EP4436599A1 - Induction of immunity to sars-cov-2 in children - Google Patents

Induction of immunity to sars-cov-2 in children

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Publication number
EP4436599A1
EP4436599A1 EP22713843.5A EP22713843A EP4436599A1 EP 4436599 A1 EP4436599 A1 EP 4436599A1 EP 22713843 A EP22713843 A EP 22713843A EP 4436599 A1 EP4436599 A1 EP 4436599A1
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European Patent Office
Prior art keywords
seq
agent
cov
genome
utilization
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP22713843.5A
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German (de)
French (fr)
Inventor
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 Gennadevich BOTIKOV
Alina Sergeevna EROKHOVA
Fatima Magomedovna Izhaeva
Natalia Anatolievna NIKITENKO
Nadezhda Leonidovna LUBENETS
Aleksandr Sergeevich SEMIKHIN
Boris Savelievich NARODITSKY
Denis Yuryevich LOGUNOV
Aleksandr Leonidovich GINTSBURG
Sergey Vladimirovich Borisevich
Vladimir Aleksandrovich Chernetsov
Evgenii Vladimirovich Kriukov
Vladimir Fedorovich Babira
Dmitrii Anatolievich KUTAEV
Svetlana Yakovlevna Loginova
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NF Gamaleya Institute of Epidemiology and Microbiology
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NF Gamaleya Institute of Epidemiology and Microbiology
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Priority claimed from RU2021134724A external-priority patent/RU2761904C1/en
Application filed by NF Gamaleya Institute of Epidemiology and Microbiology filed Critical NF Gamaleya Institute of Epidemiology and Microbiology
Publication of EP4436599A1 publication Critical patent/EP4436599A1/en
Pending legal-status Critical Current

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    • A61K39/12—Viral antigens
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to biotechnology, immunology and virology.
  • the claimed agent can be used for prevention of diseases caused by severe acute respiratory syndrome virus SARS- CoV-2.
  • SARS-CoV-2 spread in Hubei province, People's Republic of China.
  • the epidemic disease caused by SARS-CoV-2 is called coronavirus disease- 19, abbreviated as COVID-19.
  • the given disease can proceed both in an asymptomatic and mild form and in a severe form that can be accompanied with sepsis and multiple organ system failure.
  • the World Health Organization declared the SARS-CoV-2-related outbreak to be a public health emergency of international concern and in March it described the spread of the disease as a pandemic.
  • July 28, 2021 over 195 million cases of illness were confirmed and 4 million people died.
  • the pharmaceutical company Pfizer in cooperation with the biotechnology company BioNTech, developed the BNT162b2 vaccine (tozinameran).
  • the given vaccine represents lipid nanoparticles with encapsulated modified mRNA encoding the SARS-CoV-2 S-protein mutant form. At the moment it is allowed to use this vaccine for adults and children aged 12 years and older (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).
  • the pharmaceutical company Modema in cooperation with the National Institute of Health (USA), developed the mRNA-1273 vaccine.
  • the active component of this vaccine is mRNA encoding SARS-CoV-2 S protein, which is surrounded with a lipid coating.
  • mRNA encoding SARS-CoV-2 S protein which is surrounded with a lipid coating.
  • SerBASE a clinical study is under way that is called KidCOVE, in which children aged 6 months - 12 years are immunized (L. A. Jackson etal. 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 cooperation with the pharmaceutical company AstraZeneca, developed the ChAdOxl nCoV-19 vector vaccine (AZD1222).
  • the active component of this vaccine is chimpanzee adenovirus ChAdOxl, including the codon-optimized encoding sequence of full-length S protein of SARS-CoV-2 vims (GenBank MN908947) with the tissue plasminogen activator leader sequence.
  • the vaccination protocol includes double immunization with an interval of 28 days (M. Voysey et al. Safety and efficacy of the ChAdOxl nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. TheLancet. Vol. 397, Issue 10269, P99-111, 2021).
  • CanSino Company developed a vector vaccine against COVID-19, based on recombinant human adenovirus serotype 5 (Ad5) expressing SARS-CoV-2 full-length S -glycoprotein. At present, the vaccine is intended for urgent use in adults aged 18 years or 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 randomised, double-blind, placebo-controlled, phase 2 trial. The Lancet. Vol. 369, Issue 10249, P479-488, 2020).
  • Ad26COV2.S Ad26COVSl
  • the active component of this vaccine is recombinant adenovirus vector serotype 26 with deletion of El and E3 region, comprising the SARS-CoV-2 S-protein gene, with furin cleavage site mutation and with two proline-stabilizing mutations.
  • the vaccine may be utilized in urgent conditions for adults aged 18 years or older. Currently there are ongoing studies of vaccine utilization in teenagers and children from birth. (J. Sadoff et al.
  • Protective immunity against SARS-CoV-2 coronavirus activates several chains of the immune system. It seems that an effective vaccine against COVID-19 shall induce both humoral and cell immune response. Besides, an important element of protective immunity shall be activation of mucosal immunity (for instance, the one implemented via expression of IgA antibodies) in the nasopharynx, which is the virus penetration gate.
  • the technical task of the claimed group of inventions is creation of agents for effective induction of immune response (including mucosal immune response) against the SARS-CoV-2 virus in children aged 1 month and older.
  • the technical result consists in creation of a safe and effective agent enabling development of reactions of humoral and cell immune response against the SARS-CoV-2 virus in children aged 1 month and older.
  • a child is bom with an immature congenital and adaptive immune system that develops and acquires memory with the child's growth. From human birth till puberty the immune system undergoes several stages in its development.
  • Neonatal T-cells greatly differ from adult cells, which is a consequence of antenatal life, when the impact of nonshared antigens is significantly limited by maternal alloantigens. Therefore, the very early adaptive T-cell immunity is characterized with tolerogenic reactivity, decreased alloantigen recognition and weak responses to nonshared antigens. Newborns' B-cells greatly differ from an adult's B-cells, too.
  • Neonatal B-cells are known to have decreased TACI, BCMA and BAFF-R expression as well as decreased 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). Together, these peculiarities contribute to depression of humoral immune responses with incomplete switching of the class of immunoglobulins.
  • IgG class antibodies With the child grows, his or her contacts with the outer world increase. Gradually there is switching of immune reactions to formation of IgG class antibodies. However, the primary immune response to many antigens remains (IgM synthesis). The local immunity system still remains immature. Gradually the average blood concentration of IgG and IgM increases and reaches the level corresponding to that of adults, however the blood level of IgA still does not reach end values.
  • the virus When a child faces SARS-CoV-2, the virus, first of all, affects the mucous membranes of the respiratory tract. It means that interactions between the virus and the immune system first take place primarily on the mucous membranes of the respiratory tract and the oral cavity. Therefore, induction of mucosal immunity is an important factor influencing protective properties of a pharmaceutical agent.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 site are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • the agent induces mucosal immune response on mucous membranes of the respiratory tract
  • the agent is prepared in a liquid or lyophilized form.
  • liquid form of the agent comprises a buffer, wt. %:
  • the reduced lyophilized form of the agent comprises a buffer, wt. %:
  • a component and/or components of the agent are intended for intranasal and/or intramuscular administration.
  • the agent is intended for administration with the dose of 5*10 9 - 5*10 10 virus particles.
  • the agent components are intended for sequential administration with an interval of over 1 week or are intended for simultaneous administration.
  • agent components can be in individual packages.
  • FIG. 1 illustrates the percentage of proliferating CD4+ (A) and CD8+ (E) T lymphocytes before immunization (Day 1) and on Day 14 of the study, after immunization of mice with the developed pharmaceutical agent Ad26-CMV-S-CoV2. Dots denote the values for each animal participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbol ** denotes a statistically significant difference between values of Days 1 and 14 (p ⁇ 0.01, Mann- Whitney test).
  • FIG. 2 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 14 of the study, after immunization of mice with the developed pharmaceutical agent Ad5-CMV-S-CoV2.
  • Dots denote the values for each animal participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval.
  • the symbols * (p ⁇ 0.05) and ** (p ⁇ 0.01) denote a statistically significant difference between values of Days 1 and 14, Mann- Whitney test.
  • FIG. 3 illustrates titers of IgG antibodies specific to RBD-domain of S ARS-Cov2 virus S protein, before vaccination (Day 1) and on Days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of lxlO 10 virus particles.
  • Dots denote the values for each volunteer participating in the study.
  • the mean geometric value of the antibody titer is represented with a black line for each group of data. Deviations denote 95% confidence interval.
  • the statistically significant difference between values on Days 21, 28 and 42 is shown with a bracket above which the value p, Wilcoxon rank sum test, is shown (#### - pO.OOOl).
  • UC means uncertain differences between the given data samples.
  • the statistically significant difference between values of Days 21, 28 and 42 comparing to the values before vaccination (Day 1) was determined by Wilcoxon rank sum test (**** - pO.OOOl).
  • FIG. 4 illustrates titers of IgG antibodies specific to RBD-domain of S ARS-Cov2 virus S protein, before vaccination (Day 1) and on Days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of 2xl0 10 virus particles.
  • Dots denote the values for each volunteer participating in the study.
  • the mean geometric value of the antibody titer is represented with a black line for each group of data. Deviations denote 95% confidence interval.
  • the statistically significant difference between values on Days 21, 28 and 42 is shown with a bracket above which the value p, Wilcoxon rank sum test, is shown (#### - pO.OOOl).
  • UC means uncertain differences between the given data samples.
  • the statistically significant difference between values of Days 21, 28 and 42 comparing to the values before vaccination (Day 1) was determined by Wilcoxon rank sum test (**** - p O.OOOl).
  • FIG. 5 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 28 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of lxlO 10 virus particles.
  • Dots denote the values for each volunteer participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval.
  • the symbol **** denotes the statistically significant difference between values of Days 1 and 28 (p ⁇ 0.0001, according to Wilcoxon rank sum test).
  • FIG. 6 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 28 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of 2x10 10 virus particles.
  • Dots denote the values for each volunteer participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbol **** denotes the statistically significant difference between values of Days 1 and 28 (pO.0001, according to Wilcoxon rank sum test).
  • the first stage in the development of an immunobiological agent against the severe acute respiratory syndrome virus SARS-CoV-2 was the selection of a vaccine antigen.
  • the literature search was performed which demonstrated that the coronavirus S protein was the most promising antigen for creating a candidate vaccine.
  • This is Type 1 transmembrane glycoprotein responsible for virus particles binding, fusion and entry into the cells.
  • 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).
  • the expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • the CMV promoter is the promoter of early cytomegalovirus genes that enables constitutive expression in a multitude of cell types.
  • the power of target gene expression managed by the CMV promoter varies depending on the cell type.
  • the transgene expression level under control of the CMV promoter was shown to decrease with increase of cell cultivation time due to gene expression suppression, which is 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].
  • the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • the CAG-promoter is the synthetic promoter that includes the early enhancer of the CMV promoter, chicken b-actin promoter and chimeric intron (chicken b-actin and rabbit b-globin). It was experimentally shown that the transcription 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. Evaluation of three different promoters driving gene expression in developing chicken embryo by using in vivo electroporation. // Genet. Mol. Res. - 2014. - Vol. 13. - P. 1270- 1277]
  • the expression cassette SEQ ID NO:3 consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • the EF1 -promoter is the promoter of human eukaryotic translation elongation factor 1b (EF-la).
