WO2022177465A1

WO2022177465A1 - The use of the agent for inducing immunity to sars-cov-2

Info

Publication number: WO2022177465A1
Application number: PCT/RU2022/000045
Authority: WO
Inventors: Olga Vadimovna ZUBKOVA; Tatiana Andreevna OZHAROVSKAIA; Inna Vadimovna DOLZHIKOVA; Olga Popova; Dmitrii Viktorovich SHCHEBLIAKOV; Daria Mikhailovna GROUSOVA; Alina Shahmirovna DZHARULLAEVA; Amir Ildarovich TUKHVATULIN; Natalia Mikhailovna TUKHVATULINA; Dmitrii Nikolaevich SHCHERBININ; Ilias Bulatovich ESMAGAMBETOV; Elizaveta Alexandrovna TOKARSKAYA; Andrei Gennadievich Botikov; Alina Sergeevna EROKHOVA; Fatima Magomedovna Izhaeva; Natalia Anatolievna NIKITENKO; Nadezhda Leonidovna LUBENETS; Aleksandr Sergeevich SEMIKHIN; Boris Savelievich NARODITSKY; Denis Yuryevich LOGUNOV
Original assignee: Federal State Budgetary Institution "National Research Center For Epidemiology And Microbiology Named After The Honorary Academician N.F. Gamaleya" Of The Ministry Of Health Of The Russian Federation
Priority date: 2021-02-21
Filing date: 2022-02-18
Publication date: 2022-08-25
Also published as: AR124744A1; RU2744442C1

Abstract

Group of invention relates to biotechnology, immunology and virology. Described is the use of an agent containing expression vector based on strain human adenovirus serotype 26 or human adenovirus serotype 5, in which E1 and E3 regions are deleted, with integrated expression cassette selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or simian adenovirus serotype 25, in which E1 and E3 regions are deleted with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 or contains only component 2 for induction of specific immunity against severe acute respiratory syndrome Sars-CoV-2 in people over 60 and/or with chronic diseases. Also an agent can containing any two of said components. Invention provides effective induction of specific immunity against severe acute respiratory syndrome Sars-CoV-2 in people over 60 and/or with chronic diseases.

Description

THE USE OF THE AGENT FOR INDUCING IMMUNITY TO SARS-COV-2

Field of the Invention

The invention relates to biotechnology, immunology and virology. The use of the agent for prevention of the diseases caused by severe acute respiratory syndrome virus SARS-CoV-2. i.e. vaccination, is disclosed, including revaccination of subjects above 60 years of age and/or having chronic diseases.

Background of the Invention

Continuing pandemic of coronavirus infection COVID-19 has affected globally millions of people. At present more than 100 million cases of COVID-19 illnesses were recorded, and more than 2 million people died from this disease.

The studies revealed that causative agent of the disease is an RNA virus SARS-CoV-2 pertaining to Coronaviridae family, Beta-CoV B line.

It was shown that the virus is spread by direct contact and also by airborne transmission. Average latent period is 5.1 days, and then the first symptoms of the disease develop (Lauer SA, Grantz KH, Bi Q, et al. The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application. Ann Intern Med. 2020. 10.7326/M20-0504). Typical symptoms of COVID-19 include fever, dry cough, dyspnea, and fatigue. Sore throat, pain in joints, running nose, and headache occur more rarely.

COVID-19 may have mild or severe course. The most dangerous complications include pneumonia, acute respiratory distress syndrome, acute respiratory failure, acute cardiac failure, acute renal failure, septic shock, cardiomyopathy etc. Severe form of the disease more often develops in elderly people and those having chronic diseases. The Center for Disease Control And Prevention reported that lethality risk associated with COVID-19 at age 50-64 years, 65-74 years, 75-84 years, and over 85 years is 30-fold, 90-fold, 220-fold, and 630-fold higher, respectively, compared to young people (aged 18-29 years) (https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/older-adults.html^'). SARS-CoV-2 invades the human organism through binding of viral S protein with angiotensin converting enzyme 2 (ACE-2) receptor, which is expressed in lungs, endothelium, heart, kidneys, and gastrointestinal tract. It has been assumed that this specific mechanism is responsible for higher risk of infection contamination of the elderly people. According to the data of CDC, 63.1% of adults above 60 years have arterial hypertension, 38% of adults above 65 years have chronic kidney disease (CKD), and 26.8% of adults above 65 years have diabetes mellitus. Many of these patients receive ACE-2 inhibitors and angiotensin receptor blockers, which activate ACE-2 receptor. Therefore, it is assumed that elderly people having these comorbidities may be at greater risk of contracting SARS-CoV-2 infection and experience more severe course of the disease (Z. Shahid et al. COVID-19 and Older Adults: What We Know. J Am Geriatr Soc. 2020 May; 68(5): 926-929).

Therefore, the elderly people and/or people having chronic disease are particularly in need of vaccination against COVID-19.

By the present time several vaccines against Covid-19 have been developed. Modema pharmaceutical company in collaboration with National Institute of Health (USA) has developed mRNA-1273 vaccine comprising mRNA encoding S protein of SARS-CoV-2, which is protected with lipid membrane. Recently phase 3 clinical trial of this vaccine has finished. In this study more than 7000 volunteers at age above 65 years and also more than 5000 volunteers younger than 65 years having chronic diseases associated with high risk of severe course of COVID-19 (diabetes mellitus, obesity and cardiac disease) were enrolled. According to published data, the vaccine efficacy for people above 65 years was 86.4% (L. Baden et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2020 Dec 30).

Pfizer pharmaceutical company in collaboration with BioNTech biotechnological company developed BNT162b2 vaccine (tozinameran). This vaccine comprises lipid nanoparticles with incapsulated modified mRNA encoding mutated S protein of SARS-CoV-2. During the last phase of clinical development more than 18 thousand people at age from 16 to 89 years were vaccinated including 7971 people (42.3%) above 55 years old. Furthermore, it has been reported that some patients had chronic diseases (obesity etc.), which are the risk factors of poor clinical outcome of COVID-19. The vaccine efficacy was 93.7%, 94.7%, and 100% for the patients above 55 years, 65 years, and 75 years, respectively (Polack F. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec).

Oxford University in collaboration with AstraZeneca pharmaceutical company has developed a vector vaccine ChAdOxl nCoV-19 (AZD1222). In this vaccine chimpanzee adenovirus ChAdOxl containing codon optimized coding sequence of the full-length S protein of SARS-CoV-2 (GenBank MN908947) with leader sequence of tissue-type plasminogen activator serves as an active ingredient. The subjects at age 56 years and above comprising 12.2% in overall cohort (1006 [13.3%] in Great Britain and 412 [10.1%] in Brazil) were enrolled into the clinical studies of this product. The study cohort included 974 subjects at age 56-69 years and 444 subjects older than 70 years. However, the patients reported that the vaccine evaluation in the age groups above 56 years was unfeasible because of limited data (Voysey M. 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. Lancet. 2021 Jan 9;397(10269):99-111).

