EA043101B1 - MEANS FOR INDUCING SPECIFIC IMMUNE AGAINST SARS-COV-2 SEVERE ACUTE RESPIRATORY SYNDROME VIRUS IN LIQUID FORM (VERSIONS) - Google Patents

Description

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

The COVID-19 epidemic, which began in late 2019 in the People's Republic of China, spread around the world in a few months and posed an unprecedented challenge to the modern healthcare system. Currently, the number of cases of COVID-19 is more than 106 million people, and the death toll is more than 2.3 million people.

The causative agent of the disease is a single-stranded RNA virus SARS-CoV-2 belonging to the Coronaviridae family to the Beta-CoV lineage.

Coronavirus is transmitted from person to person by airborne droplets, airborne dust and contact routes. The incubation period averages 5-6 days, after which the first symptoms of the disease develop. For COVID-19, the characteristic symptoms are fever, dry cough, shortness of breath, fatigue. Less common are sore throat, joint pain, runny nose, headache. The severity of illness can range from asymptomatic infection to severe acute respiratory syndrome and death.

The rapid spread of SARS-CoV-2 and the high mortality rate have created an urgent need to develop effective means of preventing diseases caused by this virus. 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. The vaccination schedule involves two immunizations 21 days apart (F.P. Polack et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 2020; 383: 26032615).

The pharmaceutical company Modema, 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 vaccination schedule involves two immunizations with an interval of 28 days (L.A. Jackson et al. An mRNA Vaccine against SARSCoV-2 - Preliminary Report. N Engl J Med 2020; 383:1920-1931).

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 ChAdOx1 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 vaccination schedule provides for a single immunization. (GenBank YP009724390) (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 Johnson and Johnson and Janssen Pharmaceutical research teams, in collaboration with the Beth Israel Deaconess Medical Center, have developed several candidate vaccines using Janssen AdVac® 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. Two vaccination regimens are currently being investigated: single and double with an interval of 8 weeks (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).

Thus, it can be noted that most of the vaccines against COVID-19 involve a double vaccination.

Each of the presented vaccines has its own strengths and weaknesses. Thus, RNA-based vaccines have less pronounced side effects, but compared to vector vaccines, they have less immunogenicity. In addition, RNA is more fragile, sensitive to conditions

- 1 043101 storage.

Vaccines based on recombinant viral vectors are highly immunogenic. However, the disadvantage of this type of vaccine is the possibility of forming an immune response to the vector part, which makes revaccination difficult. Also, due to the fact that adenoviruses circulate in the human population, a part of the population may have a pre-existing immunity against these viruses. Expression vectors based on adenoviruses from other mammals can solve the problem of pre-existing immunity, but such vectors are less able to penetrate into human cells, and this reduces the effectiveness of vaccines.

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

containing component 1, which is an agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5, with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and also containing component 2, which is an agent in the form of an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with built-in expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

containing component 1, which is an agent in the form of an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions are delegated, 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; with built-in expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3;

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

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

It should be noted that this method of application has several disadvantages. So, for example, each of the components of a pharmaceutical agent can cause side effects and allergic reactions, therefore, with a double vaccination schedule, the number of such events will increase. Also, such an immunization scheme has a number of practical difficulties, consisting in the fact that it is necessary to ensure the appearance of patients with a certain time interval. In addition, there are a number of logistical difficulties associated with the timely delivery of the necessary components of the tool.

Thus, there is a need in the art to expand the arsenal of pharmaceutical agents capable of inducing an immune response against the SARS-CoV-2 virus in the general population.

The technical objective of the invention is the creation of products containing one active ingredient, and at the same time providing an effective induction of an immune response against the SARS-CoV2 virus in the general population.

Disclosure of the essence of the invention

The solution to the technical problem is a variant of the agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of a recombinant human adenovirus strain of the 26th serotype, in which E1 and E3 region, and the ORF6-Ad26 region is replaced with ORF6-Ad5, with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

A variant of an agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form has also been created, containing as the only active component an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted. , with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

In addition, a variant of the agent for inducing specific immunity against the virus is claimed.

- 2 043101 severe acute respiratory syndrome SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted, with an integrated expression cassette, selected of SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3.

At the same time, in a particular case of its implementation, the buffer solution of the agent for the liquid form contains, wt%:

tris from 0.1831 to 0.3432 sodium chloride from 0.3313 to 0.6212 sucrose from 3.7821 to 7.0915 magnesium chloride hexahydrate from 0.0154 to 0.0289 EDTA from 0.0029 to 0.0054 polysorbate-80 from 0.0378 to 0.0709 ethanol 95% from 0.0004 to 0.0007 water rest.

Each variant of the agent is used to induce specific immunity against the SARS-CoV-2 severe acute respiratory syndrome virus.

In this case, the agent is intended for use intranasally or intramuscularly. Also, the agent can be administered jointly and simultaneously intranasally and intramuscularly.

Moreover, in a particular case of execution, the agent is administered intranasally at a dose of 5x10 ¹⁰ to 5x1011 viral particles or intramuscularly at a dose of 5x10 ¹⁰ to 5x1011 viral particles. And joint administration intranasally and intramuscularly is carried out at a dose of 5x10 ¹⁰ to 5x1011 viral particles intramuscularly, at a dose of 5x10 ¹⁰ to 5x1011 viral particles intranasally.

Co-administration involves intranasal and intramuscular administration within the same vaccination procedure.

The technical result consists in the development of a tool that ensures the development of humoral and cellular immune responses against the SARS-CoV-2 virus in the general population.

