JP2023512381A - Use of drugs to induce specific immunity against severe acute respiratory syndrome virus SARS-COV-2 in children - Google Patents

Abstract

The group of inventions relates to biotechnology, immunology and virology, and in particular to agents for preventing disease caused by severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age. To this end, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5. component 1 in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, component 2 in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 3 and/or based on the genome of recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted, sequence Six variants of drug utilization have been developed that include component 3 in the form of an expression vector that incorporates an expression cassette selected from No. 4, SEQ ID No. 2, and SEQ ID No. 3. The claimed components may be used individually or in combination. The present invention group provides for the production of safe and effective agents that enable the generation of humoral and cellular immune responses against the SARS-CoV-2 virus in children over one month of age. In addition, the drug induces a humoral immune response that corresponds to that of adults and an enhanced mucosal response in the respiratory tract.

Description

FIELD OF THE INVENTION The present invention relates to biotechnology, immunology and virology. The claimed agents can be used to prevent disease caused by severe acute respiratory syndrome virus SARS-CoV-2.

BACKGROUND OF THE INVENTION In December 2019, a novel zoonotic coronavirus named SARS-CoV-2 broke out in Hubei Province, People's Republic of China. The epidemic caused by SARS-CoV-2 is called coronavirus disease-19, abbreviated as COVID-19. A given disease can progress in both asymptomatic and mild forms and severe forms that may be associated with sepsis and multiple organ system failure. Within months, the disease spread worldwide, affecting over 200 countries. In January 2020, the World Health Organization declared the SARS-CoV-2-related outbreak a public health emergency of international concern, and in March declared the spread of the disease a pandemic. expressed. By July 28, 2021, over 195 million cases have been confirmed and 4 million people have died.

The ongoing COVID-19 pandemic poses the ultimate threat to public health. Currently, the development of a safe and effective vaccine against SARS-CoV-2 is a top global priority.

Within the year following the start of the pandemic, various pharmaceutical companies offered variants of their candidate vaccines against COVID-19.

Pharmaceutical company Pfizer, in collaboration with biotech company BioNTech, has developed a BNT162b2 vaccine (Todinameran). A given vaccine represents lipid nanoparticles encapsulating modified mRNA encoding the SARS-CoV-2 S protein mutant. At present, this vaccine is approved for use in adults and children over the age of 12 (F.P. Polacketal. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 2020; 383: 2603- 2615; www.cdc.gov/coronavirus/2019-ncov/vaccines/recommendations/adolescents.html).

Pharmaceutical company Moderna, in collaboration with the National Institute of Health (USA), developed the mRNA-1273 vaccine. The active component of this vaccine is the mRNA encoding the SARS-CoV-2 S protein, surrounded by a lipid coating. Emergency use of this vaccine is currently approved for adults 18 years and older. Moderna is also conducting a study in children aged 12-17 that is currently under review. At present, a study called KidCOVE to immunize children aged 6 months to 12 years is underway (L. A. Jackson et al. An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med 2020; 383: 1920-1931; https://penntoday.upenn.edu/news/covid-vaccine-kids; https://clinicaltrials.gov/ct2/show/NCT04796896).

The University of Oxford, in collaboration with the pharmaceutical company AstraZeneca, has developed a ChAdOx1 nCoV-19 vector vaccine (AZD1222). The active component of this vaccine is the chimpanzee adenovirus ChAdOx1, which contains the codon-optimized coding sequence of the full-length S protein of the SARS-CoV-2 virus with the tissue plasminogen activator leader sequence (GenBank MN908947). The vaccination protocol included two immunizations 28 days apart (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 recombinant human adenovirus serotype 5 (Ad5) expressing SARS-CoV-2 full length S glycoprotein. Currently, the vaccine is targeted for emergency use in adults aged 18 years and older (GenBankYP_009724390) (Feng-Cai Zhu et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years). or older: a randomized, double-blind, placebo-controlled, phase 2 trial. The Lancet. Vol. 369, Issue 10249, P479-488, 2020).

Johnson and Johnson and the Janssen Pharmaceutical Research group, in collaboration with the Beth Israel Deaconess Medical Center, have implemented the JanssenAdVac® technology to produce several candidate vaccines. After performing safety and efficacy studies, the candidate vaccine Ad26. COV2. S(Ad26COVS1) was selected. The active component of this vaccine is a recombinant adenoviral vector serotype with deletions of the E1 and E3 regions, containing the SARS-CoV-2 S protein gene with a furin cleavage site mutation and two proline stabilizing mutations. 26. Vaccines are available in emergencies for adults 18 years and older. Studies of vaccine use in teenagers and new-born children are currently underway (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; .

Beijing Institute of Biological Products Co. has developed an inactivated vaccine against COVID-19. Emergency use of this vaccine is permitted in adults 18 years of age and older. Information has also been published on clinical studies of this vaccine in children in the age groups 3-6, 7-12 and 13-17. Research is currently underway (https://www.who.int/news/item/07-05-2021-who-lists-additional-covid-19-vaccine-for-emergency-use-and-issues -interim-policy-recommendations; https://clinicaltrials.gov/ct2/show/NCT04917523?cond=covid-19+vaccine&age_v=5&draw=2&rank=10).

Therefore, currently only one mRNA-based vaccine (Pfizer) against COVID-19 is approved for use in children. However, doctors are noticing an increasing number of young patients being hospitalized with a COVID-19 diagnosis. Mortality among the younger population is also rising (https://www.paho.org/en/news/5-5-2021-hospitalizations-and-deaths-younger-people-soar-due-covid-19 -paho-director-reports), leading to the need for vaccines for the prevention of COVID-19 in children.

Protective immunity against the SARS-CoV-2 coronavirus activates several lines of the immune system. An effective vaccine against COVID-19 would likely induce both humoral and cellular immune responses. Moreover, an important component of protective immunity will be the activation of mucosal immunity (eg, carried out by the expression of IgA antibodies) in the nasopharynx, the virus entry gate.

Therefore, in the context of the present invention, there is a need for the development of novel agents capable of inducing an immune response against SARS-CoV-2 in children, including the respiratory mucosa, which is a major gate of infection.

Execution of the Invention The technical problem of the claimed group of inventions is to create a drug for effectively inducing an immune response (including a mucosal immune response) against the SARS-CoV-2 virus in children aged 1 month or older. is.

The technical result is the creation of safe and effective agents that enable the generation of humoral and cellular immune responses against the SARS-CoV-2 virus in children over one month of age.

Children are born with immature innate and adaptive immune systems that develop as they grow and acquire memories. From birth to puberty in humans, the immune system goes through several stages in its development.

A newborn's immune system is in a suppressed state. The phagocytic system is not well developed. Neonatal T cells differ greatly from adult cells when the effects of non-shared antigens are largely limited by maternal alloantigens, which is a consequence of life within the mother. Thus, very early adaptive T cell-mediated immunity is characterized by tolerogenic reactivity, reduced alloantigen recognition, and weak responses to non-shared antigens. Neonatal B cells also differ significantly from adult B cells. Neonatal B cells are known to have reduced TACI, BCMA and BAFF-R expression, and reduced IgG and IgA production in response to CD40L and IL-10 (Kaur K, Chowdhury S, Greenspan NS, Schreiber JR. Decreased expression of tumor necrosis factor family receptors involved in humoral immune responses in preterm neonates. Blood. 2007 Oct 15;110(8):2948-54. doi: 10.1182/blood-2007-01-069245. Epub 2007 Jul 18. PMID: 17634409). Taken together, these properties, along with defective immunoglobulin class switching, contribute to suppression of the humoral immune response. B cells of neonates and infants less than 2 months of age demonstrate reduced somatic hypermutation that limits the affinity maturation of antibodies compared to adults. Also, bone marrow stromal cells at early life stages are unable to support the long-term survival and differentiation of plasmablasts into plasma cells, thus, in contrast to older children and adults, immunization may be more effective. Any IgG antibodies produced later are rapidly reduced (Pihlgren M, Friedli M, Tougne C, Rochat AF, Lambert PH, Siegrist CA. Reduced ability of neonatal and early-life bone marrow stromal cells to support plasmablast survival. J Immunol. 2006 Jan 1;176(1):165-72. doi: 10.4049/jimmunol.176.1.165. PMID: 16365407). As a result, the effectiveness of the adaptive immune system for initial responses to T-cell-dependent antigens is much lower in neonates compared to older children and adults (A.K. Simon, G.A. Hollander, A. McMichael. Evolution of 2015 Dec 22; 282(1821): 20143085. doi: 10.1098/rspb.2014.3085, PMCID: PMC4707740, PMID: 26702035).