The study groups Johnson and Johnson and Janssen Pharmaceutical in collaboration with Beth Israel Deaconess Medical Center have developed Ad26.COV2.S vaccine. The active principle in this vaccine is a recombinant vector based on genome of human adenovirus serotype 26 with El and E3 sites deleted, containing the gene encoding S protein of SARS- CoV-2 with mutation in the furin cleavage site and two proline-stabilizing mutations. Recently the results of phase 1 -2b clinical study have been published. In this study the age cohort from 18 to 55 years and age cohort of subjects above 65 years participated. The study results showed that in 90% of the subjects regardless of age group virus neutralizing antibodies could be detected on Day 29 after the first dose of the vaccine. By Day 57 the virus neutralizing antibodies were detected in all vaccinated volunteers. The second dose administration resulted in 2.6-2.9 elevation of the antibody titer (Sadoff J. Interim Results of a Phase l-2a Trial of Ad26.COV2.S Covid-19 Vaccine. N Engl J Med. 2021 Jan 13. doi: 10.1056/NEJMoa2034201. Epub ahead of print. PMID: 33440088.). This solution was chosen by the authors as a prototype. The disadvantage of the prototype is that after the first administration of the vaccine the antibodies against the vector part of the expression vector can develop, which will decrease the vaccination efficacy under administration of subsequent doses.

Therefore, the background of the invention shows a need for developing a safe agent, which can induce the immune response against SARS-CoV-2 virus in elderly people and/or in the patients having chronic diseases.

Disclosure of the invention

Technical problem of the claimed group of inventions is the development of agents providing effective induction of immune response against SARS-CoV-2 virus in subjects at age 60 years and above and in patients having chronic diseases.

Technical result is the creation of safe and efficacious agent providing development of humoral and cell immune responses against SARS-CoV-2 in subjects at age 60 years and above and in patients having chronic diseases.

As the years passed the ageing of the immune system occurs, which is characterized with impairment of qualitative and quantitative characteristics of the immune response that can impact on safety and efficacy profiles of the developed vaccines.

Quantitative changes are characterized by decrease of naive T cells population and also in the change in CD4+:CD8+ cell ratio, mainly because of considerable decrease in number of CD8+ T cells. Ageing also leads to decrease in T cell receptor repertoire and in overall worse survival of T cells. Qualitative changes include predominant production of short-living effect T cells compared to memory cell precursors thus leading to impaired immune response. B cell population is not so sensitive to the age-related changes, however the production of functional antibodies in elderly people drop because of lower expression of selected proteins. Besides the age various chronic disease can impact on the immune response development, because many of them are often associated with disturbances of the immune system functioning.

Thus, development of the agent providing effective induction of the immune response against SARS-CoV-2 including humoral and cell responses against SARS-CoV-2 for the aged people and/or people having chronic diseases is a challenging scientific task.

Said technical result is achieved through disclosure of using an agent containing component 1, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and/or containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for inducing specific immunity against severe acute respiratory syndrome SARS- CoV-2 in subjects above 60 years of age and/or having chronic diseases. In addition, the use of the agent is disclosed, said agent containing a component 1 , which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and also containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of simian adenovirus serotype 25 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, or containing only component 2 for inducing specific immunity against severe acute respiratory syndrome SARS-CoV-2 in subjects above 60 years of age and/or having chronic diseases.

The use of another agent is also disclosed, said agent containing a component 1 , which is an agent in the form of expression vector based on genome of recombinant strain of simian adenovirus serotype 25 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and also containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3for inducing specific immunity against severe acute respiratory syndrome SARS- CoV-2 in subjects above 60 years of age and/or having chronic diseases.

Furthermore, said agent is used through intranasal and/or intramuscular administration.

In addition, said agent is used through sequential administration of the components at a time interval of more than one week.

In addition, said agent is used in liquid or lyophilized form.

What is more, the liquid form of the agent when used contains buffer solution, which is composed of, % by weight: tris 0.1831 to 0.3432 sodium chloride 0.3313 to 0.6212 saccharose 3.7821 to 7.0915 magnesium chloride hexahydrate 0.0154 to 0.0289

EDTA 0.0029 to 0.0054 polysorbate-80 0.0378 to 0.0709 ethanol 95% 0.0004 to 0.0007 water balance.

In addition, reconstituted lyophilized agent when used contains buffer composed of, % by weight tris 0.0180 to 0.0338 sodium chloride 0.1044 to 0.1957 saccharose 5.4688 to 10.2539 magnesium chloride hexahydrate 0.0015 to 0.0028 EDTA 0.0003 to 0.0005 polysorbate-80 0.0037 to 0.0070 water balance.

Furthermore, when the agent is used a component 1 and a component 2 are in separate containers.

Short description of the figures

FIG. 1 illustrates the results of assessment of immunization efficacy in volunteers using a liquid form of the developed agent according to variant 1 through assessment of the percentage of proliferating CD4+ H CD8+ lymphocytes restimulated with S antigen of SARS-CoV-2.

Y-axis - An amount of proliferating cells, %.

X-axis - Days. f* - indicates % of proliferating CD4+ T cells in each volunteer on Day 0.

• - indicates % of proliferating CD4+ T cells in each volunteer on Day 28. # - indicates % of proliferating CD8+ T cells in each volunteer on Day 0.

• - indicates % of proliferating CD8+ T cells in each volunteer on Day 28.

The median value is shown as a black line for each data set, deviations correspond to 95% confidence interval. Symbol **** indicates statistically significant difference between the values obtained on Day 0 and Day 28 (pO.OOl, Mann- Whitney test).

FIG. 2 illustrates the fold increase in IFNy concentration in the culture medium of peripheral blood mononuclear cells after restimulation with coronavirus S antigen, which were collected from the volunteers at age above 60 years sequentially immunized with the agent, variant 1, before immunization (Day 0) and on Day 28 of the study

Y-axis - Interferon-gamma concentration (folds)

X-axis - Days.

• - indicates the values measured in each volunteer on Day 0.

■ - indicates the values measured in each volunteer on Day 28.

FIG. 3 illustrates the results of assessment of the antibody-mediated immune response against the antigen of SARS-CoV2 in the volunteers, who were immunized with the liquid form of the developed agent according to variant 1

Y-axis - Titer of IgG against RBD of S glycoprotein of SARS-CoV-2.

X-axis - Days S - indicates the values obtained for each volunteer. Geometric mean of antibody titer is shown as a black line for each data set. Statistically significant difference between the values obtained on Days 21, 28 and 42 is shown as a bracket with a p-value according to Wilcoxon T test above

Implementation of invention

The first stage in the development of the agent for inducing specific immunity against severe acute respiratory syndrome SARS-CoV-2 was the selection of a vaccine antigen. As a part of this process the literature search was performed which demonstrated that the coronavirus S protein was the most promising antigen for creating a candidate vaccine. This type I transmembrane glycoprotein is responsible for virus particles binding, fusion and entry into the cells. As demonstrated, it serves as inducer of neutralizing antibodies (Liang M et al, SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity. Biomed Environ Sci. 2005 Dec;18(6):363-74).

