EA043182B1 - APPLICATION OF A DRUG FOR INDUCING SPECIFIC IMMUNE AGAINST SARS-COV-2 SEVERE ACUTE RESPIRATORY SYNDROME VIRUS IN CHILDREN - Google Patents

Description

Technical field

The invention relates to biotechnology, immunology and virology. The proposed agent can be used to prevent diseases caused by the SARS-CoV-2 severe acute respiratory syndrome virus.

State of the art

In December 2019, a novel coronavirus of zoonotic origin, which was named SARS-CoV-2, spread in the Hubei province of the People's Republic of China. The epidemic disease caused by SARS-CoV-2 is called coronavirus disease-19 (coronavirus disease-19), abbreviated as COVID-19. This disease can occur both in an asymptomatic or mild form, and in a severe form, which may be accompanied by sepsis and multiple organ failure. Within months, the disease had spread throughout the world, affecting more than 200 countries. In January 2020, the World Health Organization declared the SARS-CoV-2 epidemic an international health emergency, and in March 2020 it described the spread of the disease as a pandemic. By July 28, 2021, the number of cases exceeded 195 million people, and the death toll - 4 million people.

The ongoing outbreak of COVID-19 poses an exceptional public health threat. The development of a safe and effective SARS-CoV-2 vaccine is now a top global priority.

Within a year of the start of the pandemic, various pharmaceutical companies offered their own versions of a COVID-19 vaccine candidate.

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 a mutant S form of the SARS-CoV-2 protein. This vaccine is currently approved for use in adults and children over 12 years of age (F.P. Polacketal. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 2020; 383: 2603-2615; www.cdc.gov/coronavirus/ 2019-ncov/vaccines/recommendations/adolescents.html).

Pharmaceutical company Moderna, in collaboration with the National Institutes of Health (USA), has developed the mRNA-1273 vaccine. The active component of this vaccine is mRNA encoding the S protein of SARS-CoV-2, which is surrounded by a lipid membrane. The vaccine is now approved for emergency use in adults 18 years of age and older. Moderna also conducted a study for children aged 12 to 17, which is currently under review. The KidCOVE clinical trial is currently underway, during which children from 6 months to 12 years of age are immunized (L. A. Jackson et al. An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med 2020; 383:1920193l ; https://penntoday.upenn.edu/news/covid-vaccine-kids; https://clinicaltrials.gov/ct2/show/NCT04796896).

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. The vaccination schedule involves two immunizations with an interval of 28 days (M. Voysey et al. Safety and efficacy of the ChAdOxl nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomized controlled trials in Brazil, South Africa , and the UK, The Lancet, Vol. 397, Issue 10269, P99-111, 2021).

CanSino has developed a vector vaccine against COVID-19 based on a replication-defective human adenovirus serotype 5 (Ad5) that expresses the full-length SARS-CoV-2 S glycoprotein. The vaccine is currently approved for emergency use in adults over 18 years of age. (GenBankYP_009724390) (Feng-Cai Zhu et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial The Lancet Vol 369, Issue 10249, P479-488, 2020).

The JohnsonandJohnson and JanssenPharmaceutical research teams, in collaboration with BethlsraelDeaconess Medical Center, have developed several candidate vaccines using JanssenAdVac® technology. Following safety and efficacy studies, the candidate vaccine Ad26.COV2.S (Ad26COVS1) was selected. The active component of this vaccine is a recombinant serotype 26 adenovirus vector 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 two proline-stabilizing mutations. The vaccine is approved for emergency use in adults over 18 years of age. Research is currently underway to use the vaccine in adolescents and children from birth. (J. Sadoff et al. 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; https://wwwjanssenmd.com/ janssen-covid19-vaccine/special-populations/pediatrics/use-ofjanssen-covidl9-vaccine-in-pediatric-participants).

Beijing Institute of Biological Products Co. An inactivated vaccine against COVID-19 has been developed. The vaccine is approved for emergency use in adults over 18 years of age. Information has also been published on a clinical study of this vaccine in children in the age groups of 3-6 years, 7-12

- 1 043182 years old, 13-17 years old. The study is currently ongoing (https://www.who.int/news/item/07-05-

2021 -who-lists-additional-covid-19-vaccine-for-emergency-use-and-issues-interim-policy-recommendations;

https://clinicaltrials.gov/ct2/show/NCT04917523?cond=covid-19+vaccine&age_v=5&draw=2&rank=10).

Thus, only one mRNA-based COVID-19 vaccine (Pfizer) is currently approved for use in children. Meanwhile, doctors' observations show that the number of young patients admitted to hospitals with a diagnosis of COVID-19 is increasing. The death rate among the young population is also on the rise and with it, the need for vaccines to prevent COVID-19 in children is increasing.

Protective immunity against the SARS-CoV-2 coronavirus involves several parts of the immune system. An effective COVID-19 vaccine would appear to induce both humoral and cellular immune responses. In addition, an important element of protective immunity can be the activation of mucosal immunity (for example, through the expression of IgA antibodies) in the nasopharynx, which is the gateway for virus entry.

Thus, there is a need in the state of the art to develop new agents that can induce an immune response against SARS-CoV-2 in children, including on the mucous membranes of the respiratory tract, which are the main gateways of infection.

Implementation of the invention

The technical task of the claimed group of inventions is to create tools that provide effective induction of an immune response (including mucosal immune response) against the SARS-CoV-2 virus in children older than 1 month.

The technical result is to create a safe and effective agent that ensures the development of humoral and cellular immune responses against the SARS-CoV2 virus in children older than 1 month.

A child is born with an immature innate and adaptive immune system that develops and acquires memory as it grows. From birth to puberty, a person's immune system goes through a series of stages in its development.