The main goal of vaccination is to provide effective and long-term protection against a pathogen. One way to achieve this goal is multiple vaccinations. When the human body first encounters a vaccine antigen, it leads to the activation of two main components of the adaptive immune response: B-lymphocytes and effector T-lymphocytes. Activated B-lymphocytes transform into plasma cells capable of producing antibodies, and also turn into memory B cells. Effector T-lymphocytes are divided into two main types: T-helpers (CD4+) and T-killers (CD8+). The main function of T-helpers is to support the development of a humoral or cellular immune response. The main function of T-killers is the destruction of damaged cells of their own body. T-killers are one of the main components of antiviral immunity. However, some time after vaccination, the number of antigen-specific immune cells in the body begins to decrease, then a booster dose of the vaccine is administered, which allows you to maintain the number of antigen-specific T- and B-cells at the proper level (providing protection of the body from the pathogen).

The development of a single-component agent that will cause a stable immune response after a single immunization is a complex scientific and practical task. At the same time, the significance of such a development is difficult to overestimate. A single injection can provide faster rates of mass vaccination, which is critical in a pandemic. Also, this tool can be useful for emergency vaccination, for immunization of mobile groups of citizens (nomadic peoples, etc.), etc. In addition, it can be noted that a single administration reduces the adverse effects on humans: trauma, the number of side effects and allergic reactions.

Brief description of the figures

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

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

O - IgG titer to the RBD domain of SARS-CoV-2 glycoprotein S in each volunteer participating in the study, on day 14.

□ - IgG titer to the RBD domain of the glycoprotein S of the SARS-CoV-2 virus in each volunteer participating in the study, on day 21.

A - IgG titer to the RBD domain of the glycoprotein S of the SARS-CoV-2 virus in each volunteer participating in the study, on day 28.

The geometric mean of the antibody titer is shown as a black line for each

- 3 043101 data groups. A statistically significant difference between the values of 14, 21 and 28 days is indicated by a bracket above which is the p value according to the Wilcoxon T-test.

In FIG. 2 shows the results of assessing the humoral immune response to the antigen of the virus

SARS-CoV2 in volunteers immunized with the liquid form of the developed agent according to the variant

2.

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

O - IgG titer to the RBD domain of the glycoprotein present in the study, on day 14.

□ - IgG titer to the RBD domain of the glycoprotein in the study, on day 21.

△ - IgG titer to the RBD domain of the glycoprotein in the study, on day 28.

virus virus virus

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2 Each Each Each Volunteer Site Volunteer Site Geometric mean antibody titer is represented as a black bar for each data group. A statistically significant difference between the values of 14, 21 and 28 days is indicated by a bracket above which is the p value according to the Wilcoxon T-test.

In FIG. Figure 3 shows the results of evaluating the effectiveness of immunization of volunteers with a liquid form of the developed agent according to option 1 in assessing the proportion of proliferating CD8+ (A) and CD4+ (B) lymphocytes restimulated by SARS-CoV-2 S antigen.

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

• - indicates the proportion of proliferating CD8+ in each volunteer on day 0.

- the proportion of proliferating CD8+ in each volunteer on day 14 is indicated

A - indicates the proportion of proliferating CD8+ in each volunteer on day 28.

• - indicates the proportion of proliferating CD4+ in each volunteer on day 0.

- the proportion of proliferating CD4+ in each volunteer on day 14 is indicated

A - indicates the proportion of proliferating CD4+ in each volunteer on day 28.

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

In FIG. Figure 4 shows the results of evaluating the effectiveness of immunization of volunteers with a liquid form of the developed agent according to option 2 in assessing the proportion of proliferating CD8+ (A) and CD4+ (B) lymphocytes restimulated by SARS-CoV-2 S antigen.

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

• - indicates the proportion of proliferating CD8+ in each volunteer on day 0.

- the proportion of proliferating CD8+ in each volunteer on day 14 is indicated

A - indicates the proportion of proliferating CD8+ in each volunteer on day 28.

• - indicates the proportion of proliferating CD4+ in each volunteer on day 0.

- the proportion of proliferating CD4+ in each volunteer on day 14 is indicated

A - indicates the proportion of proliferating CD4+ in each volunteer on day 28.

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

Implementation of the invention

The active component of the developed agent is an expression vector based on the genome of a recombinant adenovirus strain with an integrated expression cassette containing the SARS-CoV-2 virus antigen gene.

Adenovirus vectors are able to penetrate various types of human cells, provide high levels of expression of the target antigen, and lead to the induction of both cellular and humoral immune responses. FGBU NITsEM them. N.F.Gamalei of the Ministry of Health of Russia developed 3 variants of expression vectors based on mammalian adenoviruses:

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;

an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted;

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

The S protein of SARS-CoV-2, which is located on the surface of the viral particle, was chosen as the antigen. This is one of the most promising antigens for inducing a strong and long-lasting immune response. Antibodies to the SARS-CoV-2 S protein have also been shown to be virus-neutralizing.

To achieve the most efficient induction of immune responses, the authors developed various variants of expression cassettes containing the S gene of the protein.

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 time of cell cultivation 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 bbb actin promoter, and a chimeric intron (chicken bbb actin and rabbit bbb 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 1α (EF-1a) promoter. The promoter is constitutively active in a wide range of cell types (Wang X, Xu Z, Tian Z, Zhang X, Xu D, Li Q, Zhang J, Wang T. The EF-1a promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells J Cell Mol Med 2017 Nov;21(11):3044-3054 doi: 10.1111/jcmm.l3216 Epub 2017 May 30 PMID: 28557288; PMCID: PMC5661254 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.

Thus, as a result of the work carried out, 3 variants of the tool were developed.

1) An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component 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 with ORF6-Ad5, with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

2) an agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with an embedded expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

3) an agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted, with an embedded expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3.

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

Example 1. Obtaining the active component of the agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2Ha based on the genome of the recombinant human adenovirus strain of the 26th serotype.

At the first stage of work, the design of 3 variants of expression cassettes was developed:

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.