To achieve the most effective induction of immune response against S protein of SARS- CoV-2 the authors developed various variants of expression cassettes.

Expression cassette SEQ ID NO:l comprises CMV promoter, gene encoding S protein of SARS-CoV-2 and polyadenylation signal. CMV promoter is a promoter of early cytomegalovirus genes, which provides constitutive expression in numerous cell types. However, the strength of expression of the target gene controlled by CMV promoter varies depending on the cell type. In addition, it was shown that the level of transgene expression controlled by CMV promoter decreases with longer cell cultivation time because of inhibition of the gene expression related to DNA methylation [Wang W., Jia YL., Li YC., Jing CQ., Guo X., Shang XF., Zhao CP., 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]

Expression cassette SEQ ID NO: 2 comprises CAG promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal. CAG-promoter is a synthetic promoter, which switch on the early enhancer of CMV promoter, chicken b-actin promoter and chimeric intron (chicken b-actin and rabbit b-globin). The experiments showed that transcriptional activity of CAG promoter is higher compared to CMV promotor. [Yang C.Q., Li X.Y., Li Q., Fu S.L., Li H., Guo Z.K., Lin J.T., Zhao S.T. Evaluation of three different promoters driving gene expression in developing chicken embryo by using in vivo electroporation. I I Genet. Mol. Res. - 2014. - Vol. 13. - P. 1270-1277]

Expression cassette SEQ ID NOG comprises EF1 promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal. EF1 promoter is a promoter of human eukaryotic translation elongation factor 1b (EF-la). The promoter is constitutively active in the wide range of cell types [PMID: 28557288. The EF-la promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells]. Gene EF-la encodes the elongation factor la, which is one of the most common proteins in eukaryotic cells and is expressed almost in all cell types of the mammals. This EF-la promotor is often active in the cells where the viral promoters are not able to express the controlled genes, and in the cells, where the viral promoters are gradually faded away.

Expression cassette SEQ ID NO:4 comprises CMV promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal.

Adenovirus-based vector system was selected for effective delivery of the gene encoding S protein of SARS-CoV-2 coronavirus into the human body. Adenoviral vectors provide a number of advantages: they cannot reproduce in the human cells, enter both dividing and nondividing cells, are able to induce cell-mediated and antibody-mediated immune response, and provide high level of the target antigen expression.

The authors developed the variants of the agent containing two components, and also variants of the agents containing one component based on different adenovirus serotypes. In this event the immune response against the vector part of adenovirus, which can develop after administration of the first component of the agent or monocomponent agent is not boosted in future and does not affect the generation of antigen-specific immune response against vaccine antigen if two-component agent is administered or if repeated administration of monocomponent agent is required, because in the latter case the agent based on another adenovirus may be administered.

Furthermore, the developed agents expand armamentarium of the agents for inducing the immune response against SARS-CoV-2 coronavirus, and this will provide overcoming the difficulties arisen from the presence of preexisting immune response against some adenovirus serotypes in some part of population.

Thus, the efforts resulted in development of the following agent variants. 1. The agent for induction of specific immunity against the diseases caused by severe acute respiratory syndrome virus SARS-CoV-2, containing component 1, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and/or containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3

2. The agent for induction of specific immunity against the diseases caused by severe acute respiratory syndrome virus SARS-CoV-2, containing a component 1, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and also containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of simian adenovirus serotype 25 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, or containing only component 2.

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

In addition, the components of the agent are supplied in separate containers.

Furthermore, the authors of the invention developed liquid and lyophilized forms of the agent. In addition, the authors of the invention selected the buffer solution variants, which allow to store the developed agent both frozen at temperature below -18°C, and as lyophilizate at temperature from +2°C to +8°C.

Furhermore, the method of use of the agent for inducing specific immunity against severe acute respiratory syndrome SARS-CoV-2 in subjects above 60 years of age and/or having chronic diseases through administration of the agent into the organism in effective amount.

In addition, the agent including the agent containing only one component, can be used on one occasion.

Two-component agent can be used sequentially at a time interval of at least one week.

The agent can be administered intranasally and/or intramuscularly.

Any of the disclosed agents can be used for revaccination without consideration of the agent, which was used for vaccination.

Implementation of the invention is supported by the following examples.

Example 1

Obtaining of expression vector containing genome of recombinant strain of human adenovirus serotype 26.

At Stage 1 the authors developed the design of plasmid construct pAd26-Ends, which carries two sites homologous to genome of human adenovirus serotype 26 (two homology arms) and ampicillin resistance gene. One homology arm is a beginning of human adenovirus serotype 26 genome (from the left inverted terminal repeat to El site) and the viral genome sequence including pIX protein. The second homology arm contains the nucleotide sequence from ORF3 of E4 site to the end of genome. pAd26-Ends construct was synthesized by ZAO “Euro gene” (Moscow).

DNA of human adenovirus serotype 26 isolated from the virions was mixed with pAd26- Ends construct. Homologous recombination between pAd26-Ends and viral DNA resulted in plasmid pAd26-dlEl, which carries the genome of human adenovirus serotype 26 with El site deleted. Then in the obtained plasmid pAd26-dlEl the sequence containing open reading frame 6 (ORF6-Ad26) was replaced with analogous sequence from the human adenovirus serotype 5 using the conventional cloning methods, to enable effective replication of human adenovirus serotype 26 in the cell culture HEK293. This resulted in plasmid pAd26-dlEl-ORF6-Ad5.

Then E3 site of the adenovirus genome (about 3321 b.p. between pill gene and U-exon) was deleted from the constructed plasmid pAd26-dlEl-ORF6-Ad5 using conventional genetic engineering methods to increase the vector packing capacity. This resulted in recombinant vector pAd26-only-null based on genome of recombinant strain of human adenovirus serotype 26 containing open reading frame ORF6 of human adenovirus serotype 5 and deleted El and E3 sites of the genome. SEQ ID NO:5 was used as a maternal sequence of human adenovirus serotype 26.

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

- expression cassette SEQ ID NO:l comprises CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal;

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

- expression cassette SEQ ID NOG comprises EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal.

On the basis of plasmid construct pAd26-Ends the constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EFl-S-CoV2, containing expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively, and also bearing homology arms of the genome of human adenovirus serotype 26 were obtained using the genetic engineering methods. Then the constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26- EFl-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, each plasmid was mixed with recombinant vector pAd26-only-null. Homologous recombination resulted in plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-only-EFl- S-CoV2, carrying the genome of recombinant strain of human adenovirus serotype 26 containing open reading frame ORF6 of human adenovirus serotype 5 and deleted El and E3 sites of the genome, with expression cassette SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively. At Stage 4 the plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26- only-EFl-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector part. The obtained DNA products were used for transfection of the cell culture HEK293.