During infancy, the immune system gradually matures. A very important early defense against many infections from which the mother had previously recovered is provided by passive IgG antibodies transferred from the mother across the placenta and by milk.

The next developmental step is provided by the destruction of maternal antibodies. The primary immune response to infectious invasion is generated by the synthesis of class M immunoglobulins and leaves no immunological memory. This type of immune response also occurs in the case of vaccination against infectious diseases, and only revaccination forms a secondary immune response due to the production of IgG class antibodies.

As children grow, their contact with the outside world increases. Gradually, the immune response switches to the formation of IgG class antibodies. However, primary immune responses to many antigens remain (IgM synthesis). The local immune system remains immature. Gradually, mean blood levels of IgG and IgM increase and reach adult-equivalent levels, but blood levels of IgA still fall short of target values.

The final developmental stage of the immune system is puberty. Against the background of increased secretion of sex steroids, the volume of lymphatic organs decreases. Sex hormone secretion causes suppression of the cell-mediated immune chain (Shcheplyagina, L.A., Kruglova, I.V. Age peculiarities of immunity in children, Russian Medical Journal No. 23 of 11.11.2009, p. 1564).

Therefore, the development of drugs for use in children to effectively induce an immune response to the SARS-CoV-2 virus, including the generation of humoral and cellular immune responses to the SARS-CoV-2 virus. is a complex scientific problem.

When children face SARS-CoV-2, the virus first affects the respiratory mucosa. This means that the interaction between the virus and the immune system primarily occurs in the respiratory and oral mucosa first. Induction of mucosal immunity is therefore an important factor influencing the protective properties of pharmaceuticals.

Based on the level of technology, it can be suggested that administration of adult-targeted vaccines to children may reduce their effectiveness due to the immaturity of the child's immune system. However, studies conducted have shown that administering one-tenth the adult dose of the developed drug to children induces a humoral immune response comparable to that of an adult. In this case this is an unexpected result.

Also, in young animals, administration of the developed drug was demonstrated to cause elevated levels of IgG antibodies on respiratory mucosa. Moreover, the introduction of intranasal administration of drugs into immunization protocols leads to secretion of IgG antibodies on the mucosa. Therefore, as a result of the studies performed, drug administration patterns were developed that induce enhanced mucosal responses in the respiratory tract.

The given technical result is achieved by the claims:

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 Use of a medicament containing components in the form of an expression vector incorporating an expression cassette for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 5 with deletion of the E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a recombinant human adenovirus serotype 5 genome with deletions of the E1 and E3 sites. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a recombinant simian adenovirus serotype 25 genome with deletions of the E1 and E3 sites. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites. 1 and the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 based on the genome of recombinant human adenovirus serotype 5 with deletion of E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

In certain embodiments:
The drug induces a mucosal immune response in the respiratory mucosa.

The drug is prepared in liquid or lyophilized form.

Therein, the liquid form of the drug contains the following buffers (% by weight):
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 rest

There, the reduced lyophilized form of the drug contains the following buffers (% by weight):
Tris 0.0180. . . 0.0338
sodium chloride 0.1044. . . 0.1957
Sucrose 5.4688. . . 10.2539
Magnesium chloride hexahydrate 0.0015. . . 0.0028
EDTA 0.0003. . . 0.0005
Polysorbate-80 0.0037. . . 0.0070
water rest

In certain embodiments, the component(s) of the medicament are intended for intranasal and/or intramuscular administration.

In certain embodiments, the agent is intended for administration at a dose of 5 ^* 10 ⁹ to 5 ^* 10 ¹⁰ viral particles.

In certain embodiments, the components of the medicament are intended for sequential administration at intervals of more than one week, or are intended for simultaneous administration.

There, the components of the medicament can be in individual packages.

Brief description of the drawing
After immunizing mice with the development drug Ad26-CMV-S-CoV2, the percentage of proliferating CD4+ (A) and CD8+ (Б) T lymphocytes before immunization (day 1) and on day 14 of the study was determined. explain. Dots indicate values for each animal participating in the study. Median values are indicated by black lines for each data group. Deviations indicate 95% confidence intervals. The symbol ^** indicates a statistically significant difference between day 1 and day 14 values (p<0.01, Mann-Whitney test). Y-axis - number of proliferating cells (%) X-axis - time (days) After immunizing mice with the development drug Ad5-CMV-S-CoV2, the percentage of proliferating CD4+ (A) and CD8+ (Б) T lymphocytes before immunization (day 1) and on day 14 of the study was determined. explain. Dots indicate values for each animal participating in the study. Median values are indicated by black lines for each data group. Deviations indicate 95% confidence intervals. The symbols ^* (p<0.05) and ^** (p<0.01) indicate statistically significant differences between day 1 and day 14 values (Mann-Whitney test). Y-axis - number of proliferating cells (%) X-axis - time (days) Specific to the RBD domain of the SARS-Cov2 viral S protein before vaccination (day 1) and on days 21, 28 and 42 of the study after immunization of volunteers with the developmental drug at a dose of 1 x 10 ¹⁰ viral particles. IgG antibody titers are illustrated. Dots indicate values for each volunteer participating in the study. Geometric mean antibody titers are represented by black lines for each data group. Deviations indicate 95% confidence intervals. Statistically significant differences between values on days 21, 28 and 42 are indicated in brackets, above which values of p (Wilcoxon rank sum test) are indicated (####-p<0.0001). . UC means the uncertain difference between a given data sample. Statistical significance between values at days 21, 28 and 42 compared to values before vaccination (day 1) was determined by Wilcoxon's rank sum test ( ^*** -p<0. 0001). Y-axis - titer of antigen-specific IgG antibodies; X-axis - time (days). Specific to the RBD domain of the SARS-Cov2 viral S protein prior to vaccination (day 1) and on days 21, 28 and 42 of the study after immunization of volunteers with the developmental drug at a dose of 2x10 ¹⁰ viral particles. IgG antibody titers are illustrated. Dots indicate values for each volunteer participating in the study. Geometric mean antibody titers are represented by black lines for each data group. Deviations indicate 95% confidence intervals. Statistically significant differences between values on days 21, 28 and 42 are indicated in brackets, above which values of p (Wilcoxon rank sum test) are indicated (####-p<0.0001). . UC means the uncertain difference between a given data sample. Statistical significance between values at days 21, 28 and 42 compared to values before vaccination (day 1) was determined by Wilcoxon's rank sum test ( ^*** -p<0. 0001). Y-axis - titer of antigen-specific IgG antibodies; X-axis - time (days). After immunizing volunteers with the developmental drug at a dose of 1 x 10 ¹⁰ viral particles, the percentage of proliferating CD4+ (A) and CD8+ (Б) T lymphocytes before immunization (day 1) and on day 28 of the study was determined. explain. Dots indicate values for each volunteer participating in the study. Median values are indicated by black lines for each data group. Deviations indicate 95% confidence intervals. The symbol ^*** indicates a statistically significant difference between the day 1 and day 28 values (p<0.0001, according to Wilcoxon's rank sum test). Y-axis - number of proliferating cells (%) X-axis - time (days) After immunizing volunteers with the developmental drug at a dose of 2 x ^{10 10} viral particles, the percentage of proliferating CD4+ (A) and CD8+ (Б) T lymphocytes before immunization (day 1) and on day 28 of the study was determined. explain. Dots indicate values for each volunteer participating in the study. Median values are indicated by black lines for each data group. Deviations indicate 95% confidence intervals. The symbol ^*** indicates a statistically significant difference between the day 1 and day 28 values (p<0.0001, according to Wilcoxon's rank sum test). Y-axis - number of proliferating cells (%) X-axis - time (days)

Embodiments of the Invention The first step in the development of immunobiological agents against severe acute respiratory syndrome virus SARS-CoV-2 was the selection of vaccine antigens. A literature survey was conducted as part of this process, demonstrating that the coronavirus S protein is the most promising antigen for generating a candidate vaccine. It is a type 1 transmembrane glycoprotein involved in binding, fusion and entry of virus particles into cells. As demonstrated, it is an inducer of neutralizing antibodies (Liang M et al, SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity. BiomedEnvironSci. 2005 Dec;18(6) :363-74).