Synthesis of the S gene of the SARS-CoV-2 virus protein was carried out by ZAO Evrogen (Moscow).

To obtain a recombinant strain of human adenovirus serotype 26, we used two plasmids obtained at the N.F. 5 043101 genome of combinant human adenovirus serotype 26 with an open reading frame ORF6 of human adenovirus serotype 5 and with a deletion of E1 and E3 regions.

At the first stage of the work, plasmids pAd26-Ends-CMV-S-CoV2, pAd26-Ends-CAG-S-CoV2, pAd26-Ends-EF1-SCoV2 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. Next, the resulting plasmids were linearized according to a unique hydrolysis site, and each plasmid was mixed with the pAd26-too recombinant vector. As a result of homologous recombination, plasmids pAd26-tooCMV-S-CoV2, pAd26-too-CAG-S-CoV2, pAd26-too-EFl-S-CoV2 were obtained, carrying the genome of recombinant human adenovirus 26 serotype with an open reading frame 0RF6 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 next stage, plasmids pAd26-too-CMV-S-CoV2, pAd26-too-CAG-S-CoV2, pAd26-too-EF1S-CoV2 were digested with specific restriction endonucleases to remove the vector part. The resulting DNA preparations were transfected into HEK293 culture cells.

As a result of the work, recombinant strains of human adenovirus serotype 26 were obtained: Ad26-too-CMV-S-CoV2, Ad26-too-CAG-S-CoV2, Ad26-too-EF1-S-CoV2. A control strain of human adenovirus serotype 26 was obtained using a similar scheme: Ad26-too, which does not contain the S gene of the SARS-CoV-2 virus protein.

Thus, an expression vector was obtained containing the genome of a recombinant strain of human adenovirus serotype 26, in which the E1 and E3 regions were deleted, and the ORF6-Ad26 region was replaced with an ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, which is the active component of the developed agent.

Example 2. Obtaining the active component of an agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 based on the genome of a recombinant human adenovirus strain of the 5th serotype.

In this work, we also used 3 variants of expression cassettes:

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.

Synthesis of the S gene of the SARS-CoV-2 virus protein was carried out by ZAO Evrogen (Moscow).

To obtain a recombinant strain of human adenovirus serotype 5, we used two plasmids obtained at the N.F. Gamaleya National Research Center for Epidemiology and Microbiology:

plasmid pAd5-Ends carrying homology arms of the genome of adenovirus serotype 5 (one arm of homology represents the beginning of the genome of human adenovirus serotype 5 (from the left inverted terminal repeat to the E1 region) and the sequence of the viral genome, including the pIX protein; the second arm homology contains the nucleotide sequence after the E4 ORF3 region to the end of the genome);

plasmid pAd5-too carrying the genome of recombinant human adenovirus serotype 5 with deletion of E1 and E3 regions.

At the first stage of the work, plasmids pAd5-Ends-CMV-S-CoV2, pAd5-Ends-CAG-S-CoV2, pAd5-Ends-EF1-S-CoV2 containing expression cassettes of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, as well as carrying arms of the genome homology of adenovirus serotype 5. Next, the resulting plasmids were linearized according to a unique hydrolysis site, and each plasmid was mixed with the pAd5-too recombinant vector. As a result of homologous recombination, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-CAG-S-CoV2, pAd5-too-EF1-S-CoV2 were obtained, carrying the genome of recombinant human adenovirus 5 with the 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 next stage, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-CAG-S-CoV2, pAd5-too-EF1-SCoV2 were digested with specific restriction endonucleases to remove the vector part. The resulting DNA preparations were transfected into HEK293 culture cells.

As a result of this work, recombinant strains of human adenovirus serotype 5 were obtained: Ad5-too-CMV-S-CoV2, Ad5-too-CAG-S-CoV2, Ad5-too-EF1-S-CoV2. According to a similar scheme, a control strain of human adenovirus serotype 5 was obtained: Ad5-too, which does not contain the S gene of the SARS-CoV-2 virus protein.

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

Example 3. Obtaining the active component of the agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 based on the genome of the recombinant strain of simian adenovirus serotype 25.

In this work, 3 variants of expression cassettes were used:

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.

Synthesis of the S gene of the SARS-CoV-2 virus protein was carried out by ZAO Evrogen (Moscow).

To obtain a recombinant strain of simian adenovirus of the 25th serotype, two plasmids were used, obtained at the N.F.

plasmid pSim25-Ends carrying the homology arms of the simian adenovirus genome serotype 25;

plasmid pSim25-null carrying the genome of recombinant simian adenovirus serotype 25 with deletion of E1 and E3 regions.

At the first stage of work, using genetic engineering methods based on the pSim25-Ends plasmid, plasmids p-Sim25-Ends-CMV-S-CoV2, p-Sim25-Ends-CAG-S-CoV2, p-Sim25-Ends-EF1 were obtained -SCoV2 containing expression cassettes of SEQ ID NO: 4, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, as well as bearing homology arms of the simian adenovirus genome serotype 25. Next, the obtained plasmids were linearized according to a unique hydrolysis site, and each plasmid was mixed with the pSim25-too recombinant vector. As a result of homologous recombination, plasmids pSim25-too-CMV-S-CoV2, pSim25-too-CAG-S-CoV2, pSim25-too-EF1-S-CoV2 were obtained, carrying the genome of recombinant simian adenovirus 25 with the deletion of E1 and E3 -regions, with an expression cassette of SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

At the next stage, plasmids pSim25-too-CMV-S-CoV2, pSim25-too-CAG-S-CoV2, pSim25-tooEF1-S-CoV2 were digested with specific restriction endonucleases to remove the vector part. The resulting DNA preparations were transfected into HEK293 culture cells.