Thus, the expression vector was obtained, containing the genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3.

Example 2

Obtaining of immunobiological agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.

At this stage of the work the expression vectors obtained in Example 1 were purified using anion exchange and exclusion chromatography. Resultant suspension contained adenovirus particles in the buffer for liquid form of the agent or in the buffer for lyophilized form of the agent.

Thus, the following immunobiological agents based on genome of recombinant strain of human adenovirus serotype 26, with El and E3 sites deleted from the genome, and the site ORF6-Ad26 substituted for ORF6-Ad5 were obtained:

1. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, SEQ ID NO:l (Ad26-CMV-S- CoV2) in the buffer for liquid form of the agent.

2. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, SEQ ID NO:l (Ad26-CMV-S- CoV2) in the buffer for lyophilized form of the agent. 3. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, SEQ ID NO:2 (Ad26-CAG-S- CoV2) in the buffer for liquid form of the agent.

4. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:2 (Ad26-CAG-S- CoV2) in the buffer for lyophilized form of the agent.

5. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:3 (Ad26-EFl-S-CoV2) in the buffer for liquid form of the agent.

6. Immunobiological agent based on genome of recombinant strain of human adenovirus serotype 26, in which El and E3 sites are deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NOG (Ad26-EFl-S-CoV2) in the buffer for lyophilized form of the agent.

Each of provided immunobiological agents is a component 1 in variant 1 and in variant 2 of the developed agent.

Example 3

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

At Stage 1 the design of plasmid construct pSim25-Ends carrying two sites homologous to the genome of simian adenovirus serotype 25 (two homology arms) was developed. One homology arm is a beginning of simian adenovirus serotype 25 genome (from the left inverted terminal repeat to El site) and the sequence from the end of El site to pIVa2 protein. The second homology arm contains the end nucleotide sequence of adenovirus genome including right inverted terminal repeat. pSim25-Ends construct was synthesized by ZAO “Eurogene” (Moscow).

DNA of simian adenovirus serotype 25 isolated from the virions was mixed with pSim25- Ends. Homologous recombination between pSim25-Ends and viral DNA resulted in plasmid pSim25-dlEl, which carries the genome of simian adenovirus serotype 25 with El site deleted.

Then E3 site of adenovirus genome (3921 b.p. from the beginning of gene 12.5K to gene 14.7K) was removed from the constructed plasmid pSim25-dlEl using conventional genetic engineering methods to increase the vector packing capacity. This resulted in plasmid construct pSim25-null encoding the full-length genome of simian adenovirus serotype 25 with deleted El an E3 sites of the genome. SEQ ID NO: 6 was used as a maternal sequence of simian adenovirus serotype 25.

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

- expression cassette SEQ ID NO:4 comprises CMV promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal;

- expression cassette SEQ ID NO:2 comprises CAG promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal;

-expression cassette SEQ ID NOG comprises EF1 promoter, gene encoding S protein of SARS-CoV-2, and polyadenylation signal.

Then on the basis of plasmid construct pSim25-Ends the constructs pArms-Sim25-CMV- S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EFl-S-CoV2, containing expression cassettes SEQ ID NO:4, SEQ ID NOG or SEQ ID NOG, respectively, and also bearing homology arms of the genome of simian adenovirus serotype 25 were obtained using the genetic engineering methods. Then the constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25- CAG-S-CoV2, pArms-Sim25-EFl-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, each plasmid was mixed with recombinant vector pSim25-null. Homologous recombination resulted in recombinant plasmid vectors pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EFl-S-CoV2, containing full-length genome of simian adenovirus serotype 25 with El and E3 sites deleted, and expression cassette SEQ ID NO:4, SEQ ID NOG or SEQ ID NOG, respectively. At Stage 3 the plasmids pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EFl-S- CoV2 were hydrolyzed with specific restriction endonuclease to remove the vector part. The obtained DNA products were used for transfection of the cell culture HEK293. Resultant material was used for accumulation of recombinant adenoviruses in preparative amount.

The work resulted in obtaining the human adenoviruses serotype 25, containing the gene encoding S protein of SARS-CoV-2: simAd25-CMV-S-CoV2 (containing expression cassette SEQ ID NO:4), simAd25-CAG-S-CoV2 (containing expression cassette SEQ ID NO:2), simAd25-EFl-S-CoV2 (containing expression cassette SEQ ID NOG).

Thus, the expression vector was obtained, containing the genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, and with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG.

Example 4

Obtaining of immunobiological agent in the form of expression vector based on the genome of recombinant strain of simian adenovirus serotype 25, in which El and E3 sites are deleted from the genome and with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG.

At this stage of the work the expression vectors obtained in Example 3 were purified using anion exchange and exclusion chromatography. Resultant suspension contained adenovirus particles in the buffer for liquid form of the agent or in the buffer for lyophilized form of the agent.

Thus, the following immunobiological agents based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome were obtained:

1. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:l (simAd25-CMV-S-CoV2) in the buffer for liquid form of the agent.

2. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:l (simAd25-CMV-S-CoV2) in the buffer for lyophilized form of the agent. 3. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:2 (simAd25-CAG-S-CoV2) in the buffer for liquid form of the agent.

4. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:2 (simAd25-CAG-S-CoV2) in the buffer for lyophilized form of the agent.

5. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in the buffer for liquid form of the agent.

6. Immunobiological agent based on genome of recombinant strain of simian adenovirus serotype 25, with El and E3 sites deleted from the genome, with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in the buffer for lyophilized form of the agent.

Each of provided immunobiological agents is a component 2 in variant 1 of the develop agent and a component 1 in variant 3 of the developed agent.

Example 5

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

At Stage 1 the design of plasmid construct pAd5-Ends carrying two sites homologous to the genome of human adenovirus serotype 5 (two homology arms) was developed. One homology arm is a beginning of human adenovirus serotype 5 (from the left inverted terminal repeat to El site) and the sequence including pIX protein of the viral genome. The second homology arm contains the nucleotide sequence after ORF3 of E4 site to the end of genome. pAd5-Ends construct was synthesized by ZAO “Eurogene” (Moscow).

DNA of human adenovirus serotype 5 isolated from the virions was mixed with pAd5- Ends construct. Homologous recombination between pAd5-Ends and viral DNA resulted in plasmid pAd5-dlEl, which carries the genome of human adenovirus serotype 5 with El site deleted.

Then E3 site of the adenovirus genome (about 2685 b.p. from the end of gene 12.5K to the beginning of U-exon sequence) was deleted from the constructed plasmid pAd5-dlEl using conventional genetic engineering methods to increase the vector packing capacity. This resulted in recombinant plasmid vector pAd5-too-null based on genome of human adenovirus serotype 5 with El and E3 deleted from the genome. SEQ ID NO:7 was used as a maternal sequence of human adenovirus serotype 5.