  • the promoter is constitutively active within a wide range of cell types [PMID: 28557288.
  • the EF-la promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells].
  • the EF-la gene encodes the elongation factor- la, which is one of the most widely spread proteins in eukaryotic cells and is expressed in all types of mammalian cells. This EF-la promoter is often active in the cells in which virus promoters are incapable of expressing controlled genes and in cells in which virus promoters are gradually suppressed.
  • the expression cassette SEQ ID NO:4 consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal.
  • Adenovirus vectors have a whole range of advantages: they are incapable of proliferating in human cells, they penetrate both in proliferating and non-proliferating cells, they are capable of inducing cell and humoral immune response, they ensure a high level of target antigen expression.
  • the authors developed variants of the agent comprising two components as well as variants comprising one component based on adenoviridae of different serotypes.
  • the immune response to the adenovirus vector part which can arise after administration of the first component of the agent or single-component agent is not boostered further on and does not influence generation of antigen-specific immune responses against the vaccine antigen in case of utilization of the two-component agent or in case of the need in repeated administration of the single-component agent as in the latter case an agent based on another adenovirus can be administered.
  • the developed agents widen the range of agents for induction of immune response against the SARS-CoV-2 coronavirus, which will enable overcoming of difficulties related to the problem of presence of pre-immunity to some serotypes of adenoviridae in a part of population.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 site are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a component in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
  • the agent induces mucosal immune response on mucous membranes of the respiratory tract
  • the agent is prepared in a liquid or lyophilized form.
  • liquid form of the agent comprises a buffer, wt. %:
  • the reduced lyophilized form of the agent comprises a buffer, wt. %:
  • a component and/or components of the agent are intended for intranasal and/or intramuscular administration.
  • the agent is intended for administration with the dose of 5*10 9 - 5*10 10 virus particles.
  • the agent components are intended for sequential administration with an interval of over 1 week or are intended for simultaneous administration.
  • agent components can be in individual packages.
  • the authors developed buffer variants that enable storing both in a frozen form at temperature below -18°C and in the form of a lyophilisate at temperature from +2°C to +8°C.
  • the agent also consisting of one component, can be used once.
  • Revaccination can be performed with any of the claimed agents irrespective of the agent used for vaccination.
  • the design of the plasmid construct pAd26-Ends comprising two sites homologous to the genome of human adenovirus serotype 26 (two homology arms) and the gene of resistance to ampicillin.
  • One homology arm is the beginning of the genome of human adenovirus serotype 26 (from the left inverted terminal repeat to El site) and the sequence of the virus genome comprising pIX protein.
  • the second homology arm comprises the nucleotide sequence after ORF3 E4 site to the genome end.
  • the pAd26-Ends construct was synthesized by CJSC "Evrogen" (Moscow).
  • Human adenovirus serotype 26 DNA isolated from virions was mixed with pAd26- Ends.
  • the pAd26-dlEl plasmid was obtained that comprised the genome of human adenovirus serotype 26 with deleted El site.
  • the expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NOG consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EFl-S- CoV2 were obtained that comprised expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively, as well as homology arms of adenovirus serotype 26 genome.
  • each plasmid was mixed with the recombinant vector pAd26-only-null.
  • the pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-only-EFl-S-CoV2 plasmids were hydrolyzed with specific restriction endonucleases to delete the vector part.
  • the obtained DNA preparations were used to transfect HEK293 culture cells.
  • the expression vector was obtained that comprised the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 region was replaced with ORF6-Ad5 with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG.
  • Example 2
  • the expression vectors obtained in example 1 were purified with the method of anion-exchange and exclusion chromatography.
  • the ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad26- CMV-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad26- CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad26- CAG-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad26- CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:3 (Ad26- EFl-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:3 (Ad26- EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
  • Each of the given immunobiological agents is component 1 in variant 1 and in variant 2 of the developed agent.
  • the pSim25-Ends plasmid construct comprising two regions homologous to the genome of simian adenovirus serotype 25 (two homology arms).
  • One homology arm is the beginning of the genome of simian adenovirus serotype 25 (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 comprises the sequence of the end of adenovirus genome, including the right inverted terminal repeat.
  • the pSim25-Ends construct was synthesized by CJSC "Evrogen" (Moscow).
  • Simian adenovirus serotype 25 DNA isolated from virions was mixed with pSim25- Ends.
  • the pSim25-dlEl plasmid was obtained that comprised the genome of simian adenovirus serotype 25 with deleted El site.
  • the pSim25-dlEl plasmid E3 site of the adenovirus genome 3921 b.p. from the beginning of gene 12,5K to gene 14,7K was deleted to increase the vector packing capacity.
  • the pSim25-null plasmid construct was obtained that encoded the full genome of simian adenovirus serotype 25 with deletion of El and E3 sites.
  • the expression cassette SEQ ID NO:4 consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NOG consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EFl-S-CoV2 were obtained that comprised expression cassettes SEQ ID NO:4, SEQ ID NOG or SEQ ID NOG, respectively, as well as homology arms of simian adenovirus serotype 25.
  • the pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EFl- S-CoV2 plasmids were hydrolyzed with the specific restriction endonuclease to delete the vector part.
  • the obtained DNA preparations were used to transfect HEK293 culture cells.
  • the obtained material was used to accumulate preparatory quantities of recombinant adenoviridae.
  • recombinant human adenoviridae serotype 25 comprising the gene of SARS-CoV-2 virus S protein: simAd25-CMV-S-CoV2 (comprising the expression cassette SEQ ID NO:4), simAd25-CAG-S-CoV2 (comprising the expression cassette SEQ ID NO:2), simAd25-EFl-S-CoV2 (comprising the expression cassette SEQ ID NO:3).
  • the expression vector was obtained that comprised the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3.
  • the expression vectors obtained in example 3 were purified with the method of anion-exchange and exclusion chromatography.
  • the ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (simAd25-CMV-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (Ad26-CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (simAd25-CAG-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (simAd25-CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
  • Each of the given immunobiological agents is component 2 in variant 1 of the developed agent and component 1 in variant 3 of the developed agent.
  • the design of the pAd5-Ends plasmid construct comprising two regions homologous to the genome of human adenovirus serotype 5 (two homology arms).
  • One homology arm is the beginning of the genome of human adenovirus serotype 5 (from the left inverted terminal repeat to El site) and the sequence of the virus genome comprising pIX protein.
  • the second homology arm comprises the nucleotide sequence after ORF3 E4 site to the genome end.
  • the pAd5-Ends construct was synthesized by CJSC "Evrogen" (Moscow). Human adenovirus serotype 5 DNA isolated from virions was mixed with pAd5- Ends. As a result of homologous recombination between pAd5-Ends and virus DNA the pAd5-dlEl plasmid was obtained that comprised the genome of human adenovirus serotype 5 with deleted El site.
  • the expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
  • the expression cassette SEQ ID NO:3 consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
  • constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms- Ad5-EFl-S-CoV2 were obtained that comprised expression cassettes SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NOG, respectively, as well as homology arms of the genome of adenovirus serotype 5.
  • each plasmid was mixed with the recombinant vector pAd5-too-null.
  • the pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5- too-EFl-S-CoV2 plasmids were hydrolyzed with the specific restriction endonuclease to delete the vector part.
  • the obtained DNA preparation was used to transfect HEK293 culture cells.
  • the obtained material was used to accumulate preparatory quantities of the recombinant adenovirus.
  • recombinant human adenoviridae serotype 5 comprising the gene of S SARS-CoV-2 virus S protein: Ad5-CMV-S-CoV2 (comprising the expression cassette SEQ ID NO:l), Ad5-CAG-S-CoV2 (comprising the expression cassette SEQ ID NO:2), Ad5-EFl-S-CoV2 (comprising the expression cassette SEQ ID NO:3).
  • the expression vector was obtained that comprised the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.
  • the expression vectors obtained in example 5 were purified with the method of anion-exchange and exclusion chromatography.
  • the ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in a buffer for the liquid form of the agent.
  • the immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
  • Each of the given immunobiological agents is component 2 in variant 1 and in variant 3 of the developed agent.
  • Each component of the developed agent is an agent based on the recombinant adenovirus with an expression cassette in a buffer.
  • the authors of the invention developed a buffer composition that ensured stability of recombinant adenovirus particles.
  • the given solution comprises:
  • Tris(hydroxymethyl)aminomethane which is required to maintain pH of the solution.
  • EDTA which is used as a free-radical oxidation inhibitor.
  • Polysorbate-80 which is used as a surfactant.
  • the immunobiological agent based on the recombinant human adenovirus serotype 26 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, l* 10 10 virus particles.
  • the immunobiological agent based on the recombinant human adenovirus serotype 5 with the expression cassette comprising the CMV promoter, the gene of SARS- CoV-2 virus S protein and polyadenylation signal, l* 10 10 virus particles.
  • stability of each of the serotypes of adenoviridae included in the agent was tested.
  • the obtained agents were stored at the temperature of -18°C and -70°C during 3 months, then defrosted and the change of the titer of the recombinant adenoviridae was analyzed.
  • the developed buffer for the liquid form of the agent ensures stability of all components of the developed agent in the following range of active ingredients (wt. %):
  • Tris 0.1831 wt. % ... 0.3432 wt. %;
  • Polysorbate-80 0.0378 wt. % ... 0.0709 wt. %;
  • Ethanol 95% 0.0004 wt. % ... 0.0007 wt. %;
  • the immunobiological agent based on the recombinant human adenovirus serotype 26 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, 1*10 10 virus particles.
  • the immunobiological agent based on the recombinant human adenovirus serotype 5 with the expression cassette comprising the CMV promoter, the gene of SARS- CoV-2 virus S protein and polyadenylation signal, 1*10 10 virus particles.
  • the immunobiological agent based on the recombinant simian adenovirus serotype 25 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, 1*10 10 virus particles.
  • the developed buffer for the lyophilized form of the agent ensures stability of all components of the developed agent in the following range of active ingredients:
  • Tris 0.0180 wt. % ... 0.0338 wt. %;
  • Magnesium chloride hexahydrate 0.0015 wt. % ... 0.0028 wt. %;
  • EDTA 0.0003 wt. % ... 0.0005 wt. %;
  • Polysorbate-80 0.0037 wt. % ... 0.0070 wt. %;
  • mice of BALB/c line were used.
  • the animals were distributed by groups of 10 and the following agents were administered to them: 1) Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 5xl0 9 v.p./100 ul; in 21 days Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 5x10 9 v.p./100 ul.
  • Ad26-CMV-S-CoV2 intranasally (i/n), dose 5xl0 9 v.p./100ul; in 21 days Ad26-CMV-S-CoV2 i/n, dose 5xl0 9 v.p./100ul.
  • the antigen (recombinant RBD) was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C. 2) The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
  • TMB tetramethylbenzidine
  • the titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group.
  • the obtained results are given in Table 3.
  • an immunobiological agent was developed that is capable of inducing mucosal immune response against SARS-CoV-2 virus on the mucous membrane of the respiratory tract in children as well as a pattern of administration of this agent leading to potentiation of the mucosal immune response.
  • mice of BALB/c of different age were used. The animals were distributed into groups of 5:
  • the antigen (recombinant RBD) was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
  • TMB tetramethylbenzidine
  • the titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group.
  • the obtained results are given in Table 4.