The immune system of the newborn is in suppression. The phagocytosis system is not developed. Neonatal T cells are significantly different from adult cells, which is a consequence of intrauterine life of the fetus, where exposure to foreign antigens is largely limited to maternal alloantigens. Therefore, very early adaptive T-cell immunity is characterized by tolerogenic reactivity, reduced alloantigen recognition, and weak responses to foreign antigens. Newborn B cells are also very different from adult B cells. It is known that neonatal B cells have reduced expression of TACI, BCMA and BAFF-R, as well as reduced production of IgG and IgA in response to CD40L and IL-10 (Kaur K, Chowdhury S, Greenspan NS, Schreiber JR. Decreased expression of tumor necrosis factor family receptors involved in humoral immune responses in preterm neonates Blood 2007 Oct 15;110(8):2948-54 doi: 10.1182/blood-2007-01-069245 Epub 2007 Jul 18 PMID: 17634409). Together, these features contribute to the blunting of humoral immune responses with incomplete immunoglobulin class switching. B cells of neonates and infants under 2 months of age show reduced somatic hypermutation compared to adults, which limits antibody affinity maturation. Also, bone marrow stromal cells early in life are unable to maintain long-term plasmablast survival and differentiation into plasma cells, so any IgG antibodies produced after immunization decline rapidly, unlike older children and adults (Pihlgren M, Friedli M, Tougne C, Rochat AF, Lambert PH, Siegrist CA Reduced ability of neonatal and early-life bone marrow stromal cells to support plasmablast survival J Immunol 2006 Jan 1;176(1): 165-72 doi: 10.4049/jimmunol. 176.1.165.PMID: 16365407). Consequently, the effectiveness of the adaptive immune system in early response to T-cell-dependent antigens is markedly reduced in newborns compared with older children and adults (A.K. Simon, G.A. Hollander, A. McMichael. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci. 2015 Dec 22;282(1821): 20143085. doi: 10.1098/rspb.2014.3085, PMCID: PMC4707740, PMID: 26702035).

During infancy, the immune system gradually matures. Critical early protection against many infectious diseases previously transmitted by the mother is provided by passive IgG antibodies transmitted from the mother transplacentally and in milk.

The next stage of development is due to the destruction of maternal antibodies. The primary immune response to the penetration of infection develops due to the synthesis of class M immunoglobulins and leaves no immunological memory. This type of immune response also occurs during vaccination against infectious diseases, and only revaccination forms a secondary immune response with the production of antibodies of the IgG class.

As the child grows, his contacts with the outside world expand. Gradually, switching of immune responses to the formation of antibodies of the IgG class begins. However, the primary immune response (IgM synthesis) persists for many antigens. The local immune system is still immature. Gradually, the average concentration of IgG and IgM in the blood increases and reaches the level corresponding to adults, but the level of IgA in the blood has not yet reached the final values.

The last stage in the development of the immune system occurs during puberty. Against the background of increased secretion of sex steroids, the volume of lymphoid organs decreases. The secretion of sex hormones leads to the suppression of the cellular link of immunity (Shcheplagina L.A., Kruglova I.V. Age-related features of immunity in children, Russian Medical Journal No. 23 dated 11/11/2009 p. 1564).

Thus, the development of an agent for use in children that 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.

When a child is exposed to SARS-CoV-2, the virus primarily infects the mucous membranes of the respiratory tract. This means that the interactions of the virus with the immune system first occur mainly on the mucous membranes of the respiratory tract and oral cavity. Therefore, the induction of mucosal immunity is an important factor affecting the protective properties of a pharmaceutical agent.

Based on the prior art, it can be assumed that the administration of a vaccine to children, which is used in adults, will lead to a decrease in its effectiveness due to the immaturity of the child's immune system. However, studies have shown that the introduction to children of 1/10 of the adult dose of the developed agent induces a humoral immune response comparable to the immune response of an adult. In this case, this is an unexpected result.

Also, on young animals, it was shown that the introduction of the developed agent induces an increase in the level of IgG antibodies on the mucous membrane of the respiratory tract. Moreover, if the intranasal route of administration of the agent is included in the immunization scheme, this leads to the secretion of IgA antibodies on the mucous membrane. Thus, as a result of the work carried out, schemes for the administration of the agent were developed that provide an enhanced mucosal response in the respiratory tract.

The specified technical result is achieved by the fact that the following is proposed.

The use of an agent containing a component 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 for the induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

Application of an agent containing a component 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 to induce specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month.

The use of an agent containing a combination that is component 1 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 component 2 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 with an integrated expression cassette are deleted, 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-CoV-2 in children older than 1 month.

Application of an agent containing a component 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 to induce specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month.

The use of an agent containing a combination that is component 1 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 component 2 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 with an integrated expression cassette are deleted, selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

The use of an agent containing a combination representing component 1 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 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 from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID

- 3 043182

NO: 3 for the induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

In special cases of implementation:

the agent ensures the induction of the mucosal immune response on the mucous membranes of the respiratory tract;

the agent is in liquid or lyophilized form.

In this case, the liquid form of the agent 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 rest

At the same time, 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 rest

In a particular case, the component and/or components of the agent are intended for intranasal and/or intramuscular administration.

In a particular case, the tool is intended for administration at a dose of 5*10 ⁹ -5*10 ¹⁰ viral particles.

In a particular case, the components of the agent are intended for sequential administration with an interval of more than 1 week or are intended for simultaneous administration.

In this case, the components of the product may be in individual packages.

Brief description of the figures

In FIG. 1 shows the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on study day 14, after immunization of mice with the developed pharmaceutical agent Ad26-CMV-S-CoV2. Dots indicate the values for each animal participating in the study. The median of the values is represented as a black line for each data group. Deviations denote 95% confidence interval. The symbol ** denotes a statistically significant difference between the values of 1 and 14 days (p<0.01, according to the Mann-Whitney test).