To achieve the most effective induction of immune responses against the SARS-CoV-2S protein, the authors developed multiple variants of the expression cassette.

Expression cassette SEQ ID NO: 1 consists of the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal. The CMV promoter is a promoter of the early cytomegalovirus gene that allows constitutive expression in many cell types. However, the force of target gene expression directed by the CMV promoter differs depending on the cell type. Furthermore, transgene expression levels under the control of the CMV promoter were shown to decrease with increasing cell culture time due to suppression of gene expression associated with 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 consists of the CAG promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal. The CAG promoter is a synthetic promoter that contains the early enhancer of the CMV promoter, the chicken β-actin promoter and chimeric introns (chicken β-actin and rabbit β-globin). Experimentally, it was 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. Promoters driving gene expression in developing chicken embryo by using in vivo electroporation. // Genet. Mol. Res. - 2014. - Vol.

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

Expression cassette SEQ ID NO: 4 consists of the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.

To effectively deliver the SARS-CoV-2 coronavirus S protein gene to the human body, an adenoviridae-based vector system was selected. Adenoviral vectors have all the advantages: they cannot grow in human cells, they enter both proliferating and non-proliferating cells, they can induce cellular and humoral immune responses. and they ensure high levels of target antigen expression.

The authors developed two-component drug variants and one-component variants based on different serovars of the Adenoviridae family. Therefore, the immune response to the adenoviral vector portion that may occur after administration of the first component of the drug or the single-component drug is not previously augmented, in the case of dual-component drug applications, or in single-component drugs. It does not affect the development of antigen-specific immune responses to vaccine antigens when repeated administrations are necessary (as in the latter case another adenovirus-based agent can be administered).

Moreover, the developed agents broaden the range of agents for the induction of an immune response against the SARS-CoV-2 coronavirus, which in part of the population is pre-existing immunity against some serotypes of the Adenoviridae family. -immunity) to overcome difficulties related to the issue of presence.

Therefore, as a result of the research carried out, the following technical solutions were developed.

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 Use of a medicament containing components in the form of an expression vector incorporating an expression cassette for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 5 with deletion of the E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a recombinant human adenovirus serotype 5 genome with deletions of the E1 and E3 sites. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a recombinant simian adenovirus serotype 25 genome with deletions of the E1 and E3 sites. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce.

A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites. 1 and the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 based on the genome of recombinant human adenovirus serotype 5 with deletion of E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age.

In certain embodiments:
The drug induces a mucosal immune response in the respiratory mucosa.

The drug is prepared in liquid or lyophilized form.

Therein, the liquid form of the drug contains the following buffers (% by weight):
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 rest

There, the vacuum lyophilized form of the drug contains the following buffers (% by weight):
Tris 0.0180. . . 0.0338
sodium chloride 0.1044. . . 0.1957
Sucrose 5.4688. . . 10.2539
Magnesium chloride hexahydrate 0.0015. . . 0.0028
EDTA 0.0003. . . 0.0005
Polysorbate-80 0.0037. . . 0.0070
water rest

In certain embodiments, the component(s) of the medicament are intended for intranasal and/or intramuscular administration.

In certain embodiments, the agent is intended for administration at a dose of 5 ^* 10 ⁹ to 5 ^* 10 ¹⁰ viral particles.

In certain embodiments, the components of the medicament are intended for sequential administration at intervals of more than one week, or are intended for simultaneous administration.

There, the components of the medicament can be in individual packages.

In addition, the authors developed buffer variants that allow both storage in frozen form at temperatures below -18°C and storage in lyophilized form at temperatures between +2°C and +8°C.

We have also developed the use of a drug to induce specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age by administering it to the organism in an effective amount.

The developed drug was shown to induce a mucosal immune response in the respiratory mucosa.

In addition, it is also possible to use single-component medicaments as well.

Revaccination can be performed with any of the claimed agents, regardless of the agent used for vaccination.

Embodiments of the invention are supported by the following examples.

Example 1
Obtaining an expression vector containing the genome of recombinant human adenovirus serotype 26.
In the first phase of the study, the design of the plasmid construct pAd26-Ends containing two sites of homology to the genome of human adenovirus serotype 26 (two homology arms) and an ampicillin resistance gene. One homology arm is the start of the human adenovirus serotype 26 genome (from the left inverted terminal repeat to the E1 site) and the sequence of the viral genome including the pIX protein. The second homology arm contains the nucleotide sequence after the ORF3 E4 site to the end of the genome. The pAd26-Ends construct was synthesized by CJSC "Evrogen" (Moscow).

Human adenovirus serotype 26 DNA isolated from virus particles was mixed with pAd26-Ends. Homologous recombination between pAd26-Ends and viral DNA resulted in the pAd26-dlE1 plasmid containing the genome of human adenovirus serotype 26 with the E1 site deleted.

Standard cloning methods were then used in the resulting pAd26-dlE1 plasmid to allow the efficient propagation of human adenovirus serotype 26 in HEK293 cell culture, a sequence containing open reading frame 6 (ORF6 -Ad26) was replaced with similar sequences from the human adenovirus serotype 5 genome. The result was the pAd26-dlE1-ORF6-Ad5 plasmid.

The E3 site of the adenoviral genome (approximately 3321 bp between the gene pVIII and the U-exon) was then deleted using standard genetic engineering methods in the construction plasmid pAd26-dlE1-ORF6-Ad5. increased the packing capacity of the vector. The result is the recombinant vector pAd26-only-null, which has the open reading frame ORF6 of human adenovirus serotype 5 and is based on the genome of human adenovirus serotype 26 with deletions of the E1 and E3 sites. rice field.

Additionally, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO: 1 consists of the CMV promoter, the SARS-CoV-2 viral S protein gene and the polyadenylation signal;
- the expression cassette SEQ ID NO: 2 consists of the CAG promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal;
- The expression cassette SEQ ID NO: 3 consists of the EF1 promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal.

Based on the plasmid construct pAd26-Ends, using genetic engineering methods, the construct pArms-26- containing the expression cassette SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, and also containing the homologous arms of the adenovirus serotype 26 genome. CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EF1-S-CoV2 were obtained. The constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EF1-S-CoV2 were then linearized at the unique hydrolysis site between the homology arms to convert each plasmid to It was mixed with the recombinant vector pAd26-only-null. containing the genome of recombinant human adenovirus serotype 26 with the open reading frame ORF6 of human adenovirus serotype 5 and deletion of the E1 and E3 sites as a result of homologous recombination, the expression cassette SEQ ID NO: 1 , pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-only-EF1-S-CoV2 plasmids having SEQ ID NO: 2 or SEQ ID NO: 3, respectively, were obtained.

In the fourth step, pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-only-EF1-S-CoV2 plasmids were hydrolyzed with specific restriction endonucleases to remove vector parts. Removed. The resulting DNA preparation was used to transfect HEK293 cultured cells.

Thus, a recombinant human adenovirus serotype 26 genome with the E1 and E3 sites deleted and the ORF6-Ad26 region replaced by ORF6-Ad5, selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. An expression vector was obtained in which the expression cassette incorporated was obtained.