As a result of the work, recombinant strains of simian adenovirus serotype 25 were obtained: simAd25-too-CMV-S-CoV2, simAd25-too-CAG-S-CoV2, simAd25-too-EF1-S-CoV2. A control strain of simian adenovirus 25 serotype simAd25-too, which does not contain the S gene of the SARS-CoV-2 virus protein, was obtained according to a similar scheme.

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

Example 4 Buffer Development

The authors of the invention have chosen a water-based buffer solution that ensures the stability of recombinant adenovirus particles. Tris(hydroxymethyl)aminomethane (Tris) was added to the buffer to maintain the pH of the solution. Sodium chloride was added to achieve the required ionic strength and osmolarity. For cryoprotection, sucrose was added to the buffer. Magnesium chloride hexahydrate was added as a source of divalent cations, EDTA as a free radical oxidation inhibitor, polysorbate-80 as a surfactant, ethanol 95% as a free radical oxidation inhibitor.

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 active components of the agent was added to each of the resulting buffer solutions:

1) 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 SEQ ID NO: 1 (Ad26-CMV-S -CoV2, 1*1011 virus particles);

2) an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with an integrated expression cassette of SEQ ID NO: 1 (Ad5-CMV-S-CoV2, l*10 ⁿ viral particles);

3) an expression vector based on the genome of a recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted, with an integrated expression cassette SEQ ID NO: 4 (simAd25-CMV-S-CoV2, 1x1011 viral particles).

The obtained funds were stored at -18°C and -70°C for 3 months, then thawed and the change in the titer of recombinant adenoviruses was assessed.

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Table 1. Composition of experimental buffer solutions for the liquid form of the agent

group number Buffer composition OK Sodium chloride (mg) Sucrose (mg) Magnesium chloride hexahydrate (mg) EDTA (mg) Polysorbate-80 (mg) Ethanol 95% (mg) Water 1 0.968 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 2 1.815 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 3 1.21 1.752 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 4 1.21 3.285 25 0.102 0.019 0.25 0.0025 up to 0.5 ml 5 1.21 2.19 20 0.102 0.019 0.25 0.0025 up to 0.5 ml 6 1.21 2.19 37.5 0.102 0.019 0.25 0.0025 up to 0.5 ml 7 1.21 2.19 25 0.0816 0.019 0.25 0.0025 up to 0.5 ml 8 1.21 2.19 25 0.153 0.019 0.25 0.0025 up to 0.5 ml 9 1.21 2.19 25 0.102 0.0152 0.25 0.0025 up to 0.5 ml 10 1.21 2.19 25 0.102 0.0285 0.25 0.0025 up to 0.5 ml AND 1.21 2.19 25 0.102 0.019 0.2 0.0025 up to 0.5 ml 12 1.21 2.19 25 0.102 0.019 0.375 0.0025 up to 0.5 ml 13 1.21 2.19 25 0.102 0.019 0.25 0.002 up to 0.5 ml 14 1.21 2.19 25 0.102 0.019 0.25 0.00375 up to 0.5 ml 15 1.21 2.19 25 0.102 0.019 0.25 0.0025 up to 0.5 ml

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

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

Tris: 0.1831 to 0.3432% by weight;

Sodium chloride: 0.3313 to 0.6212% by weight;

Sucrose: 3.7821 to 7.0915% by weight;

Magnesium chloride hexahydrate: 0.0154 to 0.0289% by weight;

EDTA: 0.0029 to 0.0054% by weight;

Polysorbate-80: 0.0378 to 0.0709% by weight;

Ethanol 95%: 0.0004 to 0.0007% by weight;

Solvent: rest.

Example 5. Obtaining an agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form

The developed agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form according to option 1 contains an expression vector based on the genome of a recombinant human adenovirus strain of the 26th serotype, with an integrated expression cassette selected from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 in buffer solution.

The developed agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form according to option 2 contains an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, with an integrated expression cassette selected from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 in buffer solution.

The developed agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form according to option 3 contains an expression vector based on the genome of a recombinant strain of simian adenovirus of the 25th serotype, with an integrated expression cassette selected from SEQ ID NO: 4 , SEQ ID NO: 2, SEQ ID NO: 3 in buffer solution.

The active component is mixed with the components of the buffer solution during the technological process. The pharmaceutical product is poured into sterile vials. Storage is carried out in a place protected from light, at a temperature not exceeding minus 18°C. Before use, it is necessary to take 8 043101 out of the refrigerator and keep at room temperature until completely defrosted for no more than 30 minutes; mix before use by gently shaking the vial (ampoule). It is not allowed to shake the drug sharply. Re-freezing is not allowed.

Example 6. Toxicity of the developed agent with a single injection (acute toxicity) in mice with intravenous and intramuscular administration

In this study, the acute toxicity of an agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form was evaluated, containing as the only active component an expression vector based on the genome of a recombinant strain of human adenovirus serotype 26, in which E1 and E3 region, and the ORF6-Ad26 region is replaced by ORF6-Ad5, with an embedded expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

means for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component 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 an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

means for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of the recombinant strain simian adenovirus of the 25th serotype, in which the E1 and E3 regions are delegated, with an integrated an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3.

The study used outbred mice of both sexes, weighing 18-20 g, aged 6-8 weeks.

The calculation of the dose of funds was carried out on the basis of the immunizing dose (10 ⁸ w.h.), obtained in a preliminary experiment on a sensitive species of animals - Syrian hamsters. Doses for mice were calculated based on body weight. The minimum dose for toxicological experiments was chosen dose for mice 10 ⁸ vp, as the closest to therapeutic. No cross-species conversion factor was used for dose conversion, doses were obtained by direct conversion to body weight according to the WHO recommendation for vaccine preparations.