  • the developed immunobiological agent induces development of humoral immune response both in adult and young animals of different age. This makes it possible to suggest that the agent will be effective in utilization for people of different age categories.
  • mice (aged 21-28 days) were used for the immunogenicity study. The animals were distributed into groups of 5 to whom the following agents were administered once: 1. Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 10 10 vp/100 ul;
  • Cell immunity level was determined by assessing the quantity of proliferating CD4+ and CD8+ lymphocytes isolated from mouse spleen in the culture in vitro after repeated cell restimulation with recombinant S protein of SARS-CoV-2. Before immunization as well as after 14 days animals' spleens were harvested from which mononuclear cells were isolated by centrifugation in the ficoll solution density gradient (1.09 g/mL; PanEco). Then, the isolated cells were colored with the fluorescent dye CFSE (Invivogen, USA) and seeded on wells of a 96-well plate (2*10 5 cells/well). The repeated stimulation of lymphocytes was carried out in in vitro conditions by adding coronavirus S protein to the culture medium (final protein concentration 1 pg/ml). Intact cells to which the antigen was not added were used as the negative control.
  • CFSE fluorescent dye
  • proliferating cells were stained with antibodies to marker molecules of T lymphocytes CD3, CD4, CD8 (BDBioscienses, USA).
  • Proliferating (with a lower quantity of cell colorant CFSE) CD4+ and CD8+ T lymphocytes were determined in the cell mixture with the use of the flow cytofluorometer BD FACS Arialll (BD Biosciences, USA).
  • the resultant percentage of proliferating cells in each sample was determined by deducing the result obtained during analysis of intact cells from the result obtained during analysis of cells restimulated with coronavirus S protein antigen.
  • mice with the developed agent makes it possible to reliably increase the percentage of proliferating CD4+ and CD8+ T lymphocytes after antigen restimulation on Day 14 after immunization.
  • the antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
  • the serum samples of immunized mice were diluted by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
  • TMB tetramethylbenzidine
  • the titer of IgG antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 5.
  • Table 5- Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean Geometrical Value Of The Titer Of Antibodies)
  • the aim of the given study was assessment of humoral immune response to S protein of SARS-CoV-2 with administration of the developed agent in different doses to young animals.
  • mice of C57/B16 line (3-4 weeks) were used to study immunogenicity of the developed agent.
  • the animals were distributed by groups of 5 to whom the following agents were administered intramuscularly, twice, with an interval of 28 days:
  • Ad5-CMV-S-CoV2 5*10 10 vp/100 ul.
  • the animals' blood was drawn, blood serum was isolated and the titer of IgG antibodies to S protein of SARS-CoV-2 coronavirus was determined by the ELISA method. To do that:
  • the antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
  • the serum samples of immunized mice were diluted 100 times and then by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
  • TMB tetramethylbenzidine
  • the titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 6.
  • the developed agent demonstrates immunogenicity within the whole range of selected doses.
  • mice of C57/B16 line (3-4 weeks) were used to study immunogenicity of the developed agent.
  • the animals were distributed by groups of 5 and the following agents were administered to them:
  • the antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
  • the serum samples of immunized mice were diluted by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
  • TMB tetramethylbenzidine
  • the reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
  • the titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group.
  • the obtained results are given in Table 7.
  • the obtained results demonstrate that the developed agent provides for development of humoral immune response against SARS-CoV-2 in young animals with different administration protocols.
  • the suggested immunization protocol included sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent according to variant 1 (Ad26-CMV-S-CoV2, Ad5- CMV-S-CoV2).
  • One volunteer discontinued participation before administration of component 1 because of newly diagnosed hypertension, correspondingly, 99 volunteers started the therapy under study.
  • Component 2 was not administered to: 1 person - because of non-attendance, 1 person - because of an adverse event (leukopenia), 1 person - because of an intestinal infection of unknown etiology (of enteroviral type), 1 person - because of hospitalization with a purulent furunculus in 5 days after administration of the component, 1 person - because of hospitalization (an intestinal infection) and 1 person - because of COVID, 2 people - refused to participate.
  • both vaccine components were received by 91 volunteers.
  • Table 8 includes the number (share) of volunteers with presence of AE in each group by the system organ class (SOC) and preferred term (PT) according to the MedDRA dictionary, as well as by connection with the preparation under study and severity degree.
  • SOC system organ class
  • PT preferred term
  • AEs were connected with the preparation under study (as the connection assessment is "possible”, “probable” or “certain”): tenderness at place of injection, influenza like syndrome, fatigue, subfebrile temperature, hot flash, nasal congestion arose immediately after vaccine injection, headache, sore throat, cough, joint ache, general weakness, stomachache, panic attack, reduction of neutrophils, increase of bilirubin level.
  • connection was determined as “doubtful” or "no connection". No allergic reactions to the preparation under study were observed.
  • the humoral immunity level was determined by assessing the titer of IgG antibodies specific to RBD domain of S protein of SARS-CoV-2 virus.
  • the participants of the study were divided into two groups:
  • component 1 and component 2 of the pharmaceutical agent 1) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of lxl 0 10 virus particles/1 ml, 47 people.
  • variant 1 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2
  • component 1 and component 2 of the pharmaceutical agent sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of 2x10 10 virus particles/1 ml, 48 people.
  • variant 1 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2
  • the titer of antigen-specific IgG in the first group was assessed on Days 21, 28 and 42 of the study and in the second group - on Days 21 and 28 of the study.
  • the titer of antibodies was determined by means of a test system for ELISA that enables determining the titer of IgG to RBD domain of S antigen of SARS-CoV-2 virus.
  • the plates with pre-adsorbed RBD (100 ng/well) were washed 5 times with a washing buffer. Then, 100 ul of the positive control and 100 ul of the negative control were added to the plate wells in duplicate. A series of two-fold dilutions of the studied samples were added to other wells (two repeats per each sample). The plate was sealed with a film and incubated for an hour at +37 °C while mixing at the rate of 300 rpm. Then the wells were washed with a working solution of the washing buffer.
  • the titer of IgG was determined as maximum serum dilution at which OD450 serum value of an immunized participant exceeds the control serum value (serum of a participant before immunization) more than 2 times.
  • component 1 and component 2 of the pharmaceutical agent 1) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of lxlO 10 virus particles/1 ml, 47 people.
  • variant 1 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2
  • component 1 and component 2 of the pharmaceutical agent sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of 2xl0 10 virus particles/1 ml, 48 people.
  • variant 1 Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2
  • Cell immunity level was determined by assessing the quantity of proliferating CD4+ and CD 8+ lymphocytes of volunteers' peripheral blood in in vitro culture after repeated cell restimulation with recombinant S protein of SARS-CoV-2. Before immunization as well as after 28 days the patients' blood samples were drawn from which mononuclear cells were isolated by centrifugation in the ficoll solution density gradient (1.077 g/mL; PanEco). Then, the isolated cells were colored with the fluorescent dye CFSE (Invivogen, USA) and seeded on wells of a 96-well plate (2*10 5 cells/well).
  • CFSE fluorescent dye
  • the repeated stimulation of lymphocytes was carried out in in vitro conditions by adding coronavirus S protein to the culture medium (final protein concentration 1 pg/ml). Intact cells to which the antigen was not added were used as the negative control. In 72 hours after antigen addition % of proliferating cells was measured and the cultural medium was taken for measuring gamma interferon quantity.
  • proliferating cells were stained with antibodies to marker molecules of T lymphocytes CD3, CD4, CD8 (anti-CD3 Pe-Cy7 (BDBiosciences, SK7 clone), anti-CD4 APC (BDBiosciences, SK3 clone), anti-CD8 PerCP-Cy5.5 (BDBiosciences, SKI clone)).
  • Proliferating (with a lower quantity of cell colorant CFSE) CD4+ and CD8+ T lymphocytes were determined in the cell mixture with the use of the flow cytofluorometer BD FACS Arialll (BD Biosciences, USA). The resultant percentage of proliferating cells in each sample was determined by deducing the result obtained during analysis of intact cells from the result obtained during analysis of cells restimulated with coronavirus S protein antigen.
  • the provided examples confirm that as a result of conducted work a safe and effective agent was created that induced development of reactions of humoral and cell immune response against SARS-CoV-2 virus in children aged 1 month and older.

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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 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.

Description

INDUCTION OF IMMUNITY TO SARS-COV-2 IN CHILDREN
Field of the invention
The invention relates to biotechnology, immunology and virology. The claimed agent can be used for prevention of diseases caused by severe acute respiratory syndrome virus SARS- CoV-2.
Background of the invention
In December 2019 a new coronavirus of zoonotic origin named SARS-CoV-2 spread in Hubei Province, People's Republic of China. The epidemic disease caused by SARS-CoV-2 is called coronavirus disease- 19, abbreviated as COVID-19. The given disease can proceed both in an asymptomatic and mild form and in a severe form that can be accompanied with sepsis and multiple organ system failure. Within several months the disease spread all over the world, affecting more than 200 countries. In January 2020, the World Health Organization declared the SARS-CoV-2-related outbreak to be a public health emergency of international concern and in March it described the spread of the disease as a pandemic. By July 28, 2021, over 195 million cases of illness were confirmed and 4 million people died.
The lasting outbreak of COVID-19 poses an ultimate threat for public health. At present, developing a safe and effective vaccine against SARS-CoV-2 is a most important global priority.
Within the year after the pandemic beginning different pharmaceutical companies offered their variants of a candidate vaccine against COVID-19.
The pharmaceutical company Pfizer, in cooperation with the biotechnology company BioNTech, developed the BNT162b2 vaccine (tozinameran). The given vaccine represents lipid nanoparticles with encapsulated modified mRNA encoding the SARS-CoV-2 S-protein mutant form. At the moment it is allowed to use this vaccine for adults and children aged 12 years and older (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). The pharmaceutical company Modema, in cooperation with the National Institute of Health (USA), developed the mRNA-1273 vaccine. The active component of this vaccine is mRNA encoding SARS-CoV-2 S protein, which is surrounded with a lipid coating. At present, urgent use of this vaccine is allowed for adults aged 18 years and older. Also, Modema carried out a study for children aged 12-17 years, which is now under review. At the moment, a clinical study is under way that is called KidCOVE, in which children aged 6 months - 12 years are immunized (L. A. Jackson etal. 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 cooperation with the pharmaceutical company AstraZeneca, developed the ChAdOxl nCoV-19 vector vaccine (AZD1222). The active component of this vaccine is chimpanzee adenovirus ChAdOxl, including the codon-optimized encoding sequence of full-length S protein of SARS-CoV-2 vims (GenBank MN908947) with the tissue plasminogen activator leader sequence. The vaccination protocol includes double immunization with an interval of 28 days (M. Voysey et al. Safety and efficacy of the ChAdOxl nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. TheLancet. Vol. 397, Issue 10269, P99-111, 2021).
CanSino Company developed a vector vaccine against COVID-19, based on recombinant human adenovirus serotype 5 (Ad5) expressing SARS-CoV-2 full-length S -glycoprotein. At present, the vaccine is intended for urgent use in adults aged 18 years or 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 randomised, double-blind, placebo-controlled, phase 2 trial. The Lancet. Vol. 369, Issue 10249, P479-488, 2020).