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

- expression cassette SEQ ID NO:l comprises CMV promoter, gene encoding S protein of SARS-CoV-2 and polyadenylation signal;

- expression cassette SEQ ID NO:2 comprises CAG promoter, gene encoding S protein of SARS-CoV-2 and polyadenylation signal;

- expression cassette SEQ ID NOG comprises EF1 promoter, gene encoding S protein of SARS-CoV-2 and polyadenylation signal.

Then on the basis of plasmid construct pAd5-Ends the constructs pArms-Ad5-CMV-S- CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EFl-S-CoV2 containing expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively, and also bearing homology arms of the genome of human adenovirus serotype 5 were obtained using the genetic engineering methods.

Then the constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5- EFl-S-CoV2 were linearized at the unique hydrolysis site between the homology arms, each plasmid was mixed with recombinant vector pAd5-too-null. Homologous recombination resulted in plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too-EFl-S- CoV2, carrying the genome of recombinant strain of human adenovirus serotype 5 with El H E3 sites deleted from the genome and expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively.

At Stage 4 the plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too- EFl-S-CoV2 were hydrolyzed with specific restriction endonuclease to remove the vector part. The obtained DNA product were used for transfection of the cell culture HEK293. Resultant material was used for accumulation of recombinant adenoviruses in preparative amounts.

The work resulted in obtaining the human adenoviruses serotype 5, containing the gene encoding S protein of SARS-CoV-2: Ad5-CMV-S-CoV2 (containing expression cassette SEQ ID NO:l), Ad5-CAG-S-CoV2 (containing expression cassette SEQ ID NO:2), Ad5-EF1-S- CoV2 (containing expression cassette SEQ ID NO:3).

Thus, the expression vector was obtained, containing the genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome, with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.

Example 6

Obtaining of immunobiological agent in the form of expression vector based on the genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome, with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.

At this stage of thfe work the expression vectors obtained in Example 5 were purified using anion exchange and exclusion chromatography. Resultant suspension contained adenovirus particles in the buffer for liquid form of the agent or in the buffer for lyophilized form of the agent.

Thus, the following immunobiological agents based on genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome:

1. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in the buffer for liquid form of the agent.

2. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in the buffer for lyophilized form of the agent. 3. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in the buffer for liquid form of the agent.

4. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing CAG promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in the buffer for lyophilized form of the agent.

5. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in the buffer for liquid form of the agent.

6. Immunobiological agent based on the genome of recombinant strain of human adenovirus serotype 5, with El and E3 sites deleted from the genome, with expression cassette containing EF1 promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in the buffer for lyophilized form of the agent.

Each of provided immunobiological agents is a component 2 in variant 1 and in variant 3 of the developed agent.

Example 7

Preparation of buffer solution.

The developed agent according to the claimed invention comprises two components supplied in separate vials. Each component is an immunobiological agent based on recombinant adenovirus with expression cassette in buffer solution.

The invention authors have identified the composition of the buffer solution capable of maintaining the stability of recombinant adenovirus particles. This solution consists of:

1. Tris(hydroxymethyl)aminomethane (Tris), which is required for maintaining pH of the solution. 2. Sodium chloride, which is added to achieve appropriate ionic strength and osmolarity.

3. Saccharose, which is used as cryoprotector.

4. Magnesium chloride hexahydrate, which is required as the source of bivalent cation.

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

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

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

8. Water, which is used as a solvent.

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

Several variants of experimental groups were obtained for determining the concentration of the compounds in the composition of buffer solution for liquid form of the agent (table 1). One of the components of the agent was added to each of the prepared buffer solutions:

1. Immunobiological agent based on recombinant human adenovirus serotype 26 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, 1 * 10¹¹ virus particles.

2. Immunobiological agent based on recombinant human adenovirus serotype 5 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, 1 * 10¹¹ virus particles.

3. Immunobiological agent based on recombinant simian adenovirus serotype 25 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, 1*10" virus particles.

Thus, the stability of each of adenovirus serotypes in the agent composition was tested. Prepared pharmaceutical products were stored at the temperature -18°C and -70°C for 3 months, followed by thawing, and the change in recombinant adenovirus titer was assessed. Table 1 - Composition of experimental buffer solutions for liquid form of the agent.

Table 1

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

Therefore, the developed buffer solution for liquid form of the agent provides stability of all components of the developed agent in the following ranges of active ingredients (% by weight):

Tris: from 0.1831 % by weight to 0.3432 % by weight;

Sodium chloride: from 0.3313 % by weight to 0.6212 % by weight;

Saccharose: from 3,7821 % by weight to 7,0915 % by weight;

Magnesium chloride hexahydrate: from 0.0154 % by weight to 0.0289 % by weight;

EDTA: from 0.0029 % by weight to 0.0054 % by weight;

Polysorbate-80: from 0.0378 % by weight to 0.0709 % by weight;

Ethanol 95%: from 0.0004 % by weight to 0.0007 % by weight;

Solvent: balance.

Several variants of experimental groups were obtained for determining the concentration of the compounds in the composition of buffer solution for lyophilized form of the agent (table 2). One of the components of the agent was added to each of the prepared buffer solutions:

1. Immunobiological agent based on recombinant human adenovirus serotype 26 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, 1*10" virus particles.

2. Immunobiological agent based on recombinant human adenovirus serotype 5 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2, and polyadenylation signal, l*10ⁿ virus particles. 3. Immunobiological agent based on recombinant simian adenovirus serotype 25 with expression cassette containing CMV promoter, the gene encoding S protein of SARS-CoV-2 and polyadenylation signal, 1*10" virus particles.

Thus, the stability of each of adenovirus serotypes in the agent composition was tested. Prepared pharmaceutical products were stored at the temperature +2 and +8°C °C for 3 months, and the change in recombinant adenovirus titer was assessed.

Table 2 - Composition of experimental buffer solutions.

Table 2

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

Therefore, the developed buffer solution for lyophilized form of the agent provides stability of all components of the developed agent in the following ranges of active ingredients (% by weight):

Tris: from 0.0180 % by weight to 0.0338 % by weight;

Sodium chloride: from 0.1044 % by weight to 0.1957 % by weight;

Saccharose: from 5,4688 % by weight to 10.2539 % by weight;

Magnesium chloride hexahydrate: from 0.0015 % by weight to 0.0028 % by weight;

EDTA: from 0.0003 % by weight to 0.0005 % by weight;

Polysorbate-80: from 0.0037 % by weight to 0.0070 % by weight;

Solvent: balance.

Example 8 The study of immunization efficacy based on assessment of humoral immune response in the aged animals vaccinated with the developed agent.

The antibody titer is one of principal characteristics of immunization efficacy. In the Example the data are presented on titer of antibodies against SARS-CoV-2 glycoprotein in laboratory animals on Day 21 after administration of the agent compared with baseline.