As a result, the following doses of the drug were chosen for administration to mice in this experiment:

10 ⁸ w.h. - close to the effective dose (ED) for mice;

10 ⁹ w.h. - increased ED for mice by 20 times;

10 ¹⁰ w.h. - increased ED for mice by 200 times;

10 ¹¹ w.h. - increased ED for mice by 2000 times;

Thus, the following experimental groups of animals were obtained:

1) Ad26-too-CMV-S-CoV2, 1x10 ⁸ v.h., 20 mice;

2) Ad26-too-CMV-S-CoV2, 1x10 ⁹ v.h., 20 mice;

3) Ad26-too-CMV-S-CoV2, 1x10 ¹⁰ v.h., 20 mice;

4) Ad26-too-CMV-S-CoV2, 1x 10 ¹¹ v.h., 20 mice;

5) Ad5-too-CMV-S-CoV2, 1x10 ⁸ v.h., 20 mice;

6) Ad5-too-CMV-S-CoV2, 1x10 ⁹ v.h., 20 mice;

7) Ad5-too-CMV-S-CoV2, 1x10 ¹⁰ v.h., 20 mice;

8) Ad5-too-CMV-S-CoV2, 1x 10 ¹¹ v.h., 20 mice;

9) simAd25-too-CMV-S-CoV2, 1x10 ⁸ v.h., 20 mice;

10) simAd25-too-CMV-S-CoV2, 1x10 ⁹ v.h., 20 mice;

11) simAd25-too-CMV-S-CoV2, 1x10 ¹⁰ v.h., 20 mice;

12) simAd25-too-CMV-S-CoV2, 1x 10 ¹¹ v.h., 20 mice;

13) placebo (buffer solution), 20 mice.

Clinical examination of each animal was carried out daily for 14 days, recording signs of intoxication and the number of dead animals.

The following parameters of the functional state of laboratory animals were recorded: activity, movement, appearance, condition of wool, eyes, ears, teeth, limbs. Physiological functions: respiration, salivation, saliva, urine, excretion. All animals remained alive throughout the experiment. In all groups, the animals looked healthy, actively ate food, responded adequately to stimuli, and showed research interest. The coat is thick, even and shiny, tightly adhering to the surface of the body; no hair loss or brittleness was detected. Muscle tone was not characterized by increased excitability. The auricles are without crusts, not inflamed, no twitching is noticed. Teeth of normal color, without breakage. The mice were of average fatness, did not suffer from exhaustion. The abdominal region was not enlarged in volume. Breathing is even, uncomplicated. Salivation is normal. Urination frequency, urine color, gastrointestinal parameters, muscle tone, reflexes corresponded to the physiological norm. The behavior of the experimental animals did not differ

- 9 043101 from control.

On the 14th day from the start of the experiment, mice were euthanized by the method of dislocation of the cervical vertebrae. During the study, animals in serious condition with signs of imminent death were not observed, there was no death of animals.

Conducted a complete necropsy of the bodies of all animals. During necropsy, the external state of the body, internal surfaces and passages, the cranial cavity, the chest, abdominal and pelvic cavities with organs and tissues located in them, the neck with organs and tissues, and the musculoskeletal system were examined.

In a macroscopic study, no effect of the agent on the state of the internal organs of mice was found, no differences were found between the control and experimental groups. There were no statistically significant differences in organ mass between the experimental and control groups. The weight gain of animals in the experimental and control groups did not differ.

Example 7. Determination of the effectiveness of immunization with the developed tool for evaluating the humoral immune response

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

Mammals were used in the experiment - mice of the BALB/c line, females 18 g. All animals were divided into 13 groups of 5 animals, which were intramuscularly injected with variants of the developed agent in liquid form at a dose of 5x101° viral particles/200 μl.

The following groups of animals were obtained:

1) Ad26-too-CMV-S-CoV2,

2) Ad26-too-CAG-S-CoV2,

3) Ad26-too-EF1-S-CoV2,

4) Ad26-too,

5) Ad5-too-CMV-S-CoV2,

6) Ad5-too-CAG-S-CoV2,

7) Ad5-too-EF1-S-CoV2,

8) Ad5-too,

9) simAd25-too-CMV-S-CoV2,

1°) simAd25-too-CAG-S-CoV2,

11) simAd25-too-EF1-S-CoV2,

12) simAd25-too,

13) placebo (buffer).

Three weeks later, the animals were bled from the tail vein and the blood serum was isolated. The antibody titer was determined by enzyme immunoassay (ELISA) according to the following protocol:

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

2) Further, to get rid of non-specific binding, the plate was plugged with 5% milk dissolved in a buffer to block a non-specific signal in a volume of 10 μl per well. Incubated on a shaker at 37°C for an hour.

3) Serum samples from immunized mice were first diluted 10°-fold and then a series of two-fold dilutions was made. 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. 2.

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Table 2. Titer of antibodies to the S protein of SARS-CoV-2 in the blood serum of mice (geometric mean of the antibody titer)

No. Animal group name Antibody titer 1 Ad26-too-CMV-S-CoV2, 2425 2 Ad26-too-CAG-S-CoV2, 2111 3 Ad26-too-EFl-S-CoV2 2786 4 Ad26-too 0 5 Ad5-too-CMV-S-CoV2, 38802 6 Ad5-too-CAG-S-CoV2, 29407 7 Ad5-too-EFl-S-CoV2 33779 8 Ad5-too 0 9 simAd25-too-CMV-S-CoV2, 14703 10 simAd25-too-CAG-S-CoV2, 16890 AND si m Ad2 5 -too-EF 1 - S -C o V2 12800 12 simAd25-too 0 13 placebo (buffer) 0

Thus, the results of the experiment show that all the developed agents induce a humoral immune response against SARS-CoV-2.

Example 8. Study of the immunogenicity of the developed agent by assessing the humoral immune response to the antigen of the SARS-CoV-2 virus in the blood of volunteers at various times after vaccination

The purpose of this experiment was to determine the intensity of the immune response to the SARS-CoV-2 virus in volunteers at various times after their immunization with various variants of the developed agent.