Johnson and Johnson and Janssen Pharmaceutical Research groups, in cooperation with Beth Israel Deaconess Medical Center, implemented the technology JanssenAdVac® to create several candidate vaccines. After conducted safety and effectiveness studies the candidate vaccine Ad26.COV2.S was selected (Ad26COVSl). The active component of this vaccine is recombinant adenovirus vector serotype 26 with deletion of El and E3 region, comprising the SARS-CoV-2 S-protein gene, with furin cleavage site mutation and with two proline-stabilizing mutations. The vaccine may be utilized in urgent conditions for adults aged 18 years or older. Currently there are ongoing studies of vaccine utilization in teenagers and children from birth. (J. Sadoff et al. 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; https://www.janssenmd.com/janssen-covidl9-vaccine/special-populations/pediatrics/use-of- janssen-covidl9-vaccine-in-pediatric-participants).
At Beijing Institute of Biological Products Co. they developed an inactivated vaccine against COVID-19. Emergency use of this vaccine is allowed in adults aged 18 years or older. Also, there is published information about a clinical study of this vaccine for children in age groups of 3-6 years, 7-12 years, 13-17 years. Currently, the study is going on (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=l 0).
Thus, at present, only one mRNA-based vaccine against COVID-19 (Pfizer) is approved for use in children. However, doctors observe that the number of young patients hospitalized with COVID-19 diagnosis increases. The mortality among young population increases, too (https://www.paho.org/en/news/5-5-2021-hospitalizations-and-deaths-vounger-people-soar- due-covid- 19-paho-director-reports). which raises the need in vaccines for prevention of COVID-19 in children.
Protective immunity against SARS-CoV-2 coronavirus activates several chains of the immune system. It seems that an effective vaccine against COVID-19 shall induce both humoral and cell immune response. Besides, an important element of protective immunity shall be activation of mucosal immunity (for instance, the one implemented via expression of IgA antibodies) in the nasopharynx, which is the virus penetration gate.
Thus, in the background of the invention there is a need in development of new agents capable of inducing the immune response against SARS-CoV-2 in children, including the mucous membranes of the respiratory tract, which are the main infection gates.
Implementation of the invention
The technical task of the claimed group of inventions is creation of agents for effective induction of immune response (including mucosal immune response) against the SARS-CoV-2 virus in children aged 1 month and older.
The technical result consists in creation of a safe and effective agent enabling development of reactions of humoral and cell immune response against the SARS-CoV-2 virus in children aged 1 month and older.
A child is bom with an immature congenital and adaptive immune system that develops and acquires memory with the child's growth. From human birth till puberty the immune system undergoes several stages in its development.
A newborn's immune system is in suppression. The phagocytosis system is not developed. Neonatal T-cells greatly differ from adult cells, which is a consequence of antenatal life, when the impact of nonshared antigens is significantly limited by maternal alloantigens. Therefore, the very early adaptive T-cell immunity is characterized with tolerogenic reactivity, decreased alloantigen recognition and weak responses to nonshared antigens. Newborns' B-cells greatly differ from an adult's B-cells, too. Neonatal B-cells are known to have decreased TACI, BCMA and BAFF-R expression as well as decreased 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). Together, these peculiarities contribute to depression of humoral immune responses with incomplete switching of the class of immunoglobulins. B-cells of newborns and infants under 2 months demonstrate reduction of somatic hypermutation comparing to adults, which limits affinity maturation of antibodies. Also, stromal cells of bone marrow at early life stages are incapable of supporting long-term survival of plasmablasts and differentiation into plasma cells, so any IgG antibodies produced after immunization quickly decrease, in distinction to children of older age and adults (Pihlgren M, Friedli M, Tougne C, Rochat AF, Lambert PH, Siegrist CA. Reduced ability of neonatal and early-life bone marrow stromal cells to support plasmablast survival. J Immunol. 2006 Jan 1 ; 176(1): 165-72. doi: 10.4049/jimmunol.l76.1.165. PMID: 16365407). As a result, effectiveness of an adaptive immune system as regards early response to T-cell-dependent antigens is much lower in neonatals comparing to children of older age and adults (A.K. Simon, G.A. Hollander, A. McMichael. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci. 2015 Dec 22; 282(1821): 20143085. doi: 10.1098/rspb.2014.3085, PMCID: PMC4707740, PMID: 26702035).
In infancy the immune system matures gradually. The critically important early protection against many infectious diseases the mother recovered from earlier is provided by passive IgG antibodies transferred from the mother transplacentally and with milk. The next development stage is provided by destruction of maternal antibodies. The primary immune response to infection penetration develops via synthesis of Class M immunoglobulins and does not leave immunological memory. Such a type of immune response takes place also in case of vaccination against infectious diseases, and only revaccination forms secondary immune response with production of IgG class antibodies.
With the child grows, his or her contacts with the outer world increase. Gradually there is switching of immune reactions to formation of IgG class antibodies. However, the primary immune response to many antigens remains (IgM synthesis). The local immunity system still remains immature. Gradually the average blood concentration of IgG and IgM increases and reaches the level corresponding to that of adults, however the blood level of IgA still does not reach end values.
The last stage of immune system development is in puberty. At the background of increasing secretion of sex steroids the volume of lymphoid organs decreases. Secretion of sex hormones causes suppression of the cell immunity chain (Shcheplyagina, L.A., Kruglova, I.V. Age peculiarities of immunity in children, Russian Medical Journal No. 23 of 11.11.2009, p. 1564).
Thus, developing an agent for utilization for children for effective induction of immune response against the SARS-CoV-2 virus, including developing reactions of humoral and cell immune response against the SARS-CoV-2 virus, is a complex scientific task.
When a child faces SARS-CoV-2, the virus, first of all, affects the mucous membranes of the respiratory tract. It means that interactions between the virus and the immune system first take place primarily on the mucous membranes of the respiratory tract and the oral cavity. Therefore, induction of mucosal immunity is an important factor influencing protective properties of a pharmaceutical agent.
Based on the level of art one can suggest that administration of an adult-intended vaccine to children will reduce its effectiveness owing to immaturity of the child's immune system. However, the conducted research showed that administration of 1/10th of the adult dose of the developed agent to a child induces humoral immune response comparable to the immune response of an adult. In this case this is an unexpected result. Also, it was demonstrated on young animals that administration of the developed agent induces an increase of the level of IgG antibodies on the mucous membrane of the respiratory tract. Besides, if the intranasal method of agent administration is introduced in the immunization protocol, this leads to secretion of IgG antibodies on the mucous membrane. Thus, as a result of work carried out there were developed agent administration patterns inducing enhanced mucosal response in the respiratory tract.
The given technical result is achieved by what is claimed:
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 site are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
In particular embodiments:
The agent induces mucosal immune response on mucous membranes of the respiratory tract;
The agent is prepared in a liquid or lyophilized form.
Therein, the liquid form of the agent comprises a buffer, wt. %:
Tris 0.1831 ... 0.3432 sodium chloride 0.3313 ... 0.6212 sucrose 3.7821 ... 7.0915 magnesium chloride hexahydrate 0.0154 ... 0.0289 EDTA 0.0029 ... 0.0054 polysorbate-80 0.0378 ... 0.0709 ethanol 95% 0.0004 ... 0.0007 water rest.
Therein, the reduced lyophilized form of the agent comprises a buffer, wt. %:
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 a particular embodiment, a component and/or components of the agent are intended for intranasal and/or intramuscular administration.
In a particular embodiment, the agent is intended for administration with the dose of 5*109- 5*1010 virus particles.
In a particular embodiment, the agent components are intended for sequential administration with an interval of over 1 week or are intended for simultaneous administration.
Therein, the agent components can be in individual packages.
Brief description of figures
FIG. 1 illustrates the percentage of proliferating CD4+ (A) and CD8+ (E) T lymphocytes before immunization (Day 1) and on Day 14 of the study, after immunization of mice with the developed pharmaceutical agent Ad26-CMV-S-CoV2. Dots denote the values for each animal participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbol ** denotes a statistically significant difference between values of Days 1 and 14 (p<0.01, Mann- Whitney test).
Y-axis - number of proliferating cells, %
X-axis - time, days
FIG. 2 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 14 of the study, after immunization of mice with the developed pharmaceutical agent Ad5-CMV-S-CoV2. Dots denote the values for each animal participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbols * (p<0.05) and ** (p<0.01) denote a statistically significant difference between values of Days 1 and 14, Mann- Whitney test.
Y-axis - number of proliferating cells, %
X-axis - time, days
FIG. 3 illustrates titers of IgG antibodies specific to RBD-domain of S ARS-Cov2 virus S protein, before vaccination (Day 1) and on Days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of lxlO10 virus particles. Dots denote the values for each volunteer participating in the study. The mean geometric value of the antibody titer is represented with a black line for each group of data. Deviations denote 95% confidence interval. The statistically significant difference between values on Days 21, 28 and 42 is shown with a bracket above which the value p, Wilcoxon rank sum test, is shown (#### - pO.OOOl). UC means uncertain differences between the given data samples. The statistically significant difference between values of Days 21, 28 and 42 comparing to the values before vaccination (Day 1) was determined by Wilcoxon rank sum test (**** - pO.OOOl).
Y-axis - titer of antigen-specific IgG antibodies;
X-axis - time, days. FIG. 4 illustrates titers of IgG antibodies specific to RBD-domain of S ARS-Cov2 virus S protein, before vaccination (Day 1) and on Days 21, 28 and 42 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of 2xl010 virus particles. Dots denote the values for each volunteer participating in the study. The mean geometric value of the antibody titer is represented with a black line for each group of data. Deviations denote 95% confidence interval. The statistically significant difference between values on Days 21, 28 and 42 is shown with a bracket above which the value p, Wilcoxon rank sum test, is shown (#### - pO.OOOl). UC means uncertain differences between the given data samples. The statistically significant difference between values of Days 21, 28 and 42 comparing to the values before vaccination (Day 1) was determined by Wilcoxon rank sum test (**** - p O.OOOl).
Y-axis - titer of antigen-specific IgG antibodies;
X-axis - time, days.
FIG. 5 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 28 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of lxlO10 virus particles. Dots denote the values for each volunteer participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbol **** denotes the statistically significant difference between values of Days 1 and 28 (p<0.0001, according to Wilcoxon rank sum test).
Y-axis - number of proliferating cells, %
X-axis - time, days
FIG. 6 illustrates the percentage of proliferating CD4+ (A) and CD8+ (B) T lymphocytes before immunization (Day 1) and on Day 28 of the study, after immunization of volunteers with the developed pharmaceutical agent with the dose of 2x1010 virus particles. Dots denote the values for each volunteer participating in the study. The median value is shown with a black line for each group of data. Deviations denote 95% confidence interval. The symbol **** denotes the statistically significant difference between values of Days 1 and 28 (pO.0001, according to Wilcoxon rank sum test).
Y-axis - number of proliferating cells, %
X-axis - time, days
Embodiment of the invention
The first stage in the development of an immunobiological agent against the severe acute respiratory syndrome virus 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 is Type 1 transmembrane glycoprotein responsible for virus particles binding, fusion and entry into the 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 reactions against SARS-CoV-2 S protein , the authors developed multiple variants of expression cassettes.
The expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal. The CMV promoter is the promoter of early cytomegalovirus genes that enables constitutive expression in a multitude of cell types. However, the power of target gene expression managed by the CMV promoter varies depending on the cell type. Moreover, the transgene expression level under control of the CMV promoter was shown to decrease with increase of cell cultivation time due to gene expression suppression, which is 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].
The expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal. The CAG-promoter is the synthetic promoter that includes the early enhancer of the CMV promoter, chicken b-actin promoter and chimeric intron (chicken b-actin and rabbit b-globin). It was experimentally shown that the transcription 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. Evaluation of three different promoters driving gene expression in developing chicken embryo by using in vivo electroporation. // Genet. Mol. Res. - 2014. - Vol. 13. - P. 1270- 1277]
The expression cassette SEQ ID NO:3 consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal. The EF1 -promoter is the promoter of human eukaryotic translation elongation factor 1b (EF-la). The promoter is constitutively active within a wide range of cell types [PMID: 28557288. The EF-la promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells]. The EF-la gene encodes the elongation factor- la, which is one of the most widely spread proteins in eukaryotic cells and is expressed in all types of mammalian cells. This EF-la promoter is often active in the cells in which virus promoters are incapable of expressing controlled genes and in cells in which virus promoters are gradually suppressed.
The expression cassette SEQ ID NO:4 consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal.
For effective delivery of SARS-CoV-2 coronavirus S protein gene into the human organism a vector system based on adenoviridae was selected. Adenovirus vectors have a whole range of advantages: they are incapable of proliferating in human cells, they penetrate both in proliferating and non-proliferating cells, they are capable of inducing cell and humoral immune response, they ensure a high level of target antigen expression.
The authors developed variants of the agent comprising two components as well as variants comprising one component based on adenoviridae of different serotypes. Thus, the immune response to the adenovirus vector part, which can arise after administration of the first component of the agent or single-component agent is not boostered further on and does not influence generation of antigen-specific immune responses against the vaccine antigen in case of utilization of the two-component agent or in case of the need in repeated administration of the single-component agent as in the latter case an agent based on another adenovirus can be administered.
Besides, the developed agents widen the range of agents for induction of immune response against the SARS-CoV-2 coronavirus, which will enable overcoming of difficulties related to the problem of presence of pre-immunity to some serotypes of adenoviridae in a part of population.
Thus, as a result of work carried out the following technical solutions were developed.
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 site are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites are deleted, while ORF6-Ad26 site is replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
In particular embodiments:
The agent induces mucosal immune response on mucous membranes of the respiratory tract;
The agent is prepared in a liquid or lyophilized form.
Therein, the liquid form of the agent comprises a buffer, wt. %:
Tris 0.1831 ... 0.3432 sodium chloride 0.3313 ... 0.6212 sucrose 3.7821 ... 7.0915 magnesium chloride hexahydrate 0.0154 ... 0.0289
EDTA 0.0029 ... 0.0054 polysorbate-80 0.0378 ... 0.0709 ethanol 95% 0.0004 ... 0.0007 water rest.
Therein, the reduced lyophilized form of the agent comprises a buffer, wt. %:
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 a particular embodiment, a component and/or components of the agent are intended for intranasal and/or intramuscular administration.
In a particular embodiment, the agent is intended for administration with the dose of 5*109- 5*1010 virus particles.
In a particular embodiment, the agent components are intended for sequential administration with an interval of over 1 week or are intended for simultaneous administration.
Therein, the agent components can be in individual packages.
Moreover, the authors developed buffer variants that enable storing both in a frozen form at temperature below -18°C and in the form of a lyophilisate at temperature from +2°C to +8°C.
Also, they developed utilization of the agent for induction of specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older by administering it in the organism in an effective quantity. The developed agent was shown to induce mucosal immune response on mucous membranes of the respiratory tract.
Moreover, the agent, also consisting of one component, can be used once.
Revaccination can be performed with any of the claimed agents irrespective of the agent used for vaccination.
Embodiment of the invention is supported with the following examples.
Example 1
Obtaining of an expression vector comprising the genome of recombinant human adenovirus serotype 26.
At the first stage of the work the design of the plasmid construct pAd26-Ends comprising two sites homologous to the genome of human adenovirus serotype 26 (two homology arms) and the gene of resistance to ampicillin. One homology arm is the beginning of the genome of human adenovirus serotype 26 (from the left inverted terminal repeat to El site) and the sequence of the virus genome comprising pIX protein. The second homology arm comprises the nucleotide sequence after ORF3 E4 site to the genome end. The pAd26-Ends construct was synthesized by CJSC "Evrogen" (Moscow).
Human adenovirus serotype 26 DNA isolated from virions was mixed with pAd26- Ends. As a result of homologous recombination between pAd26-Ends and virus DNA the pAd26-dlEl plasmid was obtained that comprised the genome of human adenovirus serotype 26 with deleted El site.
Then, in the obtained pAd26-dlEl plasmid with the use of standard cloning methods the sequence comprising open reading frame 6 (ORF6-Ad26) was replaced with the similar sequence from the genome of human adenovirus serotype 5 so that human adenovirus serotype 26 would be capable of effective propagation in HEK293 cell culture. As a result, the pAd26-dlEl-ORF6-Ad5 plasmid was obtained.
Then, with the use of standard genetic engineering methods in the constructed plasmid pAd26-dlEl-ORF6-Ad5 E3 site of the adenovirus genome (approximately 3321 b.p. between genes pVIII and U-exon) was deleted to increase vector packing capacity. As a result, the recombinant vector pAd26-only-null based on the genome of human adenovirus serotype 26 with open reading frame ORF6 of human adenovirus serotype 5 and with deletion of El and E3 sites was obtained.
Moreover, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NOG consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
On the basis of the plasmid construct pAd26-Ends, using the genetic engineering method, constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26-EFl-S- CoV2 were obtained that comprised expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively, as well as homology arms of adenovirus serotype 26 genome. Afterwards, the constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, pArms-26- EFl-S-CoV2 were linearized by the unique site of hydrolysis between homology arms, each plasmid was mixed with the recombinant vector pAd26-only-null. As a result of homologous recombination there were obtained the pAd26-only-CMV-S-CoV2, pAd26- only-CAG-S-CoV2, pAd26-only-EFl-S-CoV2 plasmids comprising the genome of recombinant human adenovirus serotype 26 with the open reading frame ORF6 of human adenovirus serotype 5 and with deletion of El and E3 sites, with the expression cassette SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively.
At the fourth stage, the pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, pAd26-only-EFl-S-CoV2 plasmids were hydrolyzed with specific restriction endonucleases to delete the vector part. The obtained DNA preparations were used to transfect HEK293 culture cells.
Thus, the expression vector was obtained that comprised the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 region was replaced with ORF6-Ad5 with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NOG, SEQ ID NOG. Example 2
Obtaining of an agent in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.
At the given stage of work the expression vectors obtained in example 1 were purified with the method of anion-exchange and exclusion chromatography. The ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
Thus, there were obtained the following immunobiological agents based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5:
1. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad26- CMV-S-CoV2) in a buffer for the liquid form of the agent.
2. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad26- CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
3. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad26- CAG-S-CoV2) in a buffer for the liquid form of the agent.
4. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad26- CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
5. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:3 (Ad26- EFl-S-CoV2) in a buffer for the liquid form of the agent.
6. The immunobiological agent based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:3 (Ad26- EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
Each of the given immunobiological agents is component 1 in variant 1 and in variant 2 of the developed agent.
Example 3
Obtaining of an expression vector comprising the genome of recombinant simian adenovirus serotype 25.
At the first stage of work there was developed the design of the pSim25-Ends plasmid construct comprising two regions homologous to the genome of simian adenovirus serotype 25 (two homology arms). One homology arm is the beginning of the genome of simian adenovirus serotype 25 (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 comprises the sequence of the end of adenovirus genome, including the right inverted terminal repeat. The pSim25-Ends construct was synthesized by CJSC "Evrogen" (Moscow).
Simian adenovirus serotype 25 DNA isolated from virions was mixed with pSim25- Ends. As a result of homologous recombination between pSim25-Ends and virus DNA the pSim25-dlEl plasmid was obtained that comprised the genome of simian adenovirus serotype 25 with deleted El site. Then, with the use of standard genetic engineering methods in the constructed pSim25-dlEl plasmid E3 site of the adenovirus genome (3921 b.p. from the beginning of gene 12,5K to gene 14,7K) was deleted to increase the vector packing capacity. As a result the pSim25-null plasmid construct was obtained that encoded the full genome of simian adenovirus serotype 25 with deletion of El and E3 sites.
Moreover, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO:4 consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NOG consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
Then, using the genetic engineering method, on the basis of the plasmid construct pSim25-Ends, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, pArms-Sim25-EFl-S-CoV2 were obtained that comprised expression cassettes SEQ ID NO:4, SEQ ID NOG or SEQ ID NOG, respectively, as well as homology arms of simian adenovirus serotype 25. Afterwards, the constructs pArms-Sim25-CMV-S-CoV2, pArms- Sim25-CAG-S-CoV2, pArms-Sim25-EFl-S-CoV2 were linearized by the unique site of hydrolysis between homology arms, each plasmid was mixed with the recombinant vector pSim25-null. As a result of homologous recombination recombinant plasmid vectors pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EFl-S-CoV2 were obtained that comprised the full genome of simian adenovirus serotype 25 with deletion of El and E3 sites and the expression cassette SEQ ID NOG, SEQ ID NOG or SEQ ID NOG, respectively.
At the third stage the pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EFl- S-CoV2 plasmids were hydrolyzed with the specific restriction endonuclease to delete the vector part. The obtained DNA preparations were used to transfect HEK293 culture cells. The obtained material was used to accumulate preparatory quantities of recombinant adenoviridae. As a result, there were obtained recombinant human adenoviridae serotype 25 comprising the gene of SARS-CoV-2 virus S protein: simAd25-CMV-S-CoV2 (comprising the expression cassette SEQ ID NO:4), simAd25-CAG-S-CoV2 (comprising the expression cassette SEQ ID NO:2), simAd25-EFl-S-CoV2 (comprising the expression cassette SEQ ID NO:3).
Thus, the expression vector was obtained that comprised the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3.
Example 4
Obtaining of an agent in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3.
At the given stage of work the expression vectors obtained in example 3 were purified with the method of anion-exchange and exclusion chromatography. The ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
Thus, there were obtained the following immunobiological agents based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites:
1. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (simAd25-CMV-S-CoV2) in a buffer for the liquid form of the agent.
2. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (Ad26-CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
3. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:4 (simAd25-CAG-S-CoV2) in a buffer for the liquid form of the agent.
4. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (simAd25-CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
5. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in a buffer for the liquid form of the agent.
6. The immunobiological agent based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (simAd25-EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
Each of the given immunobiological agents is component 2 in variant 1 of the developed agent and component 1 in variant 3 of the developed agent.
Example 5
Obtaining of an expression vector comprising the genome of recombinant human adenovirus serotype 5.
At the first stage of work there was developed the design of the pAd5-Ends plasmid construct comprising two regions homologous to the genome of human adenovirus serotype 5 (two homology arms). One homology arm is the beginning of the genome of human adenovirus serotype 5 (from the left inverted terminal repeat to El site) and the sequence of the virus genome comprising pIX protein. The second homology arm comprises the nucleotide sequence after ORF3 E4 site to the genome end. The pAd5-Ends construct was synthesized by CJSC "Evrogen" (Moscow). Human adenovirus serotype 5 DNA isolated from virions was mixed with pAd5- Ends. As a result of homologous recombination between pAd5-Ends and virus DNA the pAd5-dlEl plasmid was obtained that comprised the genome of human adenovirus serotype 5 with deleted El site.
