMV-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

2) Ad26-CMV-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

3) Ad26-CMV-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

4) Ad26-CAG-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

5) Ad26-CAG-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

6) Ad26-CAG-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

7) Ad26-EFl-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

8) Ad26-EFl-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

- 14 042672

9) Ad26-EFl-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

10) Ad26-null (component 1), Ad5-null (component 2);

11) Ad26-CMV-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2);

12) Ad26-CMV-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2);

13) Ad26-CMV-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2);

14) Ad26-CAG-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2);

15) Ad26-CAG-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2);

16) Ad26-CAG-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2);

17) Ad26-EFl-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2);

18) Ad26-EFl-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2);

19) Ad26-EFl-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2);

20) Ad26-null (component 1), simAd25-null (component 2);

21) simAd25-CMV-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

22) simAd25-CMV-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

23) simAd25-CMV-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

24) simAd25-CAG-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

25) simAd25-CAG-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

26) simAd25-CAG-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

27) simAd25-EFl-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);

28) simAd25-EFl-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);

29) simAd25-EFl-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);

30) simAd25-null (component 1), Ad5-null (component 2);

31) Ad26-CMV-S-CoV2;

32) Ad26-CAG-S-CoV2;

33) Ad26-EFl-S-CoV2;

34) Ad26-null;

35) Ad5-CMV-S-CoV2;

- 15 042672

36) Ad5-CAG-S-CoV2;

37) Ad5-EFl-S-CoV2;

38) Ad26-null;

39) simAd25-CMV-S-CoV2;

40) simAd25-CAG-S-CoV2;

41) simAd25-EFl-S-CoV2;

42) simAd25-null;

43) phosphate-buffered saline.

Three weeks later, the animals were bled from the tail vein and the blood serum was isolated. The antibody titer 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 tablet was plugged with 5% milk dissolved in TPBS in a volume of 100 μl per well. Incubated on a shaker at 37°C for an hour.

3) Serum samples from immunized mice were diluted by the 2-fold dilution method. A total of 12 dilutions of each sample were prepared.

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. 3.

- 16 042672

Table 3

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

No. Animal group name Antibody titer 1 Ad26-CMV-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 24834 2 Ad26-CMV-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 24834 3 Ad26-CMV-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 24834 4 Ad26-C AG -S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 24834 5 Ad26-C AG-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 28526 6 Ad26-C AG -S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 28526 7 Ad26-EFl-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 24834 8 Ad26-EFl-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 24834 9 Ad26-EFl-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 28526 10 Ad26- null (component 1), Ad5-null (component 2) 0 AND Ad26-CMV-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2) 37641 12 Ad26-CMV-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2) 49667

- 17 042672

13 Ad26-CMV-S-CoV2 (component 1), simAd25-EFl-S -CoV2 (component 2) 37641 14 Ad26-CAG-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2) 37641 15 Ad26-CAG-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2) 37641 16 Ad26-CAG-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2) 37641 17 Ad26-EF1-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2) 49667 18 Ad26-EF1-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2) 37641 19 Ad26-EF1-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2) 37641 20 Ad26-null (component 1), simAd25-null (component 2) 0 21 Ad25-CMV-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 28526 22 simAd25-CMV-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 28526 23 simAd25-CMV-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 32768 24 simAd25-CAG-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 28526 25 simAd25-CAG-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 32768 26 simAd25-CAG-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 28526 27 simAd25-EFl-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2) 28526 28 simAd25-EFl-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2) 32768

- 18 042672

29 simAd25-EFl-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2) 28526 thirty simAd25-null (component 1), Ad5-null (component 2) 0 31 Ad26-CMV-S-CoV2; 8192 32 Ad26-CAG-S-CoV2; 9410 33 Ad26-EFl-S-CoV2; 10809 34 Ad26-null; 0 35 Ad5-CMV-S-CoV2; 24834 36 Ad5-CAG-S-CoV2; 24834 37 Ad5-EFl-S-CoV2; 21619 38 Ad26-null; 0 39 simAd25-CMV-S-CoV2; 18820 40 simAd25-CAG-S-CoV2; 18820 41 simAd25-EFl-S-CoV2; 18820 42 simAd25-null; 0 43 phosphate buffered saline. 0

As can be seen from the presented data, all variants of the drug induce a humoral immune response to the SARS-CoV-2 glycoprotein in older animals.