Example 2
Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 Obtaining a drug in the form of an expression vector that incorporates an expression cassette.
At the given research stage, the expression vector obtained in Example 1 was purified by anion exchange and exclusion chromatography methods. The ready suspension contained adenoviral particles in a buffer for the liquid form of the drug or in a buffer for the lyophilized form of the drug.

Thus, the following immunobiological agents based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 were obtained:
1. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the CMV promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-CMV-S-CoV2) in a buffer for liquid forms of the agent comprising an expression cassette SEQ ID NO: 1 containing a polyadenylation signal.
2. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the CMV promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-CMV-S-CoV2) in a buffer for a lyophilized form of the agent comprising an expression cassette SEQ ID NO: 1 containing a polyadenylation signal.
3. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the CAG promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-CAG-S-CoV2) in a buffer for liquid forms of the agent comprising an expression cassette SEQ ID NO:2 containing a polyadenylation signal.
4. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the CAG promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-CAG-S-CoV2) in a buffer for a lyophilized form of the agent comprising an expression cassette SEQ ID NO:2 containing a polyadenylation signal.
5. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the EF1 promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-EF1-S-CoV2) in a buffer for liquid forms of the agent comprising an expression cassette SEQ ID NO:3 containing a polyadenylation signal.
6. Based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5, the EF1 promoter, the SARS-CoV-2 viral S protein gene, and An immunobiological agent (Ad26-EF1-S-CoV2) in a buffer for a lyophilized form of the agent comprising an expression cassette SEQ ID NO:3 containing a polyadenylation signal.

Each of the resulting immunobiological agents is component 1 of variant 1 and variant 2 of the development drug.

Example 3
Obtaining an expression vector containing the recombinant simian adenovirus serotype 25 genome.
In the first phase of the study, the design of a pSim25-Ends plasmid construct containing two regions of homology (two homology arms) to the simian adenovirus serotype 25 genome was developed. One homology arm is the sequence from the beginning of the simian adenovirus serotype 25 genome (from the left inverted terminal repeat to the E1 site) and from the end of the E1 site to the pIVa2 protein. The second homology arm contains sequences at the end of the adenoviral genome including the right inverted terminal repeat. The pSim25-Ends construct was synthesized by CJSC "Evrogen" (Moscow).

Simian adenovirus serotype 25 DNA isolated from virus particles was mixed with pSim25-Ends. Homologous recombination between pSim25-Ends and viral DNA resulted in the pSim25-dlE1 plasmid containing the genome of simian adenovirus serotype 25 with the E1 site deleted.

The E3 site of the adenoviral genome (3921 bp from the start of gene 12,5K to gene 14,7K) was then deleted using standard genetic engineering methods in the constructed pSim25-dlE1 plasmid. , which increased the packing capacity of the vector. The result was a pSim25-null plasmid construct encoding the entire genome of simian adenovirus serotype 25 with the E1 and E3 sites deleted.

Additionally, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO: 4 consists of the CMV promoter, the SARS-CoV-2 viral S protein gene and the polyadenylation signal;
- the expression cassette SEQ ID NO: 2 consists of the CAG promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal;
- The expression cassette SEQ ID NO: 3 consists of the EF1 promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal.

Genetic engineering methods were then used based on the plasmid construct pSim25-Ends to construct pArms containing the expression cassette SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, and also the homologous arms of simian adenovirus serotype 25. -Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EF1-S-CoV2. The constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EF1-S-CoV2 were then linearized at the unique hydrolysis site between the homology arms to generate each plasmid. It was mixed with the recombinant vector pSim25-null. Recombinant plasmid vector pSim25- containing the whole genome of simian adenovirus serotype 25 with deleted E1 and E3 sites as a result of homologous recombination and the expression cassette SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 3, respectively. CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EF1-S-CoV2 were obtained.

In the third step, the pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EF1-S-CoV2 plasmids were hydrolyzed by specific restriction endonucleases to remove the vector part. The resulting DNA preparation was used to transfect HEK293 cultured cells. The resulting material was used to accumulate preliminary quantities of recombinant adenoviridae.

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

Thus, an expression vector containing the genome of recombinant simian adenovirus serotype 25 with deleted E1 and E3 sites and an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3 is obtained. rice field.

Example 4
A drug in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3 based on the genome of recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted. Get.
At a given stage of research, anion exchange and exclusion chromatography methods were used to purify the expression vector obtained in Example 3. The ready suspension contained adenoviral particles in a buffer for the liquid form of the drug or in a buffer for the lyophilized form of the drug.

Thus, the following immunobiological agents based on the genome of recombinant simian adenovirus serotype 25 with deleted E1 and E3 sites were obtained:
1. Based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites, comprising an expression cassette SEQ ID NO: 4 containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (simAd25-CMV-S-CoV2) in buffer for liquid form of drug.
2. Based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites, comprising an expression cassette SEQ ID NO: 4 containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (Ad26-CMV-S-CoV2) in buffer for lyophilized form of drug.
3. Based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites, comprising an expression cassette SEQ ID NO: 4 containing the CAG promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (simAd25-CAG-S-CoV2) in buffer for liquid form of drug.
4. Based on the genome of recombinant simian adenovirus serotype 25 with deletion of the E1 and E3 sites, comprising an expression cassette SEQ ID NO: 2 containing the CAG promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (simAd25-CAG-S-CoV2) in buffer for lyophilized form of drug.
5. Based on the genome of a recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (simAd25-EF1-S-CoV2) in buffer for liquid form of drug.
6. Based on the genome of a recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (simAd25-EF1-S-CoV2) in buffer for lyophilized form of drug.

Each of the resulting immunobiological agents is Component 2 of Development Drug Variant 1 and Component 1 of Development Drug Variant 3.

Example 5
Obtaining an expression vector containing the recombinant human adenovirus serotype 5 genome.
In the first phase of the study, the design of a pAd5-Ends plasmid construct containing two regions of homology (two homology arms) to the human adenovirus serotype 5 genome was developed. One homology arm is the start of the human adenovirus serotype 5 genome (from the left inverted terminal repeat to the E1 site) and the sequence of the viral genome including the pIX protein. The second homology arm contains the nucleotide sequence after the ORF3 E4 site to the end of the genome. The pAd5-Ends construct was synthesized by CJSC "Evrogen" (Moscow).

Human adenovirus serotype 5 DNA isolated from virus particles was mixed with pAd5-Ends. Homologous recombination between pAd5-Ends and viral DNA resulted in the pAd5-dlE1 plasmid containing the genome of human adenovirus serotype 5 with the E1 site deleted.

The E3 site of the adenoviral genome (2685 bp from the end of gene 12,5K to the start of the sequence U-exon) was then deleted using standard genetic engineering methods in the constructed pAd5-dlE1 plasmid. increased the packing capacity of the vector. The result was a recombinant plasmid vector pAd5-too-null based on the genome of human adenovirus serotype 5 with deletions of the E1 and E3 sites of the genome. Additionally, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO: 1 consists of the CMV promoter, the SARS-CoV-2 viral S protein gene and the polyadenylation signal;
- the expression cassette SEQ ID NO: 2 consists of the CAG promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal;
- The expression cassette SEQ ID NO: 3 consists of the EF1 promoter, the gene for the SARS-CoV-2 viral S protein and the polyadenylation signal.

Then, based on the plasmid construct pAd5-Ends, genetic engineering methods were used to construct constructs containing the expression cassette SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, and also containing the homology arms of the adenovirus serotype 5 genome. pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EF1-S-CoV2 were obtained.

The constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EF1-S-CoV2 were then linearized at the unique hydrolysis site between the homology arms to convert each plasmid to It was mixed with the recombinant vector pAd5-too-null. pAd5-too-CMV containing the genome of recombinant human adenovirus serotype 5 with deletion of the E1 and E3 sites as a result of homologous recombination and carrying the expression cassette SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively -S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too-EF1-S-CoV2 plasmids were obtained.