Then, with the use of standard genetic engineering methods in the constructed pAd5- dlEl plasmid E3 site of the adenovirus genome (2685 b.p. from the end of gene 12,5K to the beginning of sequence U-exon) was deleted to increase the vector packing capacity. As a result, there was obtained the recombinant plasmid vector pAd5-too-null based on the genome of human adenovirus serotype 5 with deletion of El and E3 sites of the genome. Moreover, the authors developed several expression cassette designs:
- the expression cassette SEQ ID NO:l consists of the CMV promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NO:2 consists of the CAG promoter, gene of SARS-CoV-2 virus S protein and polyadenylation signal;
- the expression cassette SEQ ID NO:3 consists of the EF1 promoter, gene of SARS- CoV-2 virus S protein and polyadenylation signal.
Then, using the genetic engineering method, on the basis of the plasmid construct pAd5-Ends, constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms- Ad5-EFl-S-CoV2 were obtained that comprised expression cassettes SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NOG, respectively, as well as homology arms of the genome of adenovirus serotype 5.
Afterwards, the constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, pArms-Ad5-EFl-S-CoV2 were linearized by the unique site of hydrolysis between homology arms, each plasmid was mixed with the recombinant vector pAd5-too-null. As a result of homologous recombination, there were obtained pAd5-too-CMV-S-CoV2, pAd5- too-GAC-S-CoV2, pAd5-too-EFl-S-CoV2 plasmids comprising the genome of recombinant human adenovirus serotype 5 with deletion of El and E3 sites and expression cassettes SEQ ID NO:l, SEQ ID NOG or SEQ ID NOG, respectively.
At the fourth stage the pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5- too-EFl-S-CoV2 plasmids were hydrolyzed with the specific restriction endonuclease to delete the vector part. The obtained DNA preparation was used to transfect HEK293 culture cells. The obtained material was used to accumulate preparatory quantities of the recombinant adenovirus.
As a result, there were obtained recombinant human adenoviridae serotype 5 comprising the gene of S SARS-CoV-2 virus S protein: Ad5-CMV-S-CoV2 (comprising the expression cassette SEQ ID NO:l), Ad5-CAG-S-CoV2 (comprising the expression cassette SEQ ID NO:2), Ad5-EFl-S-CoV2 (comprising the expression cassette SEQ ID NO:3).
Thus, the expression vector was obtained that comprised the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.
Example 6
Obtaining of an agent in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3.
At the given stage of work the expression vectors obtained in example 5 were purified with the method of anion-exchange and exclusion chromatography. The ready suspension comprised adenovirus particles in a buffer for the liquid form of the agent or in a buffer for lyophilized form of the agent.
Thus, there were obtained the following immunobiological agents based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites:
1. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in a buffer for the liquid form of the agent.
2. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:l (Ad5-CMV-S-CoV2) in a buffer for the lyophilized form of the agent.
3. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in a buffer for the liquid form of the agent.
4. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the CAG promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:2 (Ad5-CAG-S-CoV2) in a buffer for the lyophilized form of the agent.
5. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in a buffer for the liquid form of the agent.
6. The immunobiological agent based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with the expression cassette comprising the EF1 promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NOG (Ad5-EFl-S-CoV2) in a buffer for the lyophilized form of the agent.
Each of the given immunobiological agents is component 2 in variant 1 and in variant 3 of the developed agent.
Example 7
Obtaining of a buffer.
Each component of the developed agent is an agent based on the recombinant adenovirus with an expression cassette in a buffer. The authors of the invention developed a buffer composition that ensured stability of recombinant adenovirus particles. The given solution comprises:
1. Tris(hydroxymethyl)aminomethane (Tris), which is required to maintain pH of the solution.
2. Sodium chloride, which is added to obtain the required ionic strength and osmolarity.
3. Sucrose, which is used as a cryoprotector.
4. Magnesium chloride hexahydrate, which is required as a source of divalent cations.
5. EDTA, which is used as a free-radical oxidation inhibitor.
6. Polysorbate-80, which is used as a surfactant.
7. Ethanol 95%, which is used as a free-radical oxidation inhibitor.
8. Water, which is used as a solvent.
The authors of the invention developed 2 buffer variants: for the liquid form of the agent and for the lyophilized form of the pharmaceutical agent.
To determine the concentration of substances included in the buffer for the liquid form of the agent, several variants of experimental groups were obtained (Table 1). One of the components of the agent was added to each of the obtained buffers:
1. The immunobiological agent based on the recombinant human adenovirus serotype 26 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, l* 1010virus particles.
2. The immunobiological agent based on the recombinant human adenovirus serotype 5 with the expression cassette comprising the CMV promoter, the gene of SARS- CoV-2 virus S protein and polyadenylation signal, l* 1010virus particles.
3. The immunobiological agent based on the recombinant simian adenovirus serotype 25 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, 1*1010 virus particles. Thus, stability of each of the serotypes of adenoviridae included in the agent was tested. The obtained agents were stored at the temperature of -18°C and -70°C during 3 months, then defrosted and the change of the titer of the recombinant adenoviridae was analyzed.
TABLE 1 - Composition Of Experimental Buffers For The Liquid Form Of The Agent.
TABLE 1
The results of the carried out experiment demonstrated that the titer of recombinant adenoviridae after their storage in the buffer for the liquid form of the agent at temperatures -18°C and -70 °C during 3 months did not change.
Thus, the developed buffer for the liquid form of the agent ensures stability of all components of the developed agent in the following range of active ingredients (wt. %):
Tris: 0.1831 wt. % ... 0.3432 wt. %;
Sodium chloride: 0.3313 wt. % ... 0.6212 wt. %;
Sucrose: 3.7821 wt. % ... 7.0915 wt. %; Magnesium chloride hexahydrate: 0.0154 wt. % ... 0.0289 wt. %;
EDTA: 0.0029 wt. % ... 0.0054 wt. %;
Polysorbate-80: 0.0378 wt. % ... 0.0709 wt. %;
Ethanol 95%: 0.0004 wt. % ... 0.0007 wt. %;
Solvent: the rest.
To determine the concentration of substances included in the buffer for the lyophilized form of the agent, several variants of experimental groups were obtained (table 2). One of the components of the agent was added to each of the obtained buffers:
1. The immunobiological agent based on the recombinant human adenovirus serotype 26 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, 1*1010 virus particles.
2. The immunobiological agent based on the recombinant human adenovirus serotype 5 with the expression cassette comprising the CMV promoter, the gene of SARS- CoV-2 virus S protein and polyadenylation signal, 1*1010 virus particles.
3. The immunobiological agent based on the recombinant simian adenovirus serotype 25 with the expression cassette comprising the CMV promoter, the gene of SARS-CoV-2 virus S protein and polyadenylation signal, 1*1010 virus particles.
Thus, stability of each of the serotypes of adenoviridae included in the agent was tested. The obtained agents were stored at the temperature of +2 and +8 °C during 3 months, then defrosted and the change of the titer of the recombinant adenoviridae was analyzed. Table 2 - Composition of Experimental Buffers.
TABLE 2
The results of the carried out experiment demonstrated that the titer of recombinant adenoviridae after their storage in the buffer for the lyophilized form of the agent at temperature +2°C and +8 °C during 3 months did not change.
Thus, the developed buffer for the lyophilized form of the agent ensures stability of all components of the developed agent in the following range of active ingredients:
Tris: 0.0180 wt. % ... 0.0338 wt. %;
Sodium chloride: 0.1044 wt. % ... 0.1957 wt. %;
Sucrose: 5.4688 wt. % ... 10.2539 wt. %;
Magnesium chloride hexahydrate: 0.0015 wt. % ... 0.0028 wt. %;
EDTA: 0.0003 wt. % ... 0.0005 wt. %;
Polysorbate-80: 0.0037 wt. % ... 0.0070 wt. %;
Solvent: the rest.
Example 8
Assessing of the ability of the developed agent to induce mucosal immune response upon intramuscular administration.
For this study young mice of BALB/c line (aged 21-28 days) were used. The animals were distributed by groups of 10 and the following agents were administered to them: 1) Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 5xl09 v.p./100 ul; in 21 days Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 5x109 v.p./100 ul.
2) Ad5-CMV-S-CoV2, i/m, dose 5xl09v.p./100ul; in 21 days Ad5-CMV-S-CoV2, i/m, dose 5xl09 v.p./100ul.
3) simAd25-CMV-S-CoV2, i/m, dose 5xl09v.p./100ul; in 21 days simAd25-CMV- S-CoV2, i/m, dose 5xl09 v.p./100ul
4) Ad26-null, i/m, dose 5xl09v.p./100ul; in 21 days Ad26-null, i/m, dose 5xl09 v.p./lOOul
5) Ad5-null, i/m, dose 5xl09v.p./100ul; in 21 days Ad5-null, i/m, dose 5x109 v.p./100ul
6) simAd25-null, i/m, dose 5xl09v.p./100ul; in 21 days simAd25-null, i/m, dose 5xl09 v.p./100ul
7) Ad26-CMV-S-CoV2, intranasally (i/n), dose 5xl09 v.p./100ul; in 21 days Ad26-CMV-S-CoV2 i/n, dose 5xl09 v.p./100ul.
8) Ad5-CMV-S-CoV2, i/n, dose 5x109 v.p./100ul; in 21 days Ad5-CMV-S-CoV2, i/n, dose 5xl09v.p./100ul.
9) Ad26-CMV-S-CoV2, i/m, dose 5xl09v.p./100ul; in 21 days Ad26-CMV-S- CoV2 i/n, dose 5xl09v.p./100ul.
10) Ad5-CMV-S-CoV2, i/m, dose 5xl0Vp./100ul; in 21 days Ad5-CMV-S-CoV2, i/n, dose 5xl09v.p./100ul.
11) Buffer
After 14 days after the latest immunization the titer of IgG and IgA antibodies was determined by the ELISA method in bronchoalveolar lavages (BAL) of experimental animals.
To do that:
1) The antigen (recombinant RBD) was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C. 2) The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
3) BAL samples were diluted 25 times and then by the method of 2-fold dilutions.
4) 50 ul of each diluted serum sample were added to the plate wells.
5) Then, the samples were incubated for an hour at 37°C.
6) The incubation was followed by three-fold washing of the wells with a phosphate buffer.
7) The secondary antibodies to mouse immunoglobulins conjugated with horseradish peroxidase were added.
8) Then, the samples were incubated for an hour at 37°C.
9) The incubation was followed by three-fold washing of the wells with a phosphate buffer.
10) Then tetramethylbenzidine (TMB) was added, which is a horseradish peroxidase substrate and as a result of the reaction converts to a colored compound. The reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
The titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 3.
Table 3. Mean Geometrical Value Of The Titer Of Igg And Iga Antibodies To SARS-Cov-2 Virus S Protein In BAL Lavages Of Experimental Animals
TABLE 3
According to the given results, all variants of the developed agent cause an increase of the titer of IgG antibodies on the surface of mucous membrane of the respiratory tract. Expression of IgA antibodies depends on the agent administration method. Maximum titers of IgA antibodies are induced when the animals are immunized sequentially: intramuscularly and then intranasally.
Thus, as a result of the work carried out an immunobiological agent was developed that is capable of inducing mucosal immune response against SARS-CoV-2 virus on the mucous membrane of the respiratory tract in children as well as a pattern of administration of this agent leading to potentiation of the mucosal immune response.