Example 9

Determination of the effectiveness of intranasal immunization with the components of the tool for assessing the humoral immune response.

The purpose of this study is to test the effectiveness of the developed agent when administered intranasally.

The experiment used mice of the C57/B16 line, females 18-20 g, 5 animals/group. The following groups of animals were formed.

1) Single intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5*10 ¹⁰ viral particles/dose.

2) Single intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5*10 ¹⁰ viral particles/dose.

3) Single intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5*10 ¹⁰ viral particles/dose.

4) Single intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5*1011 viral particles/dose.

5) Single intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5*1011 viral particles/dose.

6) Single intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5*1011 viral particles/dose.

7) Single intranasal administration of a buffer solution (negative control).

Three weeks later, the animals were bled from the tail vein and the blood serum was isolated.

The antibody titer was determined by enzyme immunoassay (ELISA) according to the following protocol.

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

2) Next, to get rid of non-specific binding, 5% milk in TPBS 100 μl/well was added to the wells of the tablet. Incubated on a shaker at 37°C for an hour.

3) Serum samples from immunized mice were diluted by the 2-fold dilution method. A total of 12 dilutions of each sample were prepared.

- 19 042672

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

5) Next, 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.

Table 4

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

group of animals Antibody titer Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 4915.2 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 16384.0 simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 13107.2 Ad26-too-CMV-S-CoV2, 5*10 ^pv /dose 14745.6 Ad5-too-CMV-S-CoV2, 5*10 ^pv /dose 22937.6 simAd25-too-CMV-S-CoV2, 5*10 ^pv /dose 16384.0 buffer solution 0

As can be seen from the results obtained, intranasal immunization of animals with the components of the developed pharmaceutical agent led to an increase in the titer of antibodies to the S protein of the SARS-CoV-2 virus. Thus, the results of this experiment confirm the possibility of using the developed agent to induce specific immunity to the SARS-CoV-2 virus by its intranasal administration.

Example 10

Study of the safety of the developed agent on volunteers over 60 years of age with chronic diseases.

The study included 110 volunteers over 60 years of age. The proposed immunization scheme included sequential intramuscular administration of component 1 and component 2 of the agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2). One volunteer withdrew prior to vaccination due to withdrawal of consent, so 109 volunteers started study therapy. Another volunteer received component 1 of the agent but withdrew before the introduction of component 2. Accordingly, both components of the funds according to option 1 received 108 volunteers. The sample of the full population for analysis (n=109) included 56 (51.4%) men and 53 (48.6%) women, 107 (98.2%) Caucasian volunteers and 2 (1.8%) Mongoloid race volunteers , mean volunteer age (mean (standard deviation, SD)) was 68.2 (5.96) years, age range 60 to 85. Body mass index (mean (SD)) was 28.4 (4.11) kg/m2.

Significant diseases in history were present in 106 (97.2%) volunteers, which is expected for this age group of people. The top three most common comorbidities were hypertension, obesity, and dyslipidemia.

A total of 35 adverse events (AEs) were reported in 17 (15.60%) volunteers during this study. The number of non-serious AEs was 34 AEs in 16 (14.68%) volunteers. One AE in 1 (0.92%) volunteer, atrial fibrillation, was regarded as serious according to the criterion requiring hospitalization or its extension, the relationship with the study drug was assessed as doubtful, the SAE was of moderate severity, required medical correction and ended in recovery.