In the fourth step, the pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-too-EF1-S-CoV2 plasmids were hydrolyzed with specific restriction endonucleases to remove the vector parts. Removed. The resulting DNA preparation was used to transfect HEK293 cultured cells. The resulting material was used to accumulate preliminary amounts of recombinant adenovirus.

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

Thus, an expression vector containing a recombinant human adenovirus serotype 5 genome with deleted E1 and E3 sites and an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 is obtained. rice field.

Example 6
A drug in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted. Get.
At a given stage of research, anion exchange and exclusion chromatography methods were used to purify the expression vector obtained in Example 5. The ready suspension contained adenoviral particles in a buffer for the liquid form of the drug or in a buffer for the lyophilized form of the drug.

Thus, the following immunobiological agents based on the genome of recombinant human adenovirus serotype 5 with deleted E1 and E3 sites were obtained:
1. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 1 containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agents (Ad5-CMV-S-CoV2) in buffers for drug liquid forms.
2. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 1 containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (Ad5-CMV-S-CoV2) in buffer for lyophilized form of drug.
3. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 2 containing the CAG promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agents (Ad5-CAG-S-CoV2) in buffers for drug liquid forms.
4. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 2 containing the CAG promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (Ad5-CAG-S-CoV2) in buffer for lyophilized form of drug.
5. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agents (Ad5-EF1-S-CoV2) in buffers for drug liquid forms.
6. Based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted, comprising an expression cassette SEQ ID NO: 3 containing the EF1 promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal Immunobiological agent (Ad5-EF1-S-CoV2) in buffer for lyophilized form of drug.

Each of the resulting immunobiological agents is component 2 of variant 1 and variant 3 of the development drug.

Example 7
Acquisition of buffer.
Each component of the development drug is a recombinant adenovirus-based drug containing an expression cassette in a buffer.

The authors of the present invention have developed a buffer composition that ensures the stability of recombinant adenoviral particles. A given solution contains:
1. Tris(hydroxymethyl)aminomethane (Tris) required to maintain the pH of the solution.
2. Sodium chloride added to obtain the required ionic strength and osmolarity.
3. Sucrose used as a cryoprotectant.
4. Magnesium chloride hexahydrate required as a source of divalent cations.
5. EDTA used as an inhibitor of free radical oxidation.
6. Polysorbate-80 used as a surfactant.
7. Ethanol 95% used as an inhibitor of free radical oxidation.
8. Water used as solvent.

The authors of the present invention have developed two buffer variants, one for the liquid form of the drug and one for the lyophilized form of the drug.

Several variants of the experimental groups were obtained in order to determine the concentration of substances contained in the buffer for the liquid form of the drug (Table 1). One of the drug components was added to each of the resulting buffers:
1. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on recombinant human adenovirus serotype 26 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.
2. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on a recombinant human adenovirus serotype 5 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.
3. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on recombinant simian adenovirus serotype 25 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.

Therefore, the stability of each of the Adenoviridae serotypes contained in the drug was tested. The resulting drug was stored at temperatures of −18° C. and −70° C. for 3 months, then thawed and analyzed for changes in recombinant Adenoviridae titers.

The results of the experiments performed show that the titer of recombinant Adenoviridae does not change after storage in buffers for liquid forms of the drug for 3 months at temperatures of -18°C and -70°C. Proven.

Therefore, the buffers developed for the liquid form of the drug ensure the stability of all components of the developed drug in the following active ingredient ranges (% by weight):
Tris: 0.1831% by weight. . . 0.3432% by weight;
Sodium chloride: 0.3313% by weight. . . 0.6212% by weight;
Sucrose: 3.7821% by weight. . . 7.0915% by weight;
Magnesium chloride hexahydrate: 0.0154% by weight. . . 0.0289% by weight;
EDTA: 0.0029% by weight. . . 0.0054% by weight;
Polysorbate-80: 0.0378% by weight. . . 0.0709% by weight;
Ethanol 95%: 0.0004% by weight. . . 0.0007% by weight;
Solvent: rest

Several variants of the experimental group were obtained in order to determine the concentration of substances contained in the buffer for the lyophilized form of the drug (Table 2). One of the drug components was added to each of the resulting buffers:
1. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on recombinant human adenovirus serotype 26 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.
2. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on a recombinant human adenovirus serotype 5 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.
3. An immunobiological agent, 1 ^* 10 ¹⁰ viral particles, based on recombinant simian adenovirus serotype 25 with an expression cassette containing the CMV promoter, the gene for the SARS-CoV-2 viral S protein, and the polyadenylation signal.

Therefore, the stability of each of the Adenoviridae serotypes contained in the drug was tested. The resulting drug was stored for 3 months at temperatures of +2 and +8° C., then thawed and analyzed for changes in recombinant Adenoviridae titers.

The results of the experiments performed demonstrate that the titer of recombinant Adenoviridae does not change after storage in the buffer for the lyophilized form of the drug for 3 months at temperatures of +2°C and +8°C. bottom.

Therefore, buffers developed for lyophilized forms of drugs ensure the stability of all components of the developed drug in the following range of active ingredients:
Tris: 0.0180% by weight. . . 0.0338% by weight;
Sodium chloride: 0.1044% by weight. . . 0.1957% by weight;
Sucrose: 5.4688% by weight. . . 10.2539% by weight;
Magnesium chloride hexahydrate: 0.0015% by weight. . . 0.0028% by weight;
EDTA: 0.0003% by weight. . . 0.0005% by weight;
Polysorbate-80: 0.0037% by weight. . . 0.0070% by weight;
Solvent: rest

Example 8
Evaluation of the ability of developed agents to induce mucosal immune responses upon intramuscular administration.
For this study, young mice (21-28 days old) of the BALB/c strain were used. Animals were distributed into groups of 10 and administered the following drugs:
1) Ad26-CMV-S-CoV2, intramuscular (i/m), dose 5x10 ⁹ v.p. p. 21 days later Ad26-CMV-S-CoV2, intramuscular (i/m), dose 5×10 ⁹ v.p. p. /100 ul.
2) Ad5-CMV-S-CoV2, i/m, dose 5x10 ⁹ v. p. /100 ul; 21 days later Ad5-CMV-S-CoV2, i/m, dose 5×10 ⁹ v.p. p. /100 ul.
3) simAd25-CMV-S-CoV2, i/m, dose 5×10 ⁹ v. p. /100 ul; 21 days later, simAd25-CMV-S-CoV2, i/m, dose 5×10 ⁹ v.p. p. /100ul
4) Ad26-null, i/m, dose 5×10 ⁹ v. p. /100 ul; 21 days later Ad26-null, i/m, dose 5×10 ⁹ v.p. p. /100ul
5) Ad5-null, i/m, dose 5×10 ⁹ v. p. 21 days later, Ad5-null, i/m, dose 5×10 ⁹ v.p. p. /100ul
6) simAd25-null, i/m, dose 5×10 ⁹ v. p. /100 ul; 21 days later, simAd25-null, i/m, dose 5×10 ⁹ v.p. p. /100ul
7) Ad26-CMV-S-CoV2, intranasally (i/n), dose 5×10 ⁹ v.p. p. 21 days later Ad26-CMV-S-CoV2 i/n, dose 5×10 ⁹ v./100 ul; p. /100 ul.
8) Ad5-CMV-S-CoV2, i/n, dose 5×10 ⁹ v. p. /100 ul; 21 days later Ad5-CMV-S-CoV2, i/n, dose 5×10 ⁹ v.p. p. /100 ul.
9) Ad26-CMV-S-CoV2, i/m, dose 5x10 ⁹ v. p. 21 days later Ad26-CMV-S-CoV2 i/n, dose 5×10 ⁹ v./100 ul; p. /100 ul.
10) Ad5-CMV-S-CoV2, i/m, dose 5x10 ⁹ v. p. /100 ul; 21 days later Ad5-CMV-S-CoV2, i/n, dose 5×10 ⁹ v.p. p. /100 ul.
11) buffer

IgG and IgA antibody titers were determined by ELISA in bronchoalveolar lavages (BAL) of experimental animals 14 days after the last immunization.