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

The abscissa axis is time, days.

In FIG. 2 shows the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (day 1) and on study day 14, after immunization of mice with the developed pharmaceutical agent Ad5-CMV-S-CoV2. Dots indicate the values for each animal participating in the study. The median of the values is represented as a black line for each data group. Deviations denote 95% confidence interval. Symbols * (p<0.05) and ** (p<0.01) indicate a statistically significant difference between the values of 1 and 14 days according to the Mann-Whitney test.

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

The abscissa axis is time, days.

- 4 043182

In FIG. Figure 3 shows the titers of IgG antibodies specific to the RBD-domain S of the SARS-Cov2 virus protein before vaccination (day 1) and on days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent at a dose of 1x10 ¹⁰ viral particles. Dots indicate the values for each volunteer participating in the study. The geometric mean of the antibody titer is shown as a black bar for each data group. Deviations denote 95% confidence interval. 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 (#### -p<0.0001). ND denotes non-significant differences between the specified data samples. Statistically significant difference between the values on days 21, 28 and 42 in comparison with the values before vaccination (day 1) was determined by the Wilcoxon T-test (**** -p<0.0001).

The y-axis is the titer of antigen-specific IgG antibodies.

The abscissa axis is time, days.

In FIG. Figure 4 shows the titers of IgG antibodies specific to the RBD-domain S of the SARS-Cov2 virus protein before vaccination (day 1) and on days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent at a dose of 2x10 ¹⁰ viral particles. Dots indicate the values for each volunteer participating in the study. The geometric mean of the antibody titer is shown as a black bar for each data group. Deviations denote 95% confidence interval. 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 (#### -p<0.0001). ND denotes non-significant differences between the specified data samples. Statistically significant difference between the values on days 21, 28 and 42 in comparison with the values before vaccination (day 1) was determined by the Wilcoxon T-test (**** -p<0.0001).

The y-axis is the titer of antigen-specific IgG antibodies.

The abscissa axis is time, days.

In FIG. 5 shows the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on study day 28, after immunization of volunteers with the developed pharmaceutical at a dose of 1x10 ¹⁰ viral particles. Dots indicate the values for each volunteer participating in the study. The median of the values is represented as a black line for each data group. Deviations denote 95% confidence interval. Symbol ****, denotes a statistically significant difference between the values of 1 and 28 days (p<0.0001, according to the Wilcoxon test).

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

The abscissa axis is time, days.

In FIG. 6 shows the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (day 1) and on study day 28, after immunization of volunteers with the developed pharmaceutical at a dose of 2x10 ¹⁰ viral particles. Dots indicate the values for each volunteer participating in the study. The median of the values is represented as a black line for each data group. Deviations denote 95% confidence interval. Symbol ****, denotes a statistically significant difference between the values of 1 and 28 days (p<0.0001, according to the Wilcoxon test).

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

The abscissa axis is time, days.

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. BiomedEnvironSci. 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 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 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 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 technical solutions were developed.

The use of an agent containing a component 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 for the induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

Application of an agent containing a component 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 to induce specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month.

The use of an agent containing a combination that is component 1 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 component 2 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 with an integrated expression cassette are deleted, 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-CoV-2 in children older than 1 month.

Application of an agent containing a component 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 to induce specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month.

The use of an agent containing a combination that is component 1 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 integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and component 2 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 to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

The use of an agent containing a combination representing component 1 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 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 from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month.

In particular cases of implementation:

the agent ensures the induction of the mucosal immune response on the mucous membranes of the respiratory tract;

the agent is in liquid or lyophilized form.

In this case, the liquid form of the agent 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 rest.

At the same time, 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 a particular case, the component and/or components of the agent are intended for intranasal and/or intramuscular administration.

In a particular case, the tool is intended for administration at a dose of 5*10 ⁹ -5*10 ¹⁰ viral particles.

In a particular case, the components of the agent are intended for sequential administration with an interval of more than 1 week or are intended for simultaneous administration.

In this case, the components of the product may be in individual packages.

In addition, the authors of the invention have selected options for the buffer solution, which allow you to store the developed product both frozen at temperatures below -18°C, and in the form of a lyophilisate at temperatures from +2°C to +8°C.

The use of an agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month was also developed by introducing it into the body in an effective amount.

It was shown that the developed tool provides the induction of the mucosal immune response on the mucous membranes of the respiratory tract.

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

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 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-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.

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 by 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 agent in the form of an expression vector 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 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 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 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 buffer solution for the 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 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-dlE1 was obtained, carrying the genome of monkey adenovirus serotype 25 with a deleted E1 region.

Further, 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-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.

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 this design

- 9 043182 pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EF1-S-CoV2 were linearized by a unique hydrolysis site between the homology arms, each plasmid was mixed with the pSim25-null recombinant vector . 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 with an integrated expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 are deleted .

Example 4

Obtaining 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 with an integrated expression cassette selected from SEQ ID NO: 4, 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: 4 (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: 4 (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 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 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 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 the homology contains the nucleotide sequence after the ORF3 of the E4 region until

- 10 043182 ends 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. 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).

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 agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 5, in which the E1 and E3 regions 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 were 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 bu-

- 11 043182 ferric 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 2 in option 1 and in option 3 of the developed agent.

Example 7

Obtaining a buffer solution.

Each component of the developed tool is a tool based on 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*10 ¹⁰ 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*10 ¹⁰ 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*10 ¹⁰ viral particles.

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

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

- 12 043182

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 eleven 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 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: 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*10 ¹⁰ 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*10 ¹⁰ 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*10 ¹⁰ 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 thawed and the change in the titer of recombinant adenoviruses was assessed.