According to their relationship with the study drug, all registered AEs were distributed as follows: 12 AEs - as related to the drug (definite relationship), 9 AEs - probably related, 4 AEs - possibly related, 7 AEs were with a doubtful relationship with the drug and for 3 AEs, the association is marked as unknown.

In terms of severity, all non-serious AEs were of mild severity (34 AEs). Only one AE was rated as moderate and included the above SAE.

In table. 5 shows data on the number of volunteers with AEs and the number of AEs according to the MedDRA system organ class.

Table 5

All adverse events reported during the study (SOC MedDRA)

Number of subjects (%) Number of events SOC MedDRA Population for analysis (N=109) Any AE 17 (15.60%) 35 General disorders and reactions at the injection site 15 (13.76%) 28 Nervous System Disorders 2(1.83%) 2 Heart disorders 2(1.83%) 3 Gastrointestinal disorders 1 (0.92%) 1 Laboratory and instrumental data 1 (0.92%) 1

Data are presented as X (%) Y, where X (%) is the number of volunteers with at least one event (percentage by column) and Y is the number of events.

Based on the results of the study, it can be concluded that the AEs identified during the study in volunteers over 60 years of age are characteristic of most vaccine drugs. There were no cases of severe AE development.

Example 11.

Determination of the effectiveness of immunization with the developed agent of volunteers over 60 years of age with chronic diseases, by assessing the intensity of cellular immunity.

The purpose of this experiment was to assess the intensity of cellular immunity to the SARS-CoV-2 virus by determining the proportion of proliferating T-lymphocytes and determining secreted gamma-interferon by mononuclear cells in vitro in volunteers over 60 years of age with chronic diseases after their immunization with various variants of the developed facilities.

The study included 110 volunteers over 60 years of age. The proposed immunization scheme included sequential intramuscular administration of component 1 and component 2 of the agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2). One volunteer withdrew prior to vaccination due to withdrawal of consent, so 109 volunteers started study therapy. Another volunteer received component 1 of the agent but withdrew before the introduction of component 2. Accordingly, both components of the funds according to option 1 received 108 volunteers. The sample of the full population for analysis (n=109) included 56 (51.4%) men and 53 (48.6%) women, 107 (98.2%) Caucasian volunteers and 2 (1.8%) Mongoloid race volunteers , mean volunteer age (mean (standard deviation, SD)) was 68.2 (5.96) years, age range 60 to 85. Body mass index (mean (SD)) was 28.4 (4.11) kg/m2.

Significant diseases in history were present in 106 (97.2%) volunteers, which is expected for this age group of people. The top three most common comorbidities were hypertension, obesity, and dyslipidemia.

Before immunization, as well as after 28 days, blood samples were taken from patients, from which mononuclear cells were isolated by centrifugation in a density gradient solution of ficoll (1.077 g/mL; PanEco). Next, the isolated cells were stained with CFSE fluorescent dye (Invivogen, USA) and seeded into wells of a 96 L plate (2*105 cells/well). 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 (BDBiosciences, clone SK7), anti-CD4 APC (BDBiosciences, clone SK3), anti-CD8 PerCP-Cy5 .5 (BDBiosciences, clone SK1)). Proliferating (with less CFSE cell dye) CD4+ and CD8+ T lymphocytes were determined in the cell mixture using a BD FACS AriaIII flow cytometer (BDBiosciences, USA).

- 21 042672

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.

From these results, it can be concluded that double immunization of volunteers over 60 years of age with the developed agent forms the formation of antigen-specific both CD4+ and CD8+ T-lymphocytes on the 28th day after the introduction of the first component of the vaccine. Thus, according to the median values, the % of proliferating CD4+ and CD8+ T-lymphocytes in volunteers of the age group over 60 years old was 0.7 (CI: 0.5-2.2) and 0.8 (CI: 0.3-2.2), respectively. A statistically significant difference in the values of proliferating both CD4+ and CD8+ T-lymphocytes before immunization (day 0) and 28 days after the introduction of the first component of the vaccine was p<0.001.