To do so:
1) Antigen (recombinant RBD) was adsorbed to the wells of a 96-well plate for ELISA at +4°C for 16 hours.
2) 100 ul/well of 5% milk in TPBS was added to plate wells to eliminate non-specific binding. It was incubated for 1 hour on a shaker at 37°C.
3) BAL samples were diluted 25-fold and then diluted by a 2-fold dilution method.
4) 50ul of each diluted serum sample was added to plate wells.
5) The samples were then incubated for 1 hour at 37°C.
6) After incubation, wells were washed three times with phosphate buffer.
7) A secondary antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.
8) The samples were then incubated for 1 hour at 37°C.
9) After incubation, the wells were washed three times with phosphate buffer.
10) Tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and changes to a colored compound as a result of the reaction, was then added. The reaction was stopped after 15 minutes by the addition of sulfuric acid. The optical density (OD) of the solution was then measured at a wavelength of 450 nm in each well using a spectrophotometer.

Antibody titers were determined as the final dilution at which the optical density of the solution was reliably higher than the negative control. The results obtained (geometric mean values) are shown in Table 3.

The results obtained show that all variants of the developed drug cause elevated titers of IgG antibodies on respiratory mucosal surfaces. The expression of IgA antibodies depends on the method of drug administration. Maximal titers of IgA antibodies are induced when animals are immunized sequentially intramuscularly and then intranasally.

Therefore, as a result of the studies carried out, immunobiological agents capable of inducing a mucosal immune response against the SARS-CoV-2 virus in the respiratory mucosa of children and this result in an enhanced mucosal immune response. Drug dosing patterns have been developed.

Example 9
Evaluation of the ability of developed agents to induce immune responses in mammals of various ages.
For this study, BALB/c juvenile mice of various ages were used. Animals were distributed into 5 groups:
1) 15-18 day old mice 2) 28-35 day old mice 3) 50-60 day old mice

An immunobiological agent developed based on human adenovirus serotype 5 (Ad5-CMV-S-CoV2), 10 ⁸ v. p. All animals were immunized once with a dose of /50ul. Twenty-one days after immunization, IgG antibody titers were measured in the sera of the animals. To do so:
1) Antigen (recombinant RBD) was adsorbed to the wells of a 96-well plate for ELISA at +4°C for 16 hours.
2) 100 ul/well of 5% milk in TPBS was added to plate wells to eliminate non-specific binding. It was incubated for 1 hour on a shaker at 37°C.
3) Serum samples were diluted 50-fold and then diluted by a 2-fold dilution method.
4) 50ul of each diluted serum sample was added to plate wells.
5) The samples were then incubated for 1 hour at 37°C.
6) After incubation, wells were washed three times with phosphate buffer.
7) A secondary antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.
8) The samples were then incubated for 1 hour at 37°C.
9) After incubation, the wells were washed three times with phosphate buffer.
10) Tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and changes to a colored compound as a result of the reaction, was then added. The reaction was stopped after 15 minutes by the addition of sulfuric acid. The optical density (OD) of the solution was then measured at a wavelength of 450 nm in each well using a spectrophotometer.

Antibody titers were determined as the final dilution at which the optical density of the solution was reliably higher than the negative control. The results obtained (geometric mean values) are shown in Table 4.

Thus, as shown by the data provided, the developed immunobiological agents induce the development of humoral immune responses in both mature and young animals of various ages. This makes it possible to suggest that the drug may be effective for use in people of different age categories.

Example 10
Immunogenicity studies of developed agents in young mice for evaluation of cell-mediated immune responses after a single vaccination.
Young BALB/c mice (21-28 days old) were used for immunogenicity studies. Animals were distributed into 5 groups and given one dose of the following drugs:
1. Ad26-CMV-S-CoV2, intramuscular (i/m), dose 10 ¹⁰ vp/100ul;
2. Ad5-CMV-S-CoV2, intramuscular (i/m), dose 10 ¹⁰ vp/100ul

Cell-mediated immunity levels were evaluated by assessing the amount of proliferating CD4+ and CD8+ lymphocytes isolated from mouse spleens in culture in vitro after repeated restimulation of cells with recombinant S protein of SARS-CoV-2. Decided. Spleens of animals were harvested before and 14 days after immunization and mononuclear cells were isolated therefrom by centrifugation in a ficoll solution density gradient (1.09 g/mL; PanEco). Isolated cells were then stained with the fluorescent dye CFSE (Invivogen, USA) and seeded in wells of a 96-well plate (2 ^* 10 ⁵ cells/well). Repeated stimulation of lymphocytes was performed under in vitro conditions by adding coronavirus S protein to the medium (final protein concentration 1 μg/ml). Intact cells without added antigen were used as a negative control.

To determine the % of proliferating cells, they were stained with antibodies against marker molecules of T lymphocytes CD3, CD4, CD8 (BD Biosciences, USA). A flow cytofluorometer BD FACS AriaIII (BD Biosciences, USA) was used to determine proliferative (with less cell stain CFSE) CD4+ and CD8+ T lymphocytes in the cell mixture. The percentage of proliferating cells obtained in each sample was determined by subtracting the results obtained during analysis of intact cells from those obtained during analysis of cells restimulated with coronavirus S protein antigen. .

The results of the determination of the percentage of proliferating CD4+ and CD8+ T lymphocytes (statistically processed) on day 1 (pre-immunization) and day 14 of the study are shown in Figure 1 (Ad26-CMV-S-CoV2) and It is shown in FIG. 2 (Ad5-CMV-S-CoV2).

The data obtained show that a single immunization of mice with the developed drug after antigen restimulation on day 14 post-immunization allows robust increases in the percentage of proliferating CD4+ and CD8+ T lymphocytes. rice field.

Therefore, it can be concluded that a single immunization of mice with the developed immunobiological agents can lead to the formation of high levels of post-vaccination cellular immunity.

Example 11
Immunogenicity study of developed drugs in young mice by different administration methods.
Young BALB/c mice (21-28 days old) were used for immunogenicity studies. Animals were distributed into 5 groups and administered the following drugs:
3. Ad26-CMV-S-CoV2, intramuscular (i/m), dose 10 ⁹ vp/100ul
4. Ad26-CAG-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
5. Ad26-EF1-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
6. Ad5-CMV-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
7. Ad5-CAG-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
8. Ad5-EF1-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
9. simAd25-CMV-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
10. simAd25-CAG-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
11. simAd25-EF1-S-CoV2, intramuscular, dose 10 ⁹ vp/100ul
12. Ad26-CMV-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
13. Ad26-CAG-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
14. Ad26-EF1-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
15. Ad5-CMV-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
16. Ad5-CAG-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
17. Ad5-EF1-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
18. simAd25-CMV-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
19. simAd25-CAG-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul
20. simAd25-EF1-S-CoV2, intranasal (i/n), dose 10 ⁹ vp/100ul

Animals were bled 21 days later, serum was isolated, and IgG antibody titers against the SARS-CoV-2 coronavirus S protein were determined by ELISA. To do so:
1. Antigens were adsorbed to the wells of a 96-well plate for ELISA at +4°C for 16 hours.
2. To remove non-specific binding, 100 ul/well of 5% milk in TPBS was added to the plate wells. It was incubated for 1 hour on a shaker at 37°C.
3. Serum samples from immunized mice were diluted by a 2-fold dilution method. A total of 12 dilutions of each sample were prepared.
4.50ul of each diluted serum sample was added to plate wells.
5. The samples were then incubated for 1 hour at 37°C.
6. After incubation, the wells were washed three times with phosphate buffer.
7. A secondary antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.
8. The samples were then incubated for 1 hour at 37°C.
9. After incubation, the wells were washed three times with phosphate buffer.
10. Tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and changes to a colored compound as a result of the reaction, was then added. The reaction was stopped after 15 minutes by the addition of sulfuric acid. The optical density (OD) of the solution was then measured at a wavelength of 450 nm in each well using a spectrophotometer.