- 13 043182

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:

Tris: 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: 0.0003 wt% to 0.0005 wt%;

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

solvent: rest.

Example 8

Evaluation of the ability of the developed agent to induce a mucosal immune response when administered intramuscularly.

For this study, young BALB/c mice (age 21-28 days) were used. The animals were divided into groups of 10, which were administered the following agents:

1) Ad26-CMV-S-CoV2, intramuscularly (IM), at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad26-CMV-SCoV2, intramuscularly (IM), at a dose of 5x10 ⁹ v.h./100 µl;

2) Ad5-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad5-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl;

3) simAd25-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days, simAd25-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl;

4) Ad26-null, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad26-null, IM, at a dose of 5x10 ⁹ v.h./100 µl;

5) Ad5-null, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad5-null, IM, at a dose of 5x10 ⁹ v.h./100 µl;

6) simAd25-null, IM, at a dose of 5x10 ⁹ vp/100 µl; after 21 days, simAd25-null, IM, at a dose of 5x10 ⁹ v.h./100 µl;

7) Ad26-CMV-S-CoV2, intranasally (in), at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad26-CMV-SCoV2 in, at a dose of 5x10 ⁹ v.h./100 µl;

- 14 043182

8) Ad5-CMV-S-CoV2, in, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad5-CMV-S-CoV2, in, at a dose

5x10 ⁹ w.h./100 µl;

9) Ad26-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad26-CMV-S-CoV2 in, at a dose

5x10 ⁹ w/100 µl;

10) Ad5-CMV-S-CoV2, IM, at a dose of 5x10 ⁹ v.h./100 µl; after 21 days Ad5-CMV-S-CoV2, in, at a dose of 5x10 ⁹ v.h./100 µl;

11) buffer solution.

14 days after the last immunization, the titer of IgG and IgA antibodies was determined by ELISA in broncho-veolar lavage (BAL) washes of experimental animals.

For this.

1) Antigen (recombinant RBD) was adsorbed onto 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 plate. Incubated on a shaker at 37°C for an hour.

3) BAL samples were diluted 25 times and then by the 2-fold dilution method.

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 minutes 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

Geometric mean titer of IgG and IgA antibodies to SARS-CoV-2 virus S protein in BAL swabs of experimental animals

Group name Titer of IgG antibodies Titer of IgA antibodies 1 Ad26-CMV-S-CoV2 IM; Ad26-CMV-S-CoV2 IM 162 3 2 Ad5-CMV-S-CoV2 IM, Ad5-CMV-S-CoV2 IM 325 4

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3 simAd25-CMV-S-CoV2 i/m, simAd25-CMV-S- CoV2 i/m 214 3 4 Ad26-CMV-S-CoV2 in; Ad26-CMV-S-CoV2 in 123 76 5 Ad5-CMV-S-CoV2 in, Ad5-CMV-S-CoV2 in 264 81 6 Ad26-CMV-S-CoV2 IM; Ad26-CMV-S-CoV2 in 373 283 7 Ad5-CMV-S-CoV2 i/m, Ad5-CMV-S-CoV2 in 348 303 8 Ad26-null IM; Ad26-null IM; 0 0 9 Ad5-null i/m; Ad5-null i/m; 0 0 10 simAd25-null i/m; simAd25-null i/m; 0 0 AND Ad26-null in; Ad26-null in; 0 0 12 Ad5-null in; Ad5-null in; 0 0 13 buffer solution 0 0

As can be seen from the presented results, all variants of the developed agent lead to an increase in the titer of IgG antibodies on the surface of the respiratory tract mucosa. The expression of IgA antibodies depends on the route of administration of the agent. Maximum titers of IgA antibodies are induced when animals are immunized sequentially: intramuscularly and then intranasally.

Thus, as a result of the work carried out, an immunobiological agent was developed that is capable of inducing a mucosal immune response against the SARS-CoV-2 virus on the respiratory tract mucosa in children, as well as a scheme for administering this agent, leading to potentiation of the mucosal immune response.

Example 9

Evaluation of the ability of the developed agent to induce an immune response in mammals of different ages.

For this study, young BALB/c mice of various ages were used. Animals were divided into groups of 5 pieces:

1) mice aged 15-18 days,

2) mice aged 28 -35 days,

3) mice aged 50-60 days.

All animals were immunized once with a developed immunobiological agent based on human adenovirus serotype 5 (Ad5-CMV-S-CoV2) at a dose of 108 vh/50 µl. 21 days after immunization, the titer of IgG antibodies in the blood serum of animals was measured.

For this.

1) Antigen (recombinant RBD) was adsorbed onto 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 plate. Incubated on a shaker at 37°C for an hour.

3) Serum samples were diluted 50 times and then by the 2-fold dilution method.

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 minutes 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.

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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

Geometric mean titer of IgG antibodies to the S protein of the SARS-CoV-2 virus in the blood serum of experimental animals depending on their age

No. Animal age Titer of IgG antibodies 1 15-18 days 6400 2 28-35 days 5572 3 50-60 days 4222

Thus, as can be seen from the presented data, the developed immunobiological agent induces the development of a humoral immune response in both adult and young mice of various ages. This suggests that the tool will be effective for use in people of various age categories.

Example 10

Study of the immunogenicity of the developed agent in young mice to assess the cellular immune response after a single vaccination.