At the second stage of the study, the concentration of interferon gamma (IFNy) was quantified in the culture medium of blood mononuclear cells of volunteers over 60 years of age. Mononuclear cells were isolated by density gradient centrifugation of ficoll solution (1.077 g/mL; PanEco) from blood samples of patients obtained before immunization and 28 days after immunization with the developed agent. The cells were restimulated with the S protein of SARS-CoV-2 and after 96 hours the concentration of IFNy was determined using the gamma-Interferon-ELISA-BEST kit (VECTOR-BEST, Russia) according to the manufacturer's instructions.

The increase in the concentration of IFN-gamma was determined by the formula X=Cst /Cint, where X is the increase in the concentration of IFN-gamma (times), Cst is the concentration of IFNy in the medium from stimulated cells (pg/ml), Cint is the concentration of IFNy in the medium from unstimulated (intact ) cells (pg/ml).

The results of the study are shown in Fig. 2.

As can be seen from the presented results, immunization of volunteers over 60 years of age with the developed agent forms the formation of antigen-specific interferon-secreting cells 28 days after the administration of the first component. Thus, according to the median values, the increase in interferon gamma in volunteers of the age group over 60 years old was 4.041 (CI: 2.106-6.802) on the 28th day of the study. The statistically significant difference in the increase in interferon gamma before immunization (day 0) and 28 days after the introduction of the first component of the vaccine was p<0.001. The obtained indicators indicate a pronounced formation of the cellular link of adaptive immunity in volunteers after immunization with the developed agent.

Thus, based on the above data, it can be concluded that immunization of volunteers over 60 years of age, including those with chronic diseases, with the developed agent can cause the formation of a tense antigen-specific cellular link of immunity against the SARS-CoV-2 virus, which is confirmed by a high degree of statistical significance. in measured parameters before and after immunization.

Example 12.

Determination of the effectiveness of immunization with the developed agent of volunteers over 60 years of age with chronic diseases, according to the intensity of humoral immunity.

The purpose of this experiment was to assess the intensity of humoral immunity to the SARS-CoV-2 virus by determining the antibody titer to the RBD-domain S of the SARS-CoV-2 virus protein in volunteers over 60 years of age with chronic diseases after their immunization with the developed agent.

The study included 110 volunteers over 60 years of age. The proposed immunization scheme included sequential intramuscular administration of component 1 and component 2 of the agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) with an interval of 21 days. One volunteer withdrew prior to vaccination due to withdrawal of consent, so 109 volunteers started study therapy. Another volunteer received component 1 of the agent but withdrew before the introduction of component 2. Accordingly, both components of the funds according to option 1 received 108 volunteers.

Significant diseases in history were present in 106 volunteers, which is expected for this age category of people. The top three most common comorbidities were hypertension, obesity, and dyslipidemia.

An assessment of the titer of antigen-specific IgG was carried out in 108 volunteers over 60 years old at points 21 and 28 days and in 63 volunteers on day 42 of the study.

The antibody titer was determined using a test system for enzyme immunoassay developed at the Federal State Budgetary Institution National Research Center for Epidemiology and Microbiology named after N.N. N.F.Gamalei of the Ministry of Health of Russia (RZN 2020/10393 2020-05-18), which allows you to determine the IgG titer to the RBD domain S of the SARS-CoV-2 virus antigen.