The titer of IgG antibody was determined as the final dilution at which the optical density of the solution was reliably higher than that of the negative control. The results obtained (geometric mean values) are shown in Table 5.

Based on the data obtained, a single immunization with the developed drug induces a humoral immune response against the SARS-CoV-2 glycoprotein.

Example 12
Immunogenicity studies of developed drugs when administered to young animals at various doses.
The purpose of a given study was to administer the developmental agent at various doses to young animals and assess the humoral immune response to the S protein of SARS-CoV-2.

Young mice (3-4 weeks old) of the C57/Bl6 strain were used to study the immunogenicity of the developed agents. The animals were distributed into 5 groups and the following drugs were administered intramuscularly twice with an interval of 28 days.
1) Ad5-CMV-S-CoV2, 5 ^* 10 ⁹ vp/100ul,
2) Ad5-CMV-S-CoV2, ^{10 10} vp/100ul,
3) Ad5-CMV-S-CoV2, 5 ^* 10 ¹⁰ vp/100ul.

Animals were bled 21 days later, serum was isolated, and IgG antibody titers against the SARS-CoV-2 coronavirus S protein were determined by ELISA. To do so:
1. Antigens were adsorbed to the wells of a 96-well plate for ELISA at +4°C for 16 hours.
2. To remove non-specific binding, 100 ul/well of 5% milk in TPBS was added to the plate wells. It was incubated for 1 hour on a shaker at 37°C.
3. Serum samples from immunized mice were diluted 100-fold and then diluted by a 2-fold dilution method. A total of 12 dilutions of each sample were prepared.
4.50ul of each diluted serum sample was added to plate wells.
5. The samples were then incubated for 1 hour at 37°C.
6. After incubation, the wells were washed three times with phosphate buffer.
7. A secondary antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.
8. The samples were then incubated for 1 hour at 37°C.
9. After incubation, the wells were washed three times with phosphate buffer.
10. Tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and changes to a colored compound as a result of the reaction, was then added. The reaction was stopped after 15 minutes by the addition of sulfuric acid. The optical density (OD) of the solution was then measured at a wavelength of 450 nm in each well using a spectrophotometer.

Antibody titers were determined as the final dilution at which the optical density of the solution was reliably higher than the negative control. The results obtained (geometric mean values) are shown in Table 6.

Based on the data obtained, the development drug demonstrates immunogenicity within the full range of doses selected.

Example 13
Immunogenicity study of developed drugs in young mice by different administration protocols.
Young mice (3-4 weeks old) of the C57/Bl6 strain were used to study the immunogenicity of the developed agents. Animals were distributed into 5 groups and administered the following drugs:
1) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad26-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
2) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
3) Ad26-CMV-S-CoV2, i/n, 10 ⁹ vp/100ul; Ad5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
4) Ad26-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; 3 weeks later, simAd5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul;
5) Ad5-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad5-CMV-S-CoV2 i/m, 10 ⁸ vp/100ul after 3 weeks;
6) Ad5-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad26-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
7) Ad5-CMV-S-CoV2, i/n, 10 ⁹ vp/100ul; Ad26-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
8) Ad5-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; 3 weeks later, simAd5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul;
9) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; after 3 weeks, simAd5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul;
10) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad5-CMV-S-CoV2 i/m, 10 ⁹ vp/100ul after 3 weeks;
11) simAd25-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul; Ad26-CMV-S-CoV2, i/m, 10 ⁹ vp/100ul after 3 weeks.

Animals were bled 21 days later, serum was isolated, and IgG antibody titers against the SARS-CoV-2 coronavirus S protein were determined by ELISA. To do so:
1. Antigens were adsorbed to the wells of a 96-well plate for ELISA at +4°C for 16 hours.
2. To remove non-specific binding, 100 ul/well of 5% milk in TPBS was added to the plate wells. It was incubated for 1 hour on a shaker at 37°C.
3. Serum samples from immunized mice were diluted by a 2-fold dilution method. A total of 12 dilutions of each sample were prepared.
4.50ul of each diluted serum sample was added to plate wells.
5. The samples were then incubated for 1 hour at 37°C.
6. After incubation, the wells were washed three times with phosphate buffer.
7. A secondary antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.
8. The samples were then incubated for 1 hour at 37°C.
9. After incubation, the wells were washed three times with phosphate buffer.
10. Tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and changes to a colored compound as a result of the reaction, was then added.
11. The reaction was stopped after 15 minutes by the addition of sulfuric acid. The optical density (OD) of the solution was then measured at a wavelength of 450 nm in each well using a spectrophotometer.

Antibody titers were determined as the final dilution at which the optical density of the solution was reliably higher than the negative control. The results obtained (geometric mean values) are shown in Table 7.

The results obtained demonstrate that the developed agents provide the generation of a humoral immune response against SARS-CoV-2 in young animals by different administration protocols.

Example 14
A study of the use of developed agents to induce specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children.
One hundred volunteers aged 12-17 participated in a given study. The proposed immunization protocol involved sequential intramuscular administration of components 1 and 2 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) of the drug according to variant 1. One volunteer discontinued participation prior to administration of Component 1 due to newly diagnosed hypertension, and 99 volunteers began study treatment accordingly. 1 due to absence, 1 due to an adverse event (leukopenia), 1 due to an intestinal infection of unknown etiology (enterovirus type), 1 due to purulent furuncle 5 days after administration of the components ( Purulent furunculus), 1 because of hospitalization (intestinal infection) and 1 because of COVID, Component 2 was not administered, and 2 refused to participate. Accordingly, 91 volunteers received both vaccine components.

We recorded 205 AEs that occurred in 73 volunteers (73.0%) after vaccination. Table 8 shows the number of volunteers who had an AE in each group (prevalence) by System Class (SOC) and Preferred Term (PT) according to the MedDRA dictionary and by association with preparation and severity under study. including. Between-group comparisons of AE frequency were performed using the χ2 test and, where appropriate, Fisher's exact test if the expected frequency in any cell was less than 5. Analyzes did not reveal statistically significant differences in the frequency of individual AEs between groups.

The following categories of adverse events (AEs) were observed during the study:
1) Systemic Disorders and Administration Site Conditions - 76AE:
1/10 of dose: 35 cases 1/5 of dose: 22 cases 2) Systemic reactions - 54 AE
1/10 of the dose: 29 cases 1/5 of the dose: 25 cases 3) Laboratory deviation - 76 AE
1/10 of dose: 44 cases 1/5 of dose: 32 cases

The following AEs were associated with the preparation under study (as the association rating was 'possible', 'probable' or 'certain'): Injection site tenderness, flu-like syndrome, fatigue, subfebrile temperature, hot flashes, nasal congestion immediately after vaccination, headache, sore throat, cough, arthralgias, general weakness, stomach pains, panic attacks, neutrophils decreased, increased bilirubin levels.

For other AEs, the association was determined to be 'doubtful' or 'no connection'. No allergic reactions to the preparations under study were observed.

Overall, it can be said that the adverse events uncovered during the study are characteristic of most vaccine drugs. No cases of occurrence of life-threatening adverse events were recorded.

Example 16
Determination of efficacy of immunization of children with developmental medicines based on assessment of humoral immunity levels.
Ninety-five children aged 12-18 years participated in a given study. Humoral immunity levels were determined by assessing the titer of IgG antibodies specific for the RBD domain of the SARS-CoV-2 virus S protein. Study participants were divided into two groups:
1) Sequential intramuscular administration of components 1 and 2 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) of the drug according to variant 1 at a dose of 1x10 ¹⁰ viral particles/ml (47 people) ).
2) Sequential intramuscular administration of components 1 and 2 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) of the drug according to variant 1 at a dose of 2x10 ¹⁰ viral particles/ml (48 people) ).