To study the immunogenicity of the developed agent, young BALB/c mice (21-28 days old) were used. Animals were divided into groups of 5, which were injected once with the following agents:

1. Ad26-CMV-S-CoV2, intramuscularly (IM), at a dose of 10 ¹⁰ h/100 µl;

2. Ad5-CMV-S-CoV2, intramuscularly (IM), at a dose of 10 ¹⁰ h/100 µl.

The intensity of cellular immunity was determined by assessing the number of proliferating CD4+ and CD8+ lymphocytes isolated from the spleen of mice in vitro culture after repeated restimulation of the cells with recombinant SARS-CoV-2 S protein. Before immunization, as well as 14 days later, spleens were taken from animals, from which mononuclear cells were isolated by centrifugation in a density gradient solution of ficoll (1.09 g/mL; PanEco). Next, the isolated cells were stained with fluorescent dye CFSE (Invivogen, USA) and seeded into wells of 96 l of the plate (2*10 ⁵ 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.

To determine the percentage of proliferating cells, they were stained with antibodies against marker molecules of T lymphocytes CD3, CD4, CD8 (BDBioscienses, USA). 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 (with performed statistical processing) of determining the percentage of proliferating CD4+ and CD8+ T lymphocytes on day 1 (before immunization) and on day 14 of the study are shown in Fig. 1 (Ad26-CMV-S-CoV2) and FIG. 2 (Ad5-CMV-S-CoV2).

The data obtained showed that a single immunization of mice with the developed agent can significantly increase the percentage of proliferating CD4+ and CD8+ T lymphocytes after antigen restimulation on the 14th day after immunization.

Thus, it can be concluded that a single immunization of mice with the developed immunobiological agent can lead to the formation of intense post-vaccination cellular immunity.

Example 11.

Study of the immunogenicity of the developed agent in young mice with various methods of administration.

To study the immunogenicity of the developed agent, young BALB/c mice (21-28 days old) were used. Animals were divided into groups of 5, which were administered the following agents:

3. Ad26-CMV-S-CoV2, intramuscularly (IM), at a dose of ¹⁰⁹ h/100 µl;

4. Ad26-CAG-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

5. Ad26-EF1-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

6. Ad5-CMV-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

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7. Ad5-CAG-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

8. Ad5-EF1-S-CoV2, intramuscularly, at a dose of 10 ⁹ vh/100 µl;

9. simAd25-CMV-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

10. simAd25-CAG-S-CoV2, intramuscularly, at a dose of 10 ⁹ h/100 µl;

11. simAd25-EF1-S-CoV2, intramuscularly, at a dose of ¹⁰⁹ h/100 µl;

12. Ad26-CMV-S-CoV2, intranasally (in), at a dose of 10 ⁹ h/100 µl;

13. Ad26-CAG-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

14. Ad26-EF1-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

15. Ad5-CMV-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

16. Ad5-CAG-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

17. Ad5-EF1-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

18. simAd25-CMV-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

19. simAd25-CAG-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl;

20. simAd25-EF1-S-CoV2, intranasally, at a dose of ¹⁰⁹ vh/100 µl.

After 21 days, blood was taken from the animals, the blood serum was isolated, and the titer of IgG antibodies to the S protein of the SARS-CoV-2 virus was determined by enzyme immunoassay. For this:

1. The antigen was adsorbed on the wells of a 96-well ELISA plate for 16 hours at a temperature of +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 plate. Incubated on a shaker at 37°C for an hour.

3. Serum samples from immunized mice were diluted in 2-fold dilutions. 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 minutes 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 titer of IgG antibodies 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.

Table 5

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

group of animals IgG antibody titer 1 Ad26-CMV-S-CoV2, IM 696 2 Ad26-CAG-S-CoV2, IM 528 3 Ad26-EFl-S-CoV2 IM 459 4 Ad5-CMV-S-CoV2, IM 9701 5 Ad5-CAG-S-CoV2, i/m 11143 6 Ad5-EFl-S-CoV2, i/m 14703 7 simAd25-CMV-S-CoV2, IM 3676 8 simAd25-CAG-S-CoV2, IM 5572

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9 simAd25-EFl-S-CoV2, IM 3676 10 Ad26-CMV-S-CoV2, in 230 AND Ad26-CAG-S-CoV2, in 200 12 Ad26-EFl-S-CoV2, in 174 13 Ad5-CMV-S-CoV2, in 606 14 Ad5-CAG-S-CoV2, in 459 15 Ad5-EFl-S-CoV2, in 459 16 simAd25-CMV-S-CoV2, in 459 17 simAd25-CAG-S-CoV2, in 400 18 simAd25-EFl-S-CoV2, in 303

As can be seen from the presented data, a single immunization with the developed agent induces a humoral immune response to the SARS-CoV-2 glycoprotein.

Example 12.

Study of the immunogenicity of the developed agent when administered to young animals at various doses.

The aim of this study was to evaluate the humoral immune response to the SARSCoV-2 S protein upon administration of the developed agent at various doses to young animals.

To study the immunogenicity of the developed agent, young mice of the C57/B16 line (3-4 weeks) were used. Animals were divided into groups of 5, which were injected with the following drugs intramuscularly, twice with an interval of 28 days:

1) Ad5-CMV-S-CoV2, 5*10 ⁹ vh/100 µl,

2) Ad5-CMV-S-CoV2, 10 ¹⁰ vh/100 µl,

3) Ad5-CMV-S-CoV2, 5*10 ¹⁰ vh/100 µl.

After 21 days, blood was taken from the animals, the blood serum was isolated, and the titer of IgG antibodies to the S protein of the SARS-CoV-2 virus was determined by enzyme immunoassay.

For this.

1. The antigen was adsorbed on the wells of a 96-well ELISA plate for 16 hours at a temperature of +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 plate. Incubated on a shaker at 37°C for an hour.

3. Serum samples from immunized mice were diluted 100-fold and further 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 minutes 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 IgG antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

Animal group IgG antibody titer 1 Ad5-CMV-S-CoV2, 5*10 ⁹ vh/100 µl, 22286 2 Ad5-CMV-S-CoV2, 10 ¹⁰ vh/100 µl, 44572 3 Ad5-CMV-S-CoV2, 5*10 ¹⁰ vh/100 µl. 117627

As can be seen from the presented data, the developed agent exhibits immunogenicity over the entire range of selected doses.