In this experiment female mice C57/B16 at age 20 months were used. All animals were divided into 43 groups 3 animal per group and received intramuscular injections of component 1 of the agent at the dose 10^u virus particles/IOOmI followed by component 2 at the dose 10¹¹ virus particles/IOOmI, or only component 1 at the dose 10¹¹ virus particles/100m1, or only component 2 at the dose 10ⁿ virus particles/ IOOmI. Thus, the following groups of animals were studied:

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

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

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

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

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

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

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

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

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

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

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

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

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

14) Ad26-CAG -S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2); ) Ad26- CAG-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2);) Ad26-CAG -S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2);) Ad26-EFl-S-CoV2 (component 1), simAd25-CMV-S-CoV2 (component 2);) Ad26- EFl-S-CoV2 (component 1), simAd25-CAG-S-CoV2 (component 2);) Ad26- EFl-S-CoV2 (component 1), simAd25-EFl-S-CoV2 (component 2);) Ad26- null (component 1), simAd25-null (component 2); ) simAd25-CMV-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);) simAd25-CMV-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);) simAd25-CMV-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);) simAd25- CAG -S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);) simAd25- CAG -S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);) simAd25- CAG -S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);) simAd25- EFl-S-CoV2 (component 1), Ad5-CMV-S-CoV2 (component 2);) simAd25- EFl-S-CoV2 (component 1), Ad5-CAG-S-CoV2 (component 2);) simAd25- EFl-S-CoV2 (component 1), Ad5-EFl-S-CoV2 (component 2);) simAd25- null (component 1), Ad5-null (component 2); ) Ad26-CMV-S-CoV2; ) Ad26- CAG -S-CoV2; ) Ad26-EFl-S-CoV2; ) Ad26-null; ) Ad5-CMV-S-CoV2; ) Ad5- CAG -S-CoV2; 37) Ad5-EFl-S-CoV2;

38) Ad26-null;

39) simAd25-CMV-S-CoV2;

40) simAd25- CAG -S-CoV2;

41) simAd25-EFl-S-CoV2;

42) simAd25-null;

43) phosphate-buffered saline.

In three weeks after immunization the blood from the tail vein was collected followed by serum separation. The antibody titer was determined by enzyme immunoassay (EIA) according to the following protocol:

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

2) In order to preclude non-specific binding, the plate was “locked” with 5% milk dissolved in TPBS in amount 100 pL/well. The plate was incubated on shaker at +37°C for 1 hour.

3) A series of 2-fold dilutions of the sera of immunized mice was prepared. A total of 12 dilutions were made for each sample.

4) 50 pL of each diluted serum sample was added into the plate wells.

5) Then the plate was incubated at +37°C for 1 hour.

6) After the end of incubation, the wells were washed with three portions of the phosphate buffer.

7) Then horseradish peroxidase-conjugated secondary antimouse-IgG antibodies were added.

8) Then the plate was incubated at +37°C for 1 hour. 9) After the end of incubation, the wells were washed with three portions of the phosphate buffer.

10) Then tetramethylbenzidine (TMB) solution was added, which is a horseradish substrate and turns into colored compound in the course of reaction. Within 15 minutes sulfuric acid was added to stop the reaction. Then optical density (OD) of solution was measured at wave length 450 nm in each well using spectrophotometer.

The antibody titer was determined as the highest dilution showing the solution optical density significantly greater than that in the negative control group. The results (geometrical means) are shown in Table 3.

Table 3 - Titer of anti-S protein antibodies in the murine serum (geometrical mean antibody titer).

Table 3

The presented data show that all variants of the agent induce humoral immune response against SARS-CoV-2 glycoprotein in the aged animals.

Example 9

The study of efficacy of intranasal immunization with the agent components based on assessment of humoral immune response

The objective of this study was to assess efficacy of the developed agent after intransal administration.

In this experiment female mice C57/B16 with the body weight 18-20 g, 5 animals per group. The following group of animals were studied:

1) Single intranasal dose of the agent based on genome of recombinant human adenovirus serotype 26 (Ad26-too-CMV-S-CoV2) in a liquid form, 5*10¹⁰ virus particles/dose. 2) Single intranasal dose of the agent based on genome of recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in a liquid form, 5*10¹⁰ virus particles/dose.

3) Single intranasal dose of the agent based on genome of recombinant simian adenovirus serotype 25 (simAd25-too-CMV-S-CoV2) in a liquid form, 5*10¹⁰ virus particles/dose.

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

5) Single intranasal dose of the agent based on genome of recombinant human adenovirus serotype 5 (Ad5-too-CMV-S-CoV2) in a liquid form, 5*10" virus particles/dose.

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

7) Single intranasal dose of buffer solution (negative control).

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

4) 50 pL of each diluted serum sample was added into the plate wells.

5) Then the plate was incubated at +37°C for 1 hour. 6) After the end of incubation, the wells were washed with three portions of the phosphate buffer.

8) Then the plate was incubated at +37°C for 1 hour.

9) After the end of incubation, the wells were washed with three portions of the phosphate buffer.

The antibody titer was determined as the highest dilution showing the solution optical density significantly greater than that in the negative control group. The results (geometrical means) are shown in Table 4.

Table 4 - Titer of anti-S protein antibodies in the murine serum (geometrical mean antibody titer).

Table 4

The presented data demonstrate that intranasal immunization of the animals with the developed pharmaceutical product resulted in increase in antibody titer against S protein of SARS-CoV-2. Thus, the results of this experiment support the use of the developed agent by intranasal route for inducing specific immunity SARS-CoV-2 virus.

Example 10

Study of safety of the developed agent in the volunteers above 60 years of age having chronic diseases.

110 volunteers above 60 years of age were enrolled to the study. Proposed immunization regimen involved sequential intramuscular administration of component 1 and component 2 of the agent, variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2). One volunteer discontinued before vaccination because of the consent withdrawal, and 109 volunteers started the study therapy. Another volunteer received component 1 of the agent but discontinued before administration of component 2. Thus, 108 volunteers received both components of the agent. The sample based on Full Analysis Set (n=109) included 56 (51.4%) males and 53 (48.6%) females, 107 (98.2%) white race volunteers and 2 (1.8%) Asian race volunteers with average age 68.2 (5.96) years ranging from 60 to 85 years. Body weight index (mean (SD)) was 28.4 (4.11) kg/m2.

As expected for this age cohort, significant morbidity was reported in the history of 106 (97.2%). The most common concomitant diseases were arterial hypertension, obesity and dyslipidemia.

During the study a total of 35 adverse events (AE) were reported in 17 (15.60%) volunteers. 16 (14.68%) volunteers experienced 34 non-serious AE. One AE in 1 (0.92%) volunteer (atrial fibrillation) was judged serious as meeting the criterion “requires hospitalization or prolongation of existing hospitalization”, the causative relation to the study agent was assessed as doubtful, the SAE of moderate severity required medical treatment and the outcome was recovery. Based on relation to the study product the distribution of all reported AE was as follows: 12 AE were considered related to the product (definite relation), 89 AE were considered probably related, 4 AE were considered possibly related, for 7 AE the relation was judged as doubtful, and for 3 AE as “unknown”.