Example 9
Assessing of the ability of the developed agent to induce immune response in mammals of different age.
For this study young mice of BALB/c of different age were used. The animals were distributed into groups of 5:
1) Mice aged 15-18 days
2) Mice aged 28 -35 days
3) Mice aged 50-60 days
All the animals were immunized once with the developed immunobiological agent based on human adenovirus serotype 5 (Ad5-CMV-S-CoV2) with the dose of 108 v.p./50 ul. On Day 21 after immunization the titer of IgG antibodies in the animal blood serum was measured. To do that:
1) The antigen (recombinant RBD) was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
2) The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
3) The serum samples were diluted 50 times and then by the method of 2-fold dilutions.
4) 50 ul of each diluted serum sample were added to the plate wells.
5) Then, the samples were incubated for an hour at 37°C.
6) The incubation was followed by three-fold washing of the wells with a phosphate buffer.
7) The secondary antibodies to mouse immunoglobulins conjugated with horseradish peroxidase were added.
8) Then, the samples were incubated for an hour at 37°C. 9) The incubation was followed by three-fold washing of the wells with a phosphate buffer.
10) Then tetramethylbenzidine (TMB) was added, which is a horseradish peroxidase substrate and as a result of the reaction converts to a colored compound. The reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
The titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 4.
Table 4. The Mean Geometrical Value Of The Titer Of Igg Antibodies To SARS- Cov-2 Virus S Protein In The Blood Serum Of Experimental Animals Depending On Their Age.
TABLE 4
Thus, as it is shown from the provided data, the developed immunobiological agent induces development of humoral immune response both in adult and young animals of different age. This makes it possible to suggest that the agent will be effective in utilization for people of different age categories.
Example 10
Study of immunogenicity of the developed agent on young mice for assessment of cell immune response after single vaccination.
Young BALB/c mice (aged 21-28 days) were used for the immunogenicity study. The animals were distributed into groups of 5 to whom the following agents were administered once: 1. Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 1010vp/100 ul;
2. Ad5-CMV-S-CoV2, intramuscularly (i/m), dose 1010 vp/100 ul
Cell immunity level was determined by assessing the quantity of proliferating CD4+ and CD8+ lymphocytes isolated from mouse spleen in the culture in vitro after repeated cell restimulation with recombinant S protein of SARS-CoV-2. Before immunization as well as after 14 days animals' spleens were harvested from which mononuclear cells were isolated by centrifugation in the ficoll solution density gradient (1.09 g/mL; PanEco). Then, the isolated cells were colored with the fluorescent dye CFSE (Invivogen, USA) and seeded on wells of a 96-well plate (2*105 cells/well). The repeated stimulation of lymphocytes was carried out in in vitro conditions by adding coronavirus S protein to the culture medium (final protein concentration 1 pg/ml). Intact cells to which the antigen was not added were used as the negative control.
To determine % of proliferating cells they were stained with antibodies to marker molecules of T lymphocytes CD3, CD4, CD8 (BDBioscienses, USA). Proliferating (with a lower quantity of cell colorant CFSE) CD4+ and CD8+ T lymphocytes were determined in the cell mixture with the use of the flow cytofluorometer BD FACS Arialll (BD Biosciences, USA). The resultant percentage of proliferating cells in each sample was determined by deducing the result obtained during analysis of intact cells from the result obtained during analysis of cells restimulated with coronavirus S protein antigen.
The results (with conducted statistical processing) of determination of the percentage of proliferating CD4+ and CD8+ T lymphocytes on Day 1 (before immunization) and on Day 14 of the study are given in Fig. 1 (Ad26-CMV-S-CoV2) and Fig. 2 (Ad5-CMV-S-CoV2).
The obtained data showed that single immunization of mice with the developed agent makes it possible to reliably increase the percentage of proliferating CD4+ and CD8+ T lymphocytes after antigen restimulation on Day 14 after immunization.
Thus, one can conclude that single immunization of mice with the developed immunobiological agent is capable of causing formation of high-level post- vaccination cell immunity. Example 11
Study of immunogenicity of the developed agent on young mice with different administration methods.
Young B ALB/c mice (aged 21-28 days) were used for the immunogenicity study. The animals were distributed by groups of 5 and the following agents were administered to them:
3. Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 109vp/100 ul
4. Ad26-CAG-S-CoV2, intramuscularly, dose 109vp/100 ul
5. Ad26-EFl-S-CoV2, intramuscularly, dose 10yvp/100 ul
6. Ad5-CMV-S-CoV2, intramuscularly, dose 109vp/100 ul
7. Ad5-CAG-S-CoV2, intramuscularly, dose 109vp/100 ul
8. Ad5-EFl-S-CoV2, intramuscularly, dose 109vp/100 ul
9. simAd25-CMV-S-CoV2, intramuscularly, dose 109vp/100 ul
10. simAd25-CAG-S-CoV2, intramuscularly, dose 109vp/100 ul
11. simAd25-EFl-S-CoV2, intramuscularly, dose 109vp/100 ul
12. Ad26-CMV-S-CoV2,intranasally (i/n),dose 109vp/100 ul
13. Ad26-CAG-S-CoV2,intranasally (i/n),dose 109vp/100 ul
14. Ad26-EFl-S-CoV2,intranasally (i/n),dose 109vp/100 ul
15. Ad5-CMV-S-CoV2,intranasally (i/n),dose 109vp/100 ul
16. Ad5-CAG-S-CoV2,intranasally (i/n),dose 109vp/100 ul
17. Ad5-EFl-S-CoV2,intranasally (i/n),dose 109vp/100 ul
18. simAd25-CMV-S-CoV2,intranasally (i/n),dose 109vp/100 ul
19. simAd25-CAG-S-CoV2,intranasally (i/n),dose 109vp/100 ul
20. simAd25-EFl-S-CoV2,intranasally (i/n),dose 109vp/100 ul In 21 days the animals' blood was drawn, blood serum was isolated and the titer of IgG antibodies to S protein of SARS-CoV-2 coronavirus was determined by the ELISA method. To do that:
1. The antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
2. The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
3. The serum samples of immunized mice were diluted by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
4. 50 ul of each diluted serum sample were added to the plate wells.
5. Then, the samples were incubated for an hour at 37°C.
6. The incubation was followed by three-fold washing of the wells with a phosphate buffer.
7. The secondary antibodies to mouse immunoglobulins conjugated with horseradish peroxidase were added.
8. Then, the samples were incubated for an hour at 37°C.
9. The incubation was followed by three-fold washing of the wells with a phosphate buffer.
10. Then tetramethylbenzidine (TMB) was added, which is a horseradish peroxidase substrate and as a result of the reaction converts to a colored compound. The reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
The titer of IgG antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 5. Table 5- Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean Geometrical Value Of The Titer Of Antibodies)
TABLE 5
Based on the given data, single immunization with the developed agent induces humoral immune response to SARS-CoV-2 glycoprotein.
Example 12
Study of immunogenicity of the developed agent upon administration to young animals in different doses.
The aim of the given study was assessment of humoral immune response to S protein of SARS-CoV-2 with administration of the developed agent in different doses to young animals.
Young mice of C57/B16 line (3-4 weeks) were used to study immunogenicity of the developed agent. The animals were distributed by groups of 5 to whom the following agents were administered intramuscularly, twice, with an interval of 28 days:
1) Ad5-CMV-S-CoV2, 5*109vp/100 ul,
2) Ad5-CMV-S-CoV2, 10lovp/100 ul,
3) Ad5-CMV-S-CoV2, 5*1010vp/100 ul. In 21 days the animals' blood was drawn, blood serum was isolated and the titer of IgG antibodies to S protein of SARS-CoV-2 coronavirus was determined by the ELISA method. To do that:
1. The antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
2. The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
3. The serum samples of immunized mice were diluted 100 times and then by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
4. 50 ul of each diluted serum sample were added to the plate wells.
5. Then, the samples were incubated for an hour at 37°C.
6. The incubation was followed by three-fold washing of the wells with a phosphate buffer.
7. The secondary antibodies to mouse immunoglobulins conjugated with horseradish peroxidase were added.
8. Then, the samples were incubated for an hour at 37°C.
9. The incubation was followed by three-fold washing of the wells with a phosphate buffer.
10. Then tetramethylbenzidine (TMB) was added, which is a horseradish peroxidase substrate and as a result of the reaction converts to a colored compound. The reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
The titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 6. Table 6- Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean Geometrical Value Of The Titer Of Antibodies)
TABLE 6
Based on the given data, the developed agent demonstrates immunogenicity within the whole range of selected doses.
Example 13
Study of immunogenicity of the developed agent on young mice with different administration protocols.
Young mice of C57/B16 line (3-4 weeks) were used to study immunogenicity of the developed agent. The animals were distributed by groups of 5 and the following agents were administered to them:
1) Ad26-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeksAd26-CMV-S-CoV2i/m, 109vp/100 ul;
2) Ad26-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeksAd5-CMV-S-CoV2i/m, 109vp/100 ul;
3) Ad26-CMV-S-CoV2, i/n, 109vp/100 ul; in 3 weeksAd5-CMV-S-CoV2i/m, 109vp/100 ul;
4) Ad26-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeks simAd5-CMV-S-CoV2i/m, 109vp/100 ul; 5) Ad5-CMV-S-CoV2, i/m, 109vp/100 uj; jn 3 WeeksAd5-CMV-S-CoV2 i/m,
108vp/100 ul;
6) Ad5-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeksAd26-CMV-S-CoV2i/m, 109vp/100 ul;
7) Ad5-CMV-S-CoV2, i/n, 109vp/100 ul; in 3 weeksAd26-CMV-S-CoV2i/m,
109vp/100 ul;
8) Ad5-CMV-S-CoV2, i/m, 109vp/100 ul;in 3 weekssimAd5-CMV-S-CoV2i/m, 109vp/100 ul;
9) simAd25-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weekssimAd5-CMV-S-CoV2i/m, 109vp/100 ul;
10) simAd25-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeks Ad5-CMV-S-CoV2 i/m, 109vp/100 ul;
11) simAd25-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeks Ad26-CMV-S-CoV2, i/m, 109vp/100 ul.
In 21 days the animals' blood was drawn, blood serum was isolated and the titer of IgG antibodies to S protein of SARS-CoV-2 coronavirus was determined by the ELISA method. To do that:
1. The antigen was adsorbed on the wells of a 96-well plate for ELISA during 16 hours at +4°C.
2. The, to get rid of non-specific binding, 5 % milk in TPBS 100 ul/well was added to the plate wells. It was incubated on a shaker at 37°C for an hour.
3. The serum samples of immunized mice were diluted by the method of 2-fold dilutions. In total, 12 dilutions of each sample were prepared.
4. 50 ul of each diluted serum sample were added to the plate wells.
5. Then, the samples were incubated for an hour at 37°C.
6. The incubation was followed by three-fold washing of the wells with a phosphate buffer. 7. The secondary antibodies to mouse immunoglobulins conjugated with horseradish peroxidase were added.
8. Then, the samples were incubated for an hour at 37°C.
9. The incubation was followed by three-fold washing of the wells with a phosphate buffer.
10. Then tetramethylbenzidine (TMB) was added, which is a horseradish peroxidase substrate and as a result of the reaction converts to a colored compound.