The study included healthy volunteers aged 18 to 60 years. All study participants were divided into several groups.

1) An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component 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 changed to ORF6-Ad5, with an embedded expression cassette of SEQ ID NO: 1, 10 ¹¹ virus particles/dose, 9 people.

2) An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form containing, as the only active component, 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, and ORF6-Ad26 region changed to ORF6-Ad5, with built-in expression cassette SEQ ID NO: 1, 10 ¹¹ virus particles/dose, 9 people.

Volunteers were once intramuscularly immunized with the appropriate agent. Before immunization, as well as after 14, 21, 28 and 42 days, blood samples were taken from patients. From the obtained samples, blood serum was isolated, which was subsequently used to determine the titer of antibodies to the S antigen of the SARS-CoV-2 virus.

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

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

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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 min in a dark place at a temperature of +20°C. After that, the reaction was stopped by adding µl of stop reagent (1M sulfuric acid solution) to each well. The result was taken into account within 10 min after stopping the reaction by measuring the optical density on a spectrophotometer at a wavelength of 450 nm.

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

The results of the analysis of the antibody titer to the antigen of the SARS-CoV-2 virus in the blood serum of volunteers after the introduction of various variants of the developed agent are shown in Fig. 12.

As can be seen from the presented data, the immunization of volunteers with both variants of the developed agent makes it possible to form a pronounced (statistically significantly different from the parameters of the control unimmunized group of volunteers) humoral immunity, characterized by an increase in the titer of antibodies to the S protein of the SARS-CoV-2 virus. At the same time, an increase in the intensity of the humoral immune response is noted with an increase in days after immunization.

Example 9. Study of the immunogenicity of the developed agent by assessing the cellular immune response to the antigen of the SARS-CoV-2 virus in the blood of volunteers at various times after vaccination

The purpose of this experiment was to determine the intensity of cellular immunity to the SARS-CoV-2 virus in volunteers after their immunization with various variants of the developed agent.

The study included healthy volunteers aged 18 to 60 years. All study participants were divided into several groups.

1) An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form containing, as the only active component, 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 ORF6-Ad26 region changed to ORF6-Ad5, with built-in expression cassette SEQ ID NO: 1, 10 ¹¹ virus particles/dose, 9 people.

2) An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form containing, as the only active component, 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, and ORF6-Ad26 region changed to ORF6-Ad5, with built-in expression cassette SEQ ID NO: 1, 10 ¹¹ virus particles/dose, 9 people.

Volunteers were once intramuscularly immunized with the appropriate agent. Before immunization, as well as after 14 and 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 fluorescent dye CFSE (Invivogen, USA) and seeded into wells of a 96 L plate (2x105 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 obtained are presented in Fig. 3, 4.

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

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

Example 10. Evaluation of adverse events in volunteers after single and double immunization with variants of the developed agent

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The purpose of this experiment was to determine the side effects in volunteers after they were immunized with various variants of the developed agent.

The study included healthy volunteers aged 18 to 60 years. All study participants were divided into several groups.

1) Single intramuscular injection of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 10 ¹¹ viral particles/dose, 9 people.

2) Single intramuscular injection of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 10 ¹¹ viral particles/dose, 9 people.

3) Two-time immunization regimen in which the agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) is first administered in liquid form 10 ¹¹ viral particles / dose, and after 21 days the agent based on recombinant human adenovirus is administered 5 serotypes (Ad5-too-CMV-S-CoV2) in liquid form 10 ¹¹ virus particles/dose, 20 people.

In table. 3 includes data on the main adverse events recorded from the start of the study and up to the visit (telephone contact) of the 180th day of the study.

Table 3. The main adverse events observed after a single injection of the developed drug compared with a double injection

Number of subjects (%) Number of events Group 1 (Ad26-tooCMV-SCoV2) Group 2 (Ad5-tooCMV-SCoV2) Group 3 (Ad26-tooCMV-SCoV2+ Ad5-tooCMV-SCoV2) Laboratory and instrumental data Increase in the number of T-lymphocytes 3 (33.33%) 3 6 (66.67%) 6 7 (35.00%) 7 Reducing the number of natural killer cells 5 (55.56%) 5 4 (44.44%) 4 7 (35.00%) 8 Increase in the number of B-lymphocytes 3 (33.33%) 3 5 (55.56%) 5 5 (25.00%) 5 Increase in the number of monocytes 5 (55.56%) 5 0 (0.00%) 0 1 (5.00%) 1 Increase in the number of CD4 lymphocytes 2 (22.22%) 2 2 (22.22%) 2 6 (30.00%) 6 Reducing the percentage of CD8 lymphocytes 4 (44.44%) 4 0 (0.00%) 0 5 (25.00%) 5 Increase in the number of CD8 lymphocytes 1 (I.11%) 1 2 (22.22%) 2 4 (20.00%) 4 Increased levels of immunoglobulin E (IgE) in the blood 1 (11.11%) 1 2 (22.22%) 2 3 (15.00%) 3 Increased erythrocyte sedimentation rate 1 (11.11%) 1 1 (11.11%) 1 2 (10.00%) 2 Increasing the number of natural killer cells 0 (0.00%) 0 2 (22.22%) 2 4 (20.00%) 4 Decreased CD4/CD8 ratio 1 (11.11%) 1 1 (11.11%) 1 4 (20.00%) 4 Increase in the number of leukocytes 1 (11.11%) 1 0 (0.00%) 0 1 (5.00%) 1 Increase in the number of platelets 1 (11.11%) 1 0 (0.00%) 0 1 (5.00%) 1