Example 13.

Study of the immunogenicity of the developed agent in young mice with various administration schemes.

Young mice (34 weeks old) were used to study the immunogenicity of the developed agent. Animals were divided into groups of 5, which were administered the following agents:

1) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad26-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

2) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad5-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

3) Ad26-CMV-S-CoV2, in, 10 ⁹ hs/100 µl; after 3 weeks Ad5-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

4) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks simAd5-CMV-S-CoV2 i/m, 10 ⁹ vh/100 µl;

5) Ad5-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad5-CMV-S-CoV2 IM, 10 ⁸ vh/100 µl;

6) Ad5-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad26-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

7) Ad5-CMV-S-CoV2, in, 10 ⁹ hs/100 µl; after 3 weeks Ad26-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

8) Ad5-CMV-S-CoV2, IM, ¹⁰⁹ hs/100 µl; after 3 weeks simAd5-CMV-S-CoV2, IM, 10 ⁹ hs/100 µl;

9) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks simAd5-CMV-S-CoV2 i/m, 10 ⁹ vh/100 µl;

10) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad5-CMV-S-CoV2 IM, 10 ⁹ h/100 µl;

11) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl; after 3 weeks Ad26-CMV-S-CoV2, i/m, 10 ⁹ vh/100 µl.

After 21 days, blood was taken from the animals, the blood serum was isolated, and the titer of IgG antibodies to the S protein of the SARS-CoV-2 virus was determined by enzyme immunoassay.

For this.

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

11. The reaction was stopped after 15 minutes 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 7.

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Table 7

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

group of animals Titer of IgG antibodies 1 Ad26-CMV-S-CoV2 IM, Ad26-CMV-S-CoV2 IM 11143 2 Ad26-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2, i/m, 29407 3 Ad26-CMV-S-CoV2, in, Ad5-CMV-S-CoV2 i/m, 22286 4 Ad26-CMV-S-CoV2, i/m, simAd5-CMV-S-CoV2, i/m, 14703 5 Ad5-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2, i/m, 16890 6 Ad5-CMV-S-CoV2, i/m, Ad26-CMV-S-CoV2, i/m, 33779 7 Ad5-CMV-S-CoV2, in, Ad26-CMV-S-CoV2 i/m, 25600 8 Ad5-CMV-S-CoV2, i/m, simAd5-CMV-S-CoV2, i/m, 14703 9 simAd5-CMV-S-CoV2, IM, simAd5-CMV-S-CoV2, IM, 12800 10 simAd5-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2, i/m, 16890 eleven simAd5-CMV-S-CoV2, IM, Ad26-CMV-S-CoV2, IM 14703

The results obtained show that the developed agent ensures the development of a humoral immune response against SARS-CoV-2 in young animals with various administration schemes.

Example 14

Study of the safety of using the developed agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 for children.

This study involved 100 volunteers aged 12 to 17 years. The proposed immunization scheme included sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2). One volunteer withdrew prior to the administration of component 1 due to newly diagnosed hypertension, and 99 volunteers entered study therapy accordingly. Also, component 2 was not received: 1 person - due to absenteeism, 1 person - due to an undesirable phenomenon (leukopenia), 1 person - due to an intestinal infection of unknown etiology (enteroviral type), 1 person - due to hospitalization with a purulent boil through 5 days after the introduction of the component, 1 person due to hospitalization (intestinal infection) and 1 person due to covid, 2 people refused to participate. Accordingly, 91 volunteers received both components of the vaccine.

There were 205 AEs that developed in 73 volunteers (73.0%) after vaccine administration. Table 8 lists the number (proportion) of volunteers with AEs in each group by organ system class (SOC) and preferred term (PT) according to MedDRA, as well as by relationship to study drug and severity. An intergroup comparison of the incidence of AE was carried out using the x2 test and, if necessary, Fisher's exact test, if the expected frequency in any of the cells was less than 5. The analysis did not reveal statistically significant differences in the incidence of individual AEs between groups.

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Table 8

Adverse events registered during the analyzed period, by classes of organ systems, preferred terms and groups (FAS-population)

Description Group 1 (dosage 1/10) N=50 Group 2 (dosage 1/5) N=50 Total N = 100 рvalue N % N % N % Any RT 37 74.0 36 72.0 73 73.0 0.822 General disorders and reactions at the injection site (78 episodes of AE) Pain at the injection site 15 30.0 AND 22.0 26 26.0 0.362 Pain (1) 7 14.0 7 14.0 14 14.0 1,000 Hyperthermia 5 10.0 3 6.0 8 8.0 0.715 Flu-like illness 3 6.0 5 10.0 8 8.0 0.715 Erythema at the injection site 4 8.0 0 oh oh 4 4.0 0.117 Itching at the injection site 1 2.0 0 oh oh 1 1.0 1,000 Local temperature increase at the injection site 1 2.0 0 oh oh 1 1.0 1,000 Chills 1 2.0 0 oh oh 1 1.0 1,000 Swelling at the injection site 1 2.0 0 oh oh 1 1.0 1,000 pyrexia 0 ο,θ 1 2.0 1 1.0 1,000 Total in the organ system 29 58.0 23 46.0 52 52.0 0.230