Plates with pre-adsorbed RBD (100 ng/well) were washed 5 times with wash buffer. Then, 100 µl of the positive control was added to the wells of the plate in 2 repetitions, as well as in 2 repetitions of 100 µl of the negative control. A series of two-fold dilutions of the studied samples was added to the remaining wells of the tablet (two repetitions for each sample). The plate was sealed with a film and incubated for 1 h at a temperature of +37°C with stirring at a speed of 300 rpm. Then the wells were washed 5 times with the working solution of the buffer for washing. Then in each

- 22 042672 the well was added to 100 μl of the working solution of the conjugate of monoclonal antibodies, the tablet was covered with adhesive film and incubated for 1 hour at a temperature of +37°C with stirring at a speed of 300 rpm. Then the wells were washed 5 times with the working solution of the buffer for washing. Then, 100 μl of chromogen-substrate solution was added to each well and incubated for 15 minutes in a dark place at a temperature of +20°C. After that, the reaction was stopped by adding 50 μl of stop reagent (1M sulfuric acid solution) to each well. The result was taken into account within 10 min after stopping the reaction by measuring the optical density on a spectrophotometer at a wavelength of 450 nm.

The IgG titer was defined as the maximum serum dilution at which the OD450 value of the immunized participant's serum exceeds the value of the control serum (participant's pre-immunization serum) by more than 2 times.

The results of the analysis of the antibody titer to the antigen of the SARS-CoV-2 virus in the blood serum of volunteers over 60 years old after the administration of various variants of the developed agent are shown in Fig. 3.

As can be seen from the presented data, the increase in antibody titer caused by the sequential immunization of volunteers with the developed agent is observed with an increase in days after immunization. Class G antibodies specific to the RBD-domain S of the SARS-Cov2 virus protein (titer higher than 1:50) were detected in 53.2% on day 21 (before the administration of the 2nd component), and on day 28 (7 days after the administration 2 components) in 98.1% of volunteers. At the same time, on day 28 (geometric mean titer value -1719.9), a significant increase in the titer of antigen-specific IgG antibodies was noted, compared with day 21 (geometric mean titer value - 33.8). At day 42, at the time of reporting, 63 out of 108 volunteers were examined. In the studied volunteers, the antibody titer (geometric mean titer value - 2926.2), in blood serum, significantly increased relative to the titer values in the same volunteers on the 28th day of the study (geometric mean titer value - 1773.5). It is worth noting that on day 42 antigen-specific antibodies of class G were detected in 100% of volunteers who donated blood.

Summarizing the results obtained in the evaluation of antibody titers in the blood serum of volunteers, we can draw the following conclusions.

1. A single immunization with 1 component of the agent can lead to the formation of antigen-specific IgG antibodies in 53.2% of volunteers in the age group over 60 years old on day 21 after immunization.

2. Immunization of volunteers in the age group over 60 years old with both components of the developed agent can lead to the formation of antigen-specific IgG antibodies in 98.1% of volunteers over 60 years old on day 28 after immunization.

3. When immunizing volunteers in the age group over 60 with the developed agent, antibody titers continue to rise by the 42nd day after the start of immunization. The data obtained suggest that the titers of antigen-specific IgG antibodies continue to increase after 42 days from the start of immunization.

Example 13

Evaluation of the effectiveness and safety of immunization with the developed tool for persons with chronic diseases.

As part of clinical trials, a study of the developed agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) was conducted on 35963 volunteers. Of these, 19866 people were immunized with both components of the agent (dose 1x1011 v.h. intramuscularly in the deltoid muscle of the shoulder, with an interval of 21 days).

The study included volunteers aged 18 to 92 years. The mean age was 45.3±12.0 years in group I (IP) and 45.3±11.9 years in group II (placebo).

Analysis of data on comorbidities showed that 3687 volunteers (24.7%) in the ILP group and 1235 volunteers (25.3%) in the placebo group had comorbidities (Table 6).