A sample from the same volunteer taken on day 1 of the study prior to vaccination was used as a reference.

Antigen-specific IgG titers for the first group were assessed on days 21, 28 and 42 of the study, and for the second group on days 21 and 28 of the study.

Antibody titers were determined using a test system for ELISA that allows determination of IgG titers against the RBD domain of the S antigen of the SARS-CoV-2 virus.

Plates pre-adsorbed with RBD (100 ng/well) were washed 5 times with wash buffer. 100 ul of positive control and 100 ul of negative control were then added to plate wells in duplicate. Serial two-fold dilutions of study samples were added to other wells (two replicates for each sample). The plate was sealed with film and incubated at +37° C. for 1 hour with mixing at a speed of 300 rpm. The wells were then washed with a working solution of wash buffer. 100 ul of working solution of monoclonal antibody conjugate was then added to each well and the plate was sealed with adhesive film and incubated at +37° C. for 1 hour with mixing at a speed of 300 rpm. The wells were then washed with a working solution of wash buffer. Next, 100 ul of chromogen substrate solution was added to each well and incubated for 15 minutes at temperatures from +20° C. in the dark. The reaction was then stopped by adding 50ul of stop reagent to each well (1M sulfuric acid solution). Results were determined 10 minutes after stopping the reaction by measuring the optical density in a spectrophotometer at a wavelength of 450 nm.

IgG titers were determined as the highest serum dilution at which the OD450 serum value of an immunized participant was more than twice the control serum value (the participant's serum prior to immunization).

The results of titration of RBD-specific IgG antibodies in the first group (1×10 ¹⁰ viral particles/dose) are shown in FIG. According to the data provided, the titer of antibodies specific for the RBD domain of the S protein of the SARS-Cov2 virus increases gradually after vaccination, reaching a maximum at 42 days. The reciprocal mean geometrical value of a given point is 13,192. By day 42 of the study, seroconversion was observed in all 47 volunteers comprising a given group.

The results of titration of RBD-specific IgG antibodies in the second group (2×10 ¹⁰ viral particles/dose) are shown in FIG. As can be seen from the data provided, the titers of antibodies specific for the RBD domain of the S protein of the SARS-Cov2 virus gradually increased after vaccination with 1/5 of the total therapeutic dose of the adult vaccine,42 reaches a maximum on day 1. The log-geometric mean value for a given point is 19,292. By day 42 of the study, seroconversion was observed in all 49 volunteers comprising a given group.

Thus, the results of the studies performed showed that vaccination of children with the developed immunobiological agents can induce the formation of high levels of post-vaccination humoral immunity.

Example 15
Determination of efficacy of immunization of children with developmental medicines based on assessment of cell-mediated immunity levels.
Ninety-five children aged 12-18 years participated in a given study. Study participants were divided into two groups:
1) Sequential intramuscular administration of components 1 and 2 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) of the drug according to variant 1 at a dose of 1x10 ¹⁰ viral particles/ml (47 people) ).
2) Sequential intramuscular administration of components 1 and 2 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) of the drug according to variant 1 at a dose of 2x10 ¹⁰ viral particles/ml (48 people) ).

Cell-mediated immunity levels were determined by assessing the amount of proliferating CD4+ and CD8+ lymphocytes in peripheral blood of volunteers in in vitro culture after repeated restimulation of cells with recombinant S protein of SARS-CoV-2. Patient blood samples were taken before and 28 days after immunization and mononuclear cells were isolated therefrom by centrifugation on a ficoll solution density gradient (1.077 g/mL; PanEco). Isolated cells were then stained with the fluorescent dye CFSE (Invivogen, USA) and seeded in wells of a 96-well plate (2 ^* 10 ⁵ cells/well). Repeated stimulation of lymphocytes was performed under in vitro conditions by adding coronavirus S protein to the medium (final protein concentration 1 μg/ml). Intact cells without added antigen were used as a negative control. Seventy-two hours after antigen addition, the percentage of proliferating cells was measured and the cultural medium was harvested for gamma interferon levels.

To determine the % of proliferating cells, these were treated with T lymphocytes CD3, CD4, CD8 (anti-CD3 Pe-Cy7 (BDBiosciences, SK7 clone), anti-CD4 APC (BDBiosciences, SK3 clone), anti-CD8 PerCP-Cy5). .5 (BDBiosciences, SK1 clone)). A flow cytofluorometer BD FACS AriaIII (BD Biosciences, USA) was used to determine proliferative (with less cell stain CFSE) CD4+ and CD8+ T lymphocytes in the cell mixture. The percentage of proliferating cells obtained in each sample was determined by subtracting the results obtained during analysis of intact cells from those obtained during analysis of cells restimulated with coronavirus S protein antigen. .

The results of the determination of the percentage of proliferating CD4+ and CD8+ T lymphocytes (statistically processed) on day 1 (pre-immunization) and day 28 of the study are shown in FIGS.

The data obtained demonstrated that immunization of children with the development drug at both selected doses after antigen restimulation on day 28 post-immunization resulted in a robust increase in the percentage of proliferating CD4+ and CD8+ T lymphocytes. showed that it is possible.

Based on the data obtained, it can be concluded that two vaccinations of children with the developed immunological agents can induce the formation of high levels of post-vaccination cellular immunity.

Thus, the examples provided demonstrate that, as a result of studies performed, safe and effective methods for inducing the development of humoral and cell-mediated immune responses to the SARS-CoV-2 virus in children over one month of age. Confirm that the drug was created.

Industrial Applicability All the provided examples demonstrate the efficacy and industrial applicability of the developed drug that effectively induces an immune response against the SARS-CoV-2 virus in children aged 1 month or older. To support.

Claims (15)

Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 Use of a medicament containing components in the form of an expression vector incorporating an expression cassette for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age. A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 5 in which the E1 and E3 sites have been deleted. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age. Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a recombinant human adenovirus serotype 5 genome in which the E1 and E3 sites have been deleted. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce. A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age. Selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant human adenovirus serotype 26 in which the E1 and E3 sites have been deleted and the ORF6-Ad26 sites have been replaced by ORF6-Ad5 From SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on component 1 in the form of an expression vector with an integrated expression cassette and a genome of recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted. specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month or older Use to induce. A component in the form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3, based on the genome of recombinant simian adenovirus serotype 25 in which the E1 and E3 sites have been deleted. 1 and a form of an expression vector incorporating an expression cassette selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 based on the genome of recombinant human adenovirus serotype 5 with deleted E1 and E3 sites. for inducing specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children over one month of age. Use according to any one of claims 1 to 6, wherein said agent is capable of inducing a mucosal immune response in respiratory mucosa. Use according to any one of claims 1 to 6, wherein the medicament is prepared in liquid or lyophilized form. The liquid form of said drug, in weight percent:
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
9. Use according to claim 8, containing water and the remainder of the buffer. The vacuum lyophilized form of said agent, in weight percent:
Tris 0.0180. . . 0.0338
sodium chloride 0.1044. . . 0.1957
Sucrose 5.4688. . . 10.2539
Magnesium chloride hexahydrate 0.0015. . . 0.0028
EDTA 0.0003. . . 0.0005
Polysorbate-80 0.0037. . . 0.0070
9. Use according to claim 8, containing water and the remainder of the buffer. Use according to any one of claims 1 to 10, wherein the component(s) of said medicament is intended for intranasal and/or intramuscular administration. Use according to any one of claims 1 to 10, wherein the medicament is intended for administration at a dose of ^5x109 to ^5x1010 viral particles. 11. Use according to any one of claims 3, 5, 6, 9, 10, wherein the constituents of said medicament are intended for continuous administration at intervals of more than one week. 11. Use according to any one of claims 3, 5, 6, 9, 10, wherein the components of said medicament are intended for simultaneous administration. 11. Use according to any one of claims 3, 5, 6, 9, 10, wherein the components are in different packages.

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