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Decrease in the number of CD4 lymphocytes 1 (11.11%) 1 0 (0.00%) 0 2(10.00%) 2 Increase in the percentage of lymphocytes 1 (11.11%) 1 0 (0.00%) 0 7(35.00%) 7 Increased CD4/CD8 ratio 0 (0.00%) 0 1 (11.11%) 1 2 (10.0%) 2 General disorders and reactions at the injection site Pain at the injection site 7 (77.78%) 7 5 (55.56%) 5 8 (40.00%) 10 Hyperthermia 8 (88.89%) 9 2 (22.22%) 2 19 (55.00%) 35 Asthenia 3 (33.33%) 3 3 (33.33%) 3 AND (55.00%) 13 Pain 3 (33.33%) 4 2 (22.22%) 2 4 (20.00%) 7 Decreased appetite 2 (22.22%) 2 0 (0.00%) 0 1 (5.00%) 1 pyrexia 0 (0.00%) 0 1 (11.11%) 1 1 (5.00%) 1 Nervous System Disorders Headache 6 (66.67%) 6 3 (33.33%) 3 AND (55.00%) 15 Respiratory, thoracic and mediastinal disorders Pain in the oropharynx (oropharyngeal) 0 (0.00%) 0 1 (11.11%) 1 1 (5.00%) 1 Rhinorrhea 0 (0.00%) 0 0 (0.00%) 0 4 (20.00%) 4 Sore throat 0 (0.00%) 0 0 (0.00%) 0 2(10.00%) 2 Nasal congestion 0 (0.00%) 0 0 (0.00%) 0 1 (5.00%) 1 sneezing 0 (0.00%) 0 0 (0.00%) 0 1 (5.00%) 1 Viral infection of the upper respiratory tract 1 (11.11%) 1 0 (0.00%) 0 0 (0.00%) 0 Gastrointestinal disorders Diarrhea 1 (11.11%) 1 0 (0.00%) 0 3 (15.00)3 Immune System Disorders Hives 1 (11.11%) 1 0 (0.00%) 0 0 (0.00%) 0

As can be seen from the presented data, a single immunization scheme with the developed agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form causes significantly fewer side effects compared to a two-fold immunization scheme.

Example 11. Determination of the effectiveness of intranasal immunization with the developed tool for evaluating the humoral immune response

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

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

1) Single intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

2) Single intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

3) Single intranasal administration of an agent based on recombinant simian adenovirus

- 14 043101 serotypes (simAd25-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ virus particles/dose.

4) Single intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x1011 viral particles/dose.

5) Single intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x1011 viral particles/dose.

6) Single intranasal administration of a recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) agent in liquid form, 5x1011 viral particles/dose.

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

Three weeks later, the animals were bled from the tail vein and the blood serum was isolated. The antibody titer was determined by enzyme immunoassay (ELISA) according to the following protocol:

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

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

3) Serum samples from immunized mice were first diluted 100-fold and then a series of two-fold dilutions was made.

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

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

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

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

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

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

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

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

Table 4. Titer of antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

group of animals Antibody titer Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 919 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 8445 simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose 6400 Ad26-too-CMV-S-CoV2, 5*10 ^pv /dose 1838 Ad5-too-CMV-S-CoV2, 5*10 ^pv /dose 19401 simAd25-too-CMV-S-CoV2, 5*10 ^pv /dose 12800 buffer solution 0

As can be seen from the results obtained, intranasal immunization of animals with the developed agent led to an increase in the titer of antibodies to the S protein of the SARS-CoV-2 virus. Thus, the results of this experiment confirm the possibility of using the developed agent for inducing specific immunity against the severe acute respiratory syndrome virus SARSCoV-2 in liquid form by intranasal administration.

Example 12. Evaluation of the immunogenicity of the developed agent during joint intramuscular and intranasal immunization

The purpose of this study is to test the effectiveness of the developed agent for combined intranasal and intramuscular administration.

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

1) Intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles / dose and simultaneously intramuscular administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-

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CMV-S-CoV2) in liquid form, 5x10 ¹⁰ virus particles/dose.

2) Intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

3) Intramuscular administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

4) Intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles / dose and at the same time intramuscular administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too- CMV-S-CoV2) in liquid form, 5x10 ¹⁰ virus particles/dose.

5) Intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

6) Intramuscular administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

7) Intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles / dose and simultaneously intramuscular administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-tooCMV- S-CoV2) in liquid form, 5x10 ¹⁰ virus particles/dose.

8) Intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

9) Intramuscular administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5x10 ¹⁰ viral particles/dose.

10) Intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x1011 viral particles / dose and simultaneously intramuscular administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-tooCMV-S -CoV2) in liquid form, ^5x10 viral particles/dose.

11) Intranasal administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x 10 ¹¹ viral particles/dose.

12) Intramuscular administration of an agent based on recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in liquid form, 5x 10 ¹¹ viral particles/dose.

13) Intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x1011 viral particles / dose and simultaneously intramuscular administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMVS) -CoV2) in liquid form, 5x 10 ¹¹ virus particles/dose.

14) Intranasal administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x 10 ¹¹ viral particles/dose.

15) Intramuscular administration of an agent based on recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in liquid form, 5x 10 ¹¹ viral particles/dose.

16) Intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5x1011 viral particles / dose and simultaneously intramuscular administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-tooCMV-S -CoV2) in liquid form, 5x1011 virus particles/dose.

17) Intranasal administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5 x 10 ¹¹ viral particles/dose.

18) Intramuscular administration of an agent based on recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in liquid form, 5x 10 ¹¹ viral particles/dose.

19) Intranasal administration of a buffer solution and simultaneously intramuscular administration of a buffer solution (negative control).

20) Intranasal administration of a buffer solution (negative control).

21) Intramuscular administration of a buffer solution (negative control).

Three weeks later, the animals were bled from the tail vein and the blood serum was isolated. The antibody titer was determined by enzyme immunoassay (ELISA) according to the following protocol:

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

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

3) Serum samples from immunized mice were first diluted 100-fold and then a series of two-fold dilutions was made.