- 23 042672

Table 6

Data on the presence of comorbidities in volunteers

Availability concomitant diseases FAS Over 60 years old Group 1 (pharmaceutical agent) Group 2 (Placebo) total Group 1 (pharmaceutical agent) Group 2 (Placebo) total No 11257 (75.3%) 3657 (74.8%) 14914 (75.2%) 857 (53.2%) 287 (54.0%) 1144 (53.4%) Yes 3687 (24.7%) 1235 (25.3%) 4922 (24.8%) 754 (46.8%) 246 (46.1%) 1000 (46.6%) total 14944 (100.0%) 4892 (100.0%) 19836 (100.0%) 1611 (100.0%) 533 (100.0%) 2144 (100.0%) Ρ(χ ² ) 0.420 0.774

Physical examination results at the screening visit showed 823 abnormalities, 42 of which were clinically significant. Most of the deviations accounted for deviations of the cardiovascular system (487 out of 823 deviations or 59.2%) (Table 7).

Table 7

Presence of abnormalities on the results of the physical examination at the screening visit

Organ/Organ System FAS Group 1 (IP) Group 2 (Placebo) R KNZ KZ KNZ KZ Skin 12/14954 (0D%) 0/14954 (0.0%) 2/4898 (<0.1%) 1/4898 (<0.1%) 0.174 Musculoskeletal system 61/14951 (0.4%) 0/14951 (0.0%) 27/4897 (0.6%) 0/4897 (0.0%) 0.214 gastrointestinal tract 73/148950 (0.5%) 2/14950 (<0.1%) 29/4897 (0.6%) 1/4897 (<0.1%) 0.432 Respiratory system 17/14951 (0.1%) 0/14951 (0.0%) 8/4898 (0.2%) 1/4898 (<0.1%) 0.165 The cardiovascular system 337/14952 (2.3%) 23/14952 (0.2%) 116/4896 (2.4%) 11/4896 (0.2%) 0.520 Bodies urinary system 52/14946 (0.4%) 1/14946 (<0.1%) 16/4895 (0.3%) 1/4895 (<0.1%) 0.617 lymph nodes 26/14938 (0.2%) 1/14938 (<0.1%) 5/4892 (0.1%) 0/4892 (0.0%) 0.550

Data from an interim immunogenicity assay are currently available and show that the vaccine elicits an immune response in volunteers. On the 42nd day of the study in the group that received the vaccine, the geometric mean antibody titer was 8996, the seroconversion rate was 98.25%. In the placebo group, the seroconversion rate was significantly lower at 14.91% (p<0.001).

In this clinical study, the safety of the developed agent was also evaluated. IN

-

Claims (8)

To date, 16,795 cases of adverse events have been reported in 7,998 volunteers. Of these, 70 episodes were described as serious adverse events, based on the fact that in all cases the event required hospitalization of study participants. A causal relationship of these phenomena with the study drug is not seen in any case, which is confirmed by the Independent Data Monitoring Committee. In general, the adverse events identified during the study are typical for most vaccine medicinal products. There was no statistically significant difference between the experimental and control groups in the frequency of adverse events that are not characteristic of vaccine preparations. The most common side effects were flu-like illness and injection site reactions. The study confirmed the absence of severe allergic reactions caused by the developed agent. Industrial Applicability All the above examples confirm the effectiveness of agents that provide effective induction of an immune response against the SARS-CoV-2 virus in people over 60 years of age and industrial applicability. CLAIM 1. Application 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, to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV2 in individuals over 60 years of age and/or having chronic diseases. 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 a recombinant strain of simian adenovirus serotype 25, in which E1 is 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 to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in individuals over 60 years of age and/or chronically ill. 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 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, for the induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in persons over 60 years of age and/or with chronic diseases. 4. Use according to claims 1 to 3, wherein the agent is administered intranasally and/or intramuscularly. 5. Use according to claims 1-3, where the components of the agent are administered sequentially with an interval of more than 1 week. 6. Use according to claims 1 to 3, characterized in that the agent is in liquid or lyophilized form. 7. Use according to claim 6, characterized in that the liquid form of the product contains a buffer solution, 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 is the rest. 8. Use according to claim 6, characterized in that the reconstituted lyophilized form of the agent contains a buffer solution, wt.%: -