As for severity, all non-serous AE were mild (34 AE). Only one AE (the above- mentioned SAE) was assessed as of moderate severity.

The volunteers experienced AE are shown by MedDRA system-organ classes in Table 5.

Table 5. All adverse events reported in the study (SOC MedDRA).

Table 5

The data are shown as X (%) Y, where X(%) is a number of volunteers who reported at least one event (percentage column), and Y is a number of events.

Based on results of the study the one can conclude that AE observed in the volunteers above 60 years of age during the study are typical for most vaccines. No severe AE were recorded.

Example 11 The study of immunization efficacy of the developed agent based on assessment of cell- mediated immunity level in volunteers above 60 years of age.

The objective of this study was to assess the level of cell-mediated immunity against SARS-CoV-2 virus based on measuring the percentage of proliferating T cells and in vitro assay of interferon gamma secreted by mononuclear cells in volunteers above 60 years of age having chronic diseases after immunization with various variants of the developed agent.

110 volunteers above 60 years of age were enrolled to the study. Proposed immunization regimen involved sequential intramuscular administration of component 1 and component 2 of the agent, variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2). One volunteer discontinued before vaccination because of the consent withdrawal, and 109 volunteers started the study therapy. Another volunteer received component 1 of the agent but discontinued before administration of component 2. Thus 108 volunteers received both components of the agent. The sample based on Full Analysis Set (n=T09) included 56 (51.4%) males and 53 (48.6%) females, 107 (98.2%) white race volunteers and 2 (1.8%) Asian race volunteers with average age 68.2 (5.96) years ranging from 60 to 85 years. Body weight index (mean (SD)) was 28.4 (4.11) kg/m2.

Before immunization and on Day 28 after immunization the blood samples were collected from the volunteers and centrifugated in the ficcol density gradient (1,077 g/mL; PanEco) in order to isolate mononuclear cells. Then the isolated cells were stained with CFSE fluorescent dye (Invivogen, USA) and added into the wells of 96-well microtitration plate (2* 10^s cells/well). Then the lymphocytes were restimulated in vitro by addition of coronavirus S protein into the culture medium (up to final protein concentration 1 pg/mL). Intact cells without antigen addition served as a negative control. 72 hours after antigen addition the percentage of proliferating cells was measured, and the culture medium was collected for measuring the interferon gamma level.

The cells were stained with antibodies against T cell marker molecules CD3, CD4, CD8 (anti-CD3 Pe-Cy7 (BD Biosciences, clone SK7), anti-CD4 APC (BD Biosciences, clone SK3), anti-CD8 PerCP-Cy5.5 (BD Biosciences, clone SKI)) to assess the percentage of proliferating cells. Flow cytofluorimeter BD FACS Arialll (BD Biosciences, USA) was used to identify proliferating (carrying lesser amount of CFSE dye) CD4+ H CD8+ T cells in the cell mixture. The result obtained from analysis of the intact cells was subtracted from the result obtained from analysis of the cells restimulated with coronavirus antigen S in order to determine the resultant percentage of proliferating cells in each sample. Final results are shown in Fig 1.

Based on results of the study the one can conclude that immunization of volunteers above 60 years of age with the developed agent on two occasions results in generation of antigen- scpecific CD4+ and CD8+ T cells on Day 28 after administration of the first component of the vaccine. The median percentage of proliferating CD4+ and CD8+ T cells in the volunteers of the age cohort above 60 years was 0.7 (Cl: 0.5-2.2) and 0.8 (Cl: 0.3-2.2), respectively. The difference between the values of both CD4+ and CD8+ T cells measured before immunization (Day 0) and 28 days after administration of the first vaccine component was statistically significant, pO.001.

At the second stage of the study the concentration of interferon gamma (IFNy) in the culture medium of human blood mononuclear cells collected from the volunteers above 60 years of age was assayed. Mononuclear cells were isolated by centrifugation in the ficcol density gradient (1,077 g/mL; PanEco) from the blood samples, which were collected before immunization with the developed agent and 28 days after immunization. The cells were restimulated with S protein of SARS-CoV-2, and interferon gamma (IFNy) concentration was measured 96 hours after restimulation with the use of Interferon gamma EIA-BEST kit (VECTOR BEST, Russia) in accordance with instruction of manufacturer.

The increase in IFNy concentration was determined from the equation X =Cst/Cint, where X is the increase of IFNy concentration (folds), Cst - IFNy concentration in the medium of stimulated cells (pg/mL), Cint - IFNy concentration in the medium of non- stimulated (intact) cells (pg/mL).

Results of the study are shown in Fig. 2

As is evident from the presented results, immunization of volunteers above 60 years of age with the developed agent lead to generation of antigen-specific interferon-secreting cells within 29 days after administration of the first component. On Day 28 of the study median increment of interferon gamma in the volunteers above 60 years of age was 4.041 (Cl: 2.106-6.802. The difference between interferon gamma increment before immunization (Day 0) and 28 days after administration of the first vaccine component was statistically significant, p<0.001. The study results demonstrate pronounced generation of cell-mediated element of adaptive immunity in the volunteers after immunization with the developed agent.

Thus, based on the above data one can conclude that immunization with the developed agent induces strong antigen-specific cell-mediated element of immunity against SARS-CoV-2 in the volunteers above 60 years of age, and this conclusion is supported by high degree of statistical significance of the measured parameters before and after immunization.

Example 12

The study of immunization efficacy of the developed agent based on assessment of humoral immunity level in volunteers above 60 years of age.

The objective of this study was to assess the level of humoral immunity against SARS- CoV-2 virus based on measuring the antibody titer against RBD of S protein of SARS-CoV-2 in volunteers above 60 years of age having chronic diseases after immunization with the developed agent.

110 volunteers above 60 years of age were enrolled to the study. Proposed immunization regimen involved sequential intramuscular administration of component 1 and component 2 of the agent, variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) with 21-day interval between the components. One volunteer discontinued before vaccination because of the consent withdrawal, and 109 volunteers started the study therapy. Another volunteer received component 1 of the agent but discontinued before administration of component 2. Thus, 108 volunteers received both components of the agent, variant 1.

As expected for this age cohort, significant morbidity was reported in the history of 106. The most common concomitant diseases were arterial hypertension, obesity and dyslipidemia.

The titers of antigen-specific IgG were assessed on Day 21 and Day 28 in 108 volunteers above 60 years and on Day 42 in 63 volunteers.