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Laboratory and instrumental data (76 episodes of AE) Reducing the number of neutrophils 17 34.0 18 36.0 35 35.0 0.834 Positive urinalysis for red blood cells 8 16.0 5 10.0 13 13.0 0.372 An increase in the level of bilirubin in the blood 3 6.0 2 4.0 5 5.0 1,000 The presence of protein in the urine 1 2.0 2 4.0 3 h, o 1,000 Decreased hemoglobin level 1 2.0 2 4.0 3 h, o 1,000 The presence of leukocytes in the urine 1 2.0 1 2.0 2 2.0 1,000 An increase in the level of alkaline phosphatase in the blood 1 2.0 1 2.0 2 2.0 1,000 Decrease in the number of lymphocytes 1 2.0 1 2.0 2 2.0 1,000 Decreased platelet count 1 2.0 1 2.0 2 2.0 1,000 Bacteria testing (2) 0 0.0 1 2.0 1 1.0 1,000 The presence of glucose in the urine 1 2.0 0 oh oh 1 1.0 1,000 The presence of casts in the urine 0 oh oh 1 2.0 1 1.0 1,000 Increased erythrocyte sedimentation rate 1 2.0 0 oh oh 1 1.0 1,000 An increase in the level of lactate dehydrogenase in the blood 0 oh oh 1 2.0 1 1.0 1,000 Increased blood cholesterol levels 1 2.0 0 oh oh 1 1.0 1,000 Increase in the number of leukocytes 0 oh oh 1 2.0 1 1.0 1,000 Increase in the number of red blood cells 1 2.0 0 oh oh 1 1.0 1,000

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Total in the organ system 25 50.0 23 46.0 48 48.0 0.689 Nervous system disorders (18 AE episodes) Headache 5 10.0 4 8.0 9 9.0 1,000 Doubtfulness 4 8.0 1 2.0 5 5.0 0.362 Dizziness 1 2.0 1 2.0 2 2.0 1,000 Total in the organ system 9 18.0 5 10.0 14 14.0 0.249 Muscular, skeletal and connective tissue disorders (9 episodes of AE) Pain in the limb 0 0.0 3 6.0 3 h, o 0.242 Myalgia 2 4.0 1 2.0 3 h, o 1,000 Arthralgia 0 oh oh 1 2.0 1 1.0 1,000 Total in the organ system 2 4.0 5 10.0 7 7.0 0.436 Gastrointestinal disorders (6 episodes of AE) Abdominal pain 0 oh oh 3 6.0 3 h, o 0.242 Vomit 1 2.0 1 2.0 2 2.0 1,000 Dry mouth 1 2.0 0 oh oh 1 1.0 1,000 Total in the organ system 2 4.0 3 6.0 5 5.0 1,000 Respiratory, thoracic and mediastinal disorders (5 episodes of AE) Nasal congestion 3 6.0 0 oh oh 3 h, o 0.242 Pain in the oropharynx (oropharyngeal) 0 oh oh 1 2.0 1 1.0 1,000 Rhinorrhea 1 2.0 0 oh oh 1 1.0 1,000

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Total in the organ system 3 6.0 1 2.0 4 4.0 0.617 Infections and invasions (3 episodes of AE) Rotavirus infection 1 2.0 0 0.0 1 1.0 1,000 Furuncle (3) 1 2.0 0 0.0 1 1.0 1,000 Enteroviral infection 0 oh oh 1 2.0 1 1.0 1,000 Total in the organ system 2 4.0 1 2.0 3 3.0 1,000 Vascular disorders (3 episodes of AEs) Vaginal hemorrhage (4) 1 2.0 0 oh oh 1 1.0 1,000 Haemorrhoids 1 2.0 0 oh oh 1 1.0 1,000 Hyperemia 1 2.0 0 oh oh 1 1.0 1,000 Total in the organ system 3 6.0 0 0.0 3 3.0 0.242 Metabolic and nutritional disorders (1 episode of AE) Decreased appetite 1 2.0 0 0.0 1 1.0 1,000 Skin and subcutaneous tissue disorders (1 episode of AE) Rash 0 oh oh 1 2.0 1 1.0 1,000 Blood and lymphatic disorders (1 episode of AE) Leukopenia 0 oh oh 1 2.0 1 1.0 1,000 Hearing and labyrinth disorders (1 episode of AE) Ear ache 1 2.0 0 oh oh 1 1.0 1,000 Renal and urinary tract disorders (1 episode of AE) Pollakiuria 0 oh oh 1 2.0 1 1.0 1,000

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Psychiatric disorders (1 episode of AE) Panic attack 0 0.0 1 2.0 1 1.0 1,000 Trauma, intoxication and procedural complications (1 episode of AE) Food poisoning 1 2.0 0 oh oh 1 1.0 1,000 Notes: The results are presented in the form: the number of subjects who reported adverse events (AEs), the percentage of the safety population in this group. 1 Hypersensitivity at the injection site. RT "Painful sensations" corresponds to PT "Tenderness [10043224]". 2 RT “Bacteria testing” corresponds to LLT “Presence of bacteria in urine [10060857]". 3 RT "Furuncle" corresponds to LLT "Furuncle of the ear [10017556]", AE - "Furuncle auricle on the left. 4 RT "Vaginal hemorrhage" corresponds to LLT "Bleeding from the vagina [10049851].”

The study reported adverse events (AEs) in the following categories.

1) General disorders and reactions at the injection site - 76 AEs:

1/10 dose: 35 cases

1/5 dose: 22 cases.

2) Systemic reactions - 54 AEs,

1/10 dose: 29 cases,

1/5 dose: 25 cases.

3) Laboratory abnormalities - 76 AEs,

1/10 dose: 44 cases,

1/5 dose: 32 cases.

At the same time, the following AEs were associated with the study drug (because the association was assessed as possible, probable, or definite): soreness at the injection site, flu-like syndrome, fatigue, low-grade fever, feeling hot, nasal congestion occurred immediately after the injection of the vaccine, headache, sore throat, cough, aching joints, general weakness, abdominal pain, panic attack, decreased neutrophils, increased bilirubin levels.