11. The reaction was stopped in 15 minutes by adding sulfuric acid. Then, using a spectrophotometer, the optical density (OD) of the solution in each well was measured at the wavelength of 450 nm.
The titer of antibodies was determined as the latest dilution in which the optical density of the solution was reliably higher than in the negative control group. The obtained results (mean geometrical value) are given in Table 7.
Table 7 - Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean Geometrical Value Of The Titer Of Antibodies)
TABLE 7
The obtained results demonstrate that the developed agent provides for development of humoral immune response against SARS-CoV-2 in young animals with different administration protocols.
Example 14
Study of utilization of the developed agent for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 for children.
100 volunteers aged 12-17 years participated in the given study. The suggested immunization protocol included sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent according to variant 1 (Ad26-CMV-S-CoV2, Ad5- CMV-S-CoV2). One volunteer discontinued participation before administration of component 1 because of newly diagnosed hypertension, correspondingly, 99 volunteers started the therapy under study. Component 2 was not administered to: 1 person - because of non-attendance, 1 person - because of an adverse event (leukopenia), 1 person - because of an intestinal infection of unknown etiology (of enteroviral type), 1 person - because of hospitalization with a purulent furunculus in 5 days after administration of the component, 1 person - because of hospitalization (an intestinal infection) and 1 person - because of COVID, 2 people - refused to participate. Correspondingly, both vaccine components were received by 91 volunteers.
205 AEs that developed in 73 volunteers (73.0%) after vaccine administration were recorded. Table 8 includes the number (share) of volunteers with presence of AE in each group by the system organ class (SOC) and preferred term (PT) according to the MedDRA dictionary, as well as by connection with the preparation under study and severity degree. The intergroup comparison of the frequency of AE development was conducted by means of x2-test and, if required, Fisher exact test, if the expected frequency in any of the cells was below 5. The analysis did not reveal statistically significant differences by the frequency of individual AE between the groups.
Table 8. Adverse Events Recorded During The Analyzed Period By System Organ Classes, Preferred Terms And Groups (FAS-Population)
TABLE 8
During the study adverse events (AE) were observed in the following categories:
1) General disorders and administration site conditions - 76 AE:
1/10 of the dose: 35 cases
1/5 of the dose: 22 cases
2) Systemic reactions - 54 AE 1/10 of the dose: 29 cases
1/5 of the dose: 25 cases
3) Laboratory deviations - 76 AE 1/10 of the dose: 44 cases
1/5 of the dose: 32 cases.
The following AEs were connected with the preparation under study (as the connection assessment is "possible", "probable" or "certain"): tenderness at place of injection, influenza like syndrome, fatigue, subfebrile temperature, hot flash, nasal congestion arose immediately after vaccine injection, headache, sore throat, cough, joint ache, general weakness, stomachache, panic attack, reduction of neutrophils, increase of bilirubin level.
For other AE the connection was determined as "doubtful" or "no connection". No allergic reactions to the preparation under study were observed.
On the whole, once can say that adverse events revealed during the study are characteristic for most vaccine medicines. No cases of development of life-threatening adverse events were recorded.
Example 16
Determination of effectiveness of immunization of children with the developed pharmaceutical agent based on assessment of humoral immunity level.
95 children aged 12-18 years participated in the given study. The humoral immunity level was determined by assessing the titer of IgG antibodies specific to RBD domain of S protein of SARS-CoV-2 virus. The participants of the study were divided into two groups:
1) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of lxl 010 virus particles/1 ml, 47 people.
2) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of 2x1010 virus particles/1 ml, 48 people.
The samples of the same volunteers taken on Day 1 of the study before vaccination were used as the reference.
The titer of antigen-specific IgG in the first group was assessed on Days 21, 28 and 42 of the study and in the second group - on Days 21 and 28 of the study.
The titer of antibodies was determined by means of a test system for ELISA that enables determining the titer of IgG to RBD domain of S antigen of SARS-CoV-2 virus.
The plates with pre-adsorbed RBD (100 ng/well) were washed 5 times with a washing buffer. Then, 100 ul of the positive control and 100 ul of the negative control were added to the plate wells in duplicate. A series of two-fold dilutions of the studied samples were added to other wells (two repeats per each sample). The plate was sealed with a film and incubated for an hour at +37 °C while mixing at the rate of 300 rpm. Then the wells were washed with a working solution of the washing buffer. Then, 100 ul of the working solution of the conjugate of monoclonal antibodies were added to each well, the plate was sealed with an adhesive film and incubated for an hour at +37 °C while mixing at the rate of 300 rpm. Then the wells were washed with a working solution of the washing buffer. Then, 100 ul of chromogen substrate solution were added to each well and incubated during 15 minutes in a dark place at a temperature from +20°C. After that, the reaction was stopped by adding 50 ul of a stop-reagent to each well (1M sulfuric acid solution). The result was determined during 10 minutes after reaction stop by measuring optical density on the spectrophotometer at the wavelength of 450 nm.
The titer of IgG was determined as maximum serum dilution at which OD450 serum value of an immunized participant exceeds the control serum value (serum of a participant before immunization) more than 2 times.
The results of determination of the titer of RBD-specific IgG antibodies in the first group (lxlO10 virus particles/dose) are given in Fig. 3. According to the provided data, the titer of antibodies specific to RBD-domain of S protein of SARS-Cov2 virus gradually increases after vaccination, achieving maximum value on Day 42. The reciprocal mean geometrical value at the given point is 13192. By Day 42 of the study seroconversion was observed in all 47 volunteers constituting the given group.
The results of determination of the titer of RBD-specific IgG antibodies in the second group (2xl010 virus particles/dose) are shown in Fig.4. As it can be seen from the provided data, the titer of antibodies specific to RBD-domain of SARS-Cov2 virus S protein gradually increases after vaccination with l/5th of the full therapeutical dose of the vaccine for adults, achieving the maximum value on Day 42. The reciprocal mean geometrical value at the given point is 19292. By Day 42 of the study seroconversion was observed in all 49 volunteers constituting the given group.
Thus, the results of the conducted study showed that vaccination of children with the developed immunobiological agent is capable of inducing formation of high-level postvaccination humoral immunity. Example 15
Determination of effectiveness of immunization of children with the developed pharmaceutical agent based on assessment of cell immunity level.
95 children aged 12-18 years participated in the given study. The participants of the study were divided into two groups:
1) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of lxlO10 virus particles/1 ml, 47 people.
2) sequential intramuscular administration of component 1 and component 2 of the pharmaceutical agent by variant 1 (Ad26-CMV-S-CoV2, Ad5-CMV-S-CoV2) in the dose of 2xl010 virus particles/1 ml, 48 people.
Cell immunity level was determined by assessing the quantity of proliferating CD4+ and CD 8+ lymphocytes of volunteers' peripheral blood in in vitro culture after repeated cell restimulation with recombinant S protein of SARS-CoV-2. Before immunization as well as after 28 days the patients' blood samples were drawn from which mononuclear cells were isolated by centrifugation in the ficoll solution density gradient (1.077 g/mL; PanEco). Then, the isolated cells were colored with the fluorescent dye CFSE (Invivogen, USA) and seeded on wells of a 96-well plate (2*105 cells/well). The repeated stimulation of lymphocytes was carried out in in vitro conditions by adding coronavirus S protein to the culture medium (final protein concentration 1 pg/ml). Intact cells to which the antigen was not added were used as the negative control. In 72 hours after antigen addition % of proliferating cells was measured and the cultural medium was taken for measuring gamma interferon quantity.
To determine % of proliferating cells they were stained with antibodies to marker molecules of T lymphocytes CD3, CD4, CD8 (anti-CD3 Pe-Cy7 (BDBiosciences, SK7 clone), anti-CD4 APC (BDBiosciences, SK3 clone), anti-CD8 PerCP-Cy5.5 (BDBiosciences, SKI clone)). Proliferating (with a lower quantity of cell colorant CFSE) CD4+ and CD8+ T lymphocytes were determined in the cell mixture with the use of the flow cytofluorometer BD FACS Arialll (BD Biosciences, USA). The resultant percentage of proliferating cells in each sample was determined by deducing the result obtained during analysis of intact cells from the result obtained during analysis of cells restimulated with coronavirus S protein antigen.
The results (with conducted statistical processing) of determination of the percentage of proliferating CD4+ and CD8+ T lymphocytes on Day 1 (before immunization) and on Day 28 of the study are given in Fig. 5 and Fig. 6.
The obtained data showed that immunization of children with the developed agent with both selected doses makes it possible to reliably increase the percentage of proliferating CD4+ and CD8+ T lymphocytes after antigen restimulation on Day 28 after immunization.
Based on the obtained data, one can conclude that double vaccination of children with the developed immunological agent is capable of inducing formation of high-level post-vaccination cell immunity.
Thus, the provided examples confirm that as a result of conducted work a safe and effective agent was created that induced development of reactions of humoral and cell immune response against SARS-CoV-2 virus in children aged 1 month and older.
Industrial applicability
All provided examples support effectiveness of the developed agents that effectively induce immune response against SARS-CoV-2 virus in children aged 1 month and older and industrial applicability.

Claims

1. Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
2. Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
3. Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO.l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV- 2 in children aged 1 month and older.
4. Utilization of an agent comprising a component in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
5. Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 26 in which El and E3 sites were deleted, while ORF6-Ad26 site was replaced with ORF6-Ad5 with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV- 2 in children aged 1 month and older.
6. Utilization of an agent comprising a combination representing component 1 in the form of an expression vector based on the genome of recombinant simian adenovirus serotype 25 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, and component 2 in the form of an expression vector based on the genome of recombinant human adenovirus serotype 5 with deleted El and E3 sites with an integrated expression cassette selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 for induction of specific immunity against severe acute respiratory syndrome virus SARS-CoV-2 in children aged 1 month and older.
7. Utilization presented herein in claims 1-6 wherein the agent enables induction of mucosal immune response on mucous membranes of the respiratory tract.
8. Utilization presented herein in claims 1-6 wherein the agent is prepared in a liquid or lyophilized form.
9. Utilization presented herein in claim 8 wherein the liquid form of the agent contains a buffer, wt. %:
Tris 0.1831 ... 0.3432
Sodium chloride 0.3313 ... 0.6212
Sucrose 3.7821 .. 7.0915
Magnesium chloride hexahydrate 0.0154 ... 0.0289
EDTA 0.0029 ... 0.0054
Polysorbate-80 0.0378 ... 0.0709
Ethanol 95% 0.0004 ... 0.0007 Water rest.
10. Utilization presented herein in claim 8 wherein the reduced lyophilized form of the agent contains a buffer, wt. %:
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.
11. Utilization presented herein in claims 1-10 wherein the component and/or components of the agent are intended for intranasal and/or intramuscular administration.
12. Utilization presented herein in claims 1-10 wherein the agent is intended for administration in the dose of 5*109- 5* 10!0 virus particles.
13. Utilization presented herein in claims 3, 5, 6, 9, 10 wherein the components of the agent are intended for sequential administration with an interval of over 1 week.
14. Utilization presented herein in claims 3, 5, 6, 9, 10 wherein the components of the agent are intended for simultaneous administration.
15. Utilization presented herein in claims 3, 5, 6, 9, 10 wherein the components are in different packages.
EP22713843.5A 2021-11-26 2022-02-18 Induction of immunity to sars-cov-2 in children Pending EP4436599A1 (en)

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