The titer of antibodies was measured with the use of enzyme immunoassay test system developed by FGBU N.F.Gamaleya National Research Center For Epidemiology And Microbiology, Ministry of Health of Russia (RZN 2020/10393 2020-05-18), which allows to measure IgG against RBD of S protein of SARS-CoV-2. Microtitration plates with pre-adsorbed RBD (100 ng/well) were washed with 5 portions of washing buffer. Then 100 pL of positive control in two replicates and 100 pL of negative control in two replicates were added to the wells. In the other wells the series of two-fold dilutions of the test samples (two replicates of each sample) were added. Thee plate was covered with adhesive film an incubated for 1 h at temperature +37°C with continuous shaking at 300 rpm. Then the wells were washed with 5 portions of washing buffer. 100 pL of working solution of conjugated monoclonal antibodies was added into each well; the plate was covered with adhesive film and an incubated for 1 h at temperature +37 °C with continuous shaking at 300 rpm. Then the wells were washed with 5 portions of washing buffer. 100 pL of chromogen- substrate solution was added into each well, and the plate was incubated in the dark at temperature +20°C for 15 minutes. Then the reaction was stopped by addition of stop-reagent (50 pL of 1M sulfuric acid solution) into each well. The result was recorded within 10 minutes after the reaction termination by measuring the optical density at 450 nm using spectrophotometer.

The IgG titer was determined as the highest serum dilution of the immunized subject which was associated with OD450 more than 2-fold greater than the value obtained with the control serum (i.e. serum of the subject prior to immunization)

The results of the analysis of antibody titer against SARS-CoV-2 antigen in the serum of volunteers above 60 years after administration of various variants of the developed agent and shown in Fig. 3.

As is evident from the presented data, the titer of antibodies induced by consecutive immunization of volunteers with the developed agent grew with time elapsed from immunization. Class G antibodies specific to RBD of S protein of SARS-Cov2 (titer > 1:50) were detected in 53.2% of volunteers on Day 21 (before component 2 administration) and in 98.1% of volunteers on Day 28 (7 days after component 2 administration). On Day 28 (geometric mean titer 1719.9) significant increase in antigen-specific IgG titer compared to Day 21 (geometric mean titer 33.8) was observed. On Day 42, when the report was finalized, 63 of 108 volunteers were studied. In the studied volunteers the antibody titer (geometric mean titer 2926.2) significantly increased compared to the titer value on Day 28 (geometric mean titer 1773.5) measured in the same volunteers. Of note, on Day 42 antigen-specific class G antibodies were detected in 100% volunteers with the blood sample available. To sum up, the results of antibody titer assessment in the serum of volunteers suggest that:

1. Single immunization with component 1 of the agent can induce generation of antigen- specific IgG antibodies in 53.2% of volunteers in age cohort above 60 years on Day 21 after immunization.

2. Immunization of volunteers in age cohort above 60 years with both components of the agent can result in generation of antigen-specific IgG antibodies in 98.1% of volunteers in age cohort above 60 years on Day 28 after immunization.

3. In volunteers in age cohort above 60 years immunized with the developed agent the antibody titers continue growing by Day 42 after the start of immunization. The data collected suggest that antigen-specific IgG antibodies continue growing also after Day 42 from the start of immunization.

Example 13

Assessment of efficacy and safety of immunization with the developed agent for the subjects having chronic diseases.

The clinical development program included the study of the developed agent, variant 1, (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in 35963 volunteers, with 19 866 subjects immunized with both components of the agent (intramuscular dose lxlO¹¹ v.p. into deltoid muscle at 21 -day interval).

The volunteers from 18 to 92 years of age were enrolled to the study. Average age in Group 1 (study agent, SA) and Group II (Placebo) was 45.3 ± 12.0 years and 45.3 ± 11.9 years, respectively).

Analysis of concomitant diseases showed that 3687 volunteers (24.7%) in Study Agent group and 1235 volunteers (25.3%) in Placebo Group had comorbidities (Table 6).

Table 6. Comorbidities in study volunteers.

Table 6

Physical examination at Screening Visit revealed the presence of 823 abnormalities, including 42 clinically significant abnormalities. The most abnormalities involved cardiovascular system (487 of 823 abnormalities, or 59.2%) (Table 7).

Table 7. The abnormalities observed at physical examination at Screening Visit

Table 7.

At present the data of interim immunogenicity analysis are available, which showed that the vaccine induces immune response in volunteers. On Day 42 of the study geometric mean titer of antibodies was 8996, with seroconversion level 98.25%. In Placebo Group seroconversion level was significantly lower: 14.91% (p <0.001).

In this clinical study the safety of the developed agent was also assessed. By the present time 16795 adverse events in 7998 volunteers has been recorded. Among them 70 episodes were described as serious adverse events because hospitalization of the study subjects was required. In none of these episodes the causal relationship between the events and the study agent was found, as adjudicated by independent Data Monitoring Committee.

As a whole, the adverse events observed in the clinical study are typical for the most vaccine products. Regarding the rates of adverse events, which are not characteristic for the vaccine products, no statistically significant difference between experimental group and control group. Flu-like illness and the injection site reaction were the most common side effects. During the study the absence of severe allergic reactions caused by the developed agent during the study was confirmed. Industrial Use

All presented examples support the efficacy and industrial use of the agents, which provide efficacious induction of immune response against SARS-CoV-2 in the persons above 60 years.

Claims

1. The use of the agent containing component 1, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and/or containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in subjects above 60 years of age and/or having chronic diseases.

2. The use of the agent containing a component 1, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 26 with El and E3 sites deleted from the genome, and the site ORF6-Ad26 is substituted for ORF6-Ad5 with integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and also containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of simian adenovirus serotype 25 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, or containing only component 2 for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in subjects above 60 years of age and/or having chronic diseases.

3. The use of the agent containing a component 1, which is an agent in the form of expression vector based on genome of recombinant strain of simian adenovirus serotype 25 with El and E3 sites deleted from the genome with integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and also containing a component 2, which is an agent in the form of expression vector based on genome of recombinant strain of human adenovirus serotype 5 with El and E3 sites deleted from the genome with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in subjects above 60 years of age and/or having chronic diseases

4. The use according to claims 1, 2, 3, wherein the agent is administered intranasally and/or intramuscularly.

5. The use according to claims 1, 2, 3, wherein the components of the agent are administered sequentially at the time interval of more than one week.

6. The use according to claims 1, 2, 3, wherein the agent is in the liquid or lyophilized form.

7. The use according to claim 6, wherein the liquid form contains buffer composed of, % by weight: tris 0.1831 to 0.3432 sodium chloride 0.3313 to 0.6212 saccharose 3.7821 to 7.0915 magnesium chloride hexahydrate 0.0154 to 0.0289 EDTA 0.0029 to 0.0054 polysorbate 80 0.0378 to 0.0709 ethanol 95% 0.0004 to 0.0007 water balance.

8. The use according to claim 6, wherein reconstituted lyophilized agent contains buffer composed of, % by weight: tris 0.0180 to 0.0338 sodium chloride 0.1044 to 0.1957 saccharose 5.4688 to 10.2539 magnesium chloride hexahydrate 0.0015 to 0.0028 EDTA 0.0003 to 0.0005 polysorbate 80 0.0037 to 0.0070 water balance

9. The use according to the claims 1,2,3, wherein component 1 and component 2 of the agent are in separate containers.