For the rest of the AEs, the relationship was indicated as questionable or no relationship. Allergic reactions to the study drug were not observed.

In general, it can be said that the adverse events identified during the study are typical for most vaccine drugs. No life-threatening adverse events have been reported.

Example 16

Determination of the effectiveness of immunization of children with a developed pharmaceutical agent, according to the intensity of humoral immunity.

This study involved 95 children aged 12 to 18 years. The intensity of humoral immunity was determined by assessing the titer of IgG antibodies specific to the RBD domain S of the SARS-CoV-2 virus protein. The study participants were divided into two groups:

1) sequential intramuscular injection of component 1 and component 2 of the pharmaceutical agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) at a dose of 1 x 10 ¹⁰ viral particles/1 ml, 47 people;

2) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) at a dose of 2x10 ¹⁰ viral particles/1 ml, 48 people.

As a reference, samples of the same volunteers taken on the 1st day of the study before vaccination were used.

The assessment of the titer of antigen-specific IgG in the first group was carried out on days 21, 28 and 42 of the study, and in the second group on days 21 and 28 of the study.

The antibody titer was determined using a test system for enzyme immunoassay, 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 tablet 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, 100 µl of the working solution of the conjugate of monoclonal antibodies was added to each well, the plate was covered with a sticky 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 determining the titer of RBD-specific IgG antibodies in the first group (1x10 ¹⁰ virus particles/dose) are shown in FIG. 3. As can be seen from the presented data, the titer of antibodies specific to the RBD-domain S of the SARS-Cov2 virus protein gradually increases after vaccination, reaching a maximum value on day 42. The reciprocal geometric mean at this point is 13192. By the 42nd day of the study, seroconversion was observed in all 47 volunteers that make up this group.

The results of determining the titer of RBD-specific IgG antibodies in the second group (2 x 10 ¹⁰ virus particles/dose) are shown in FIG. 4. As can be seen from the presented data, the titer of antibodies specific to the RBD-domain S of the SARS-Cov2 virus protein gradually increases after vaccination with 1/5 of the full therapeutic dose of the vaccine for adults, reaching a maximum value on day 42. The reciprocal geometric mean at this point is 19292. By the 42nd day of the study, seroconversion was observed in all 49 volunteers that make up this group.

Thus, the results of the study showed that vaccination of children with the developed immunobiological agent can lead to the formation of intense post-vaccination humoral immunity.

Example 15

Determination of the effectiveness of immunization of children with a developed pharmaceutical agent, by assessing the intensity of cellular immunity.

This study involved 95 children aged 12 to 18 years. The study participants were divided into two groups:

1) sequential intramuscular injection of component 1 and component 2 of the pharmaceutical agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) at a dose of 1x10 ¹⁰ viral particles/1 ml, 47 people;

2) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent according to option 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) at a dose of 2x10 ¹⁰ viral particles/1 ml, 48 people.

The intensity of cellular immunity was determined by assessing the number of proliferating CD4+ and CD8+ lymphocytes in the peripheral blood of volunteers in in vitro culture after repeated restimulation of cells with recombinant SARS-CoV-2 S protein. 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). 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 (with statistical processing) of determining the percentage of proliferating

CD4+ and CD8+ T lymphocytes at day 1 (prior to immunization) and day 28 of the study are shown in FIG. 5 and FIG. 6.

The data obtained showed that immunization of children with the developed agent in both selected doses allows a significant increase in the percentage of proliferating CD4+ and CD8+ T lymphocytes after antigen restimulation on the 28th day after vaccination.

Based on the data obtained, it can be concluded that two-fold vaccination of children with the developed immunobiological agent can lead to the formation of intense post-vaccination cellular immunity.

Thus, the presented examples confirm that as a result of the work carried out, a safe and effective agent was created that ensures the development of humoral and cellular immune responses against the SARS-CoV-2 virus in children older than 1 month.

Industrial Applicability

All the above examples confirm the effectiveness of the developed agents that provide effective induction of an immune response against the SARS-CoV-2 virus in children older than 1 month and industrial applicability.

Claims (15)

1. Application of an agent containing a component 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, for the induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month. 2. The use of an agent containing a component 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, for the induction of specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month. 3. The use of an agent containing a combination that is component 1 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 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 component 2 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 with an integrated expression a 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-CoV-2 in children older than 1 month. 4. The use of an agent containing a component 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, for the induction of specific immunity against severe acute respiratory syndrome virus SARSCoV-2 in children older than 1 month. 5. The use of an agent containing a combination that is component 1 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 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 component 2 in the form of an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions with an integrated expression a cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children older than 1 month. 6. Use of an agent containing a combination representing component 1 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 in the form of an expression vector based on the genome of the 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 children older than 1 month. 7. Use according to claims 1 to 6, wherein the agent provides for the induction of a mucosal immune response on the mucous membranes of the respiratory tract. - 28 043182 8. Use according to claims 1 to 6, characterized in that the agent is in liquid or lyophilized form. 9. Use according to claim 8, characterized in that the liquid form of the agent 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. 10. Use according to claim 8, characterized in that 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 is the rest. 11. Use according to claims 1 to 10, where the component and/or components of the agent are intended for intranasal and/or intramuscular administration. 12. Use according to claims 1-10, where the agent is intended for administration at a dose of 5*109-5*10 ¹⁰ viral particles. 13. Use according to claims 3, 5, 6, 9, 10, where the components of the agent are intended for sequential administration with an interval of more than 1 week. 14. Use according to claims 3, 5, 6, 9, 10, where the components of the agent are intended for simultaneous administration. 15. Application according to claims 3, 5, 6, 9, 10, characterized in that the components are in different packages.