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

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

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

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

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

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

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

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

Table 5. Titer of antibodies to protein S in the blood serum of mice (geometric mean of antibody titer)

group of animals Antibody titer 1 Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/m 3200 2 Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose i/n 1056 3 Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ vh/dose IM 2111 4 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/m, 38802 5 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose i/n 8445 6 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose IM 33779 7 simAd25-too-CMV-S-CoV2, 5* ^{10 10} hs/dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/m 22286 8 simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ h/dose i/n 6400 9 simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ vh/dose IM 16890 10 Ad5-too-CMV-S-CoV2, 5* ^{10 10} hs/dose i/n, Ad26-too-CMV-S-CoV2, 5* ^{10 10} hs/dose i/m 44572 AND Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, Ad5-too-CMV-S-CoV2, 5* ^{10 10} hs/dose i/m 44572 12 Ad5-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/m 51200 13 simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, Ad5-too-CMV-S-CoV2, 5* 10 ¹⁰ hs/dose i/m 51200

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14 Ad26-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ¹⁰ hs/dose i/m 19401 15 simAd25-too-CMV-S-CoV2, 5* ^{10 10} hs/dose i/n, Ad26-too-CMV-S-CoV2, 5* 10 ¹⁰ hs/dose i/m 22286 16 Ad26-too-CMV-S-CoV2, 5*10 ⁿ hs/dose i/n, Ad26-too-CMV-S-CoV2, 5*10 ^p hs/dose i/m 3676 17 Ad26-too-CMV-S-CoV2, 5*10 ^p hv/dose i/n, 1213 18 Ad26-too-CMV-S-CoV2, 5*10 ^vh /dose i/m 2425 19 Ad5-too-CMV-S-CoV2, 5*10 ^p iv/dose i/n, Ad5-too-CMV-S-CoV2, 5*10 ^p iv/dose i/m 44572 20 Ad5-too-CMV-S-CoV2, 5*10 ^p iv/dose i/n 9701 21 Ad5-too-CMV-S-CoV2, 5*10 ^vh /dose i/m 33779 22 simAd25-too-CMV-S-CoV2, 5*10 ^pv /dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ^pvv /dose i/m 25600 23 simAd25-too-CMV-S-CoV2, 5*10 ^p.p./dose i/n 7352 24 simAd25-too-CMV-S-CoV2, 5*10 ^p hs/dose i/m 19401 25 Ad5-too-CMV-S-CoV2, 5*10 ⁿ iv/dose i/n, Ad26-too-CMV-S-CoV2, 5*10 ⁿ iv/dose i/m 51200 26 Ad26-too-CMV-S-CoV2, 5*10 ⁿ hs/dose i/n, Ad5-too-CMV-S-CoV2, 5*10 ^p hs/dose i/m 51200 27 Ad5-too-CMV-S-CoV2, 5*10 ⁿ sv/dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ⁿ sv/dose i/m 51200 28 simAd25-too-CMV-S-CoV2, 5*10 ^sv /dose i/n, Ad5-too-CMV-S-CoV2, 5* 10 ¹¹ iv/dose i/m 58813 29 Ad26-too-CMV-S-CoV2, 5*10 ^pv /dose i/n, simAd25-too-CMV-S-CoV2, 5*10 ^pv /dose i/m 22286 thirty simAd25-too-CMV-S-CoV2, 5*10 ^sv /dose i/n, Ad26-too-CMV-S-CoV2, 5* 10 ¹¹ iv/dose i/m 25600 31 Buffer solution i/n Buffer solution i/m 0 32 Buffer solution i/n 0 33 Buffer solution i/m 0

As can be seen from the results obtained, joint intranasal and intramuscular immunization of animals with the developed agent led to a more powerful humoral immune response, compared with immunization by only one method. Thus, the results of this experiment confirm the possibility of using the developed agent to induce specific immunity to the SARS-CoV-2 virus by joint and simultaneous intramuscular and intranasal administration.

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Industrial Applicability

All of the above examples confirm the effectiveness of pharmaceutical agents that provide effective induction of an immune response against the SARS-CoV-2 virus and industrial applicability.

Claims (8)

1. An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component 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 with ORF6-Ad5, with an inserted expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. 2. An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of a recombinant human adenovirus strain of the 5th serotype, in which the E1 and E3 regions are deleted, with an embedded expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. 3. An agent for the induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in liquid form, containing as the only active component an expression vector based on the genome of the recombinant strain of simian adenovirus serotype 25, in which the E1 and E3 regions are deleted, with an embedded expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3. 4. The agent according to claims 1-3, characterized in that it additionally contains a buffer solution containing, wt.%: tris 0.1831-0.3432, sodium chloride 0.3313-0.6212, sucrose 3.7821-7, 0915, magnesium chloride hexahydrate 0.0154-0.0289, EDTA 0.0029-0.0054, polysorbate-80 0.0378-0.0709, ethanol 95% 0.0004-0.0007, water - the rest. 5. The use of an agent according to claims 1 to 3 for inducing an immune response against the SARS-CoV-2 virus, where said agent is intended for intranasal or intramuscular administration or combined intranasal and intramuscular administration. 6. Use according to claim 5, characterized in that said agent is intended for intranasal administration at a dose of 5x10 ¹⁰ -5x10 ¹¹ viral particles. 7. Use according to claim 5, characterized in that said agent is intended for intramuscular administration at a dose of 5x10 ¹⁰ -5x10 ¹¹ viral particles. 8. The use according to claim 5, characterized in that when administered intranasally and intramuscularly, the indicated agent is administered intramuscularly at a dose of 5x10 ¹⁰ -5x10 ¹¹ viral particles, and intranasally at a dose of 5x10 ¹⁰ -5x10 ¹¹ viral particles.