Note: Descriptions are shown in the official language in which they were submitted.
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UTILIZATION OF AN AGENT FOR INDUCTION OF SPECIFIC IMMUNITY
AGAINST SEVERE ACUTE RESPIRATORY SYNDROME VIRUS 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
Med 2020; 383: 2603-2615;
www.cdc.govicoronavirus/2019-
ncov/vaccinestrecommendations/adolescents.html).
Date Recue/Date Received 2022-04-06
The pharmaceutical company Modena, 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, Modena 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.govict2/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 virus (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
ChAdOx 1 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 (Ad26COVS1). 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 1-2a Trial of Ad26.COV2.S Covid-
19 Vaccine. N
2
Date Recue/Date Received 2022-04-06
Engl J Med, 2021 Jan 13.D01: 10.1056
NEJMoa2034201;
haps ://www.janssenmd .com/j anssen-covid19-vaccine/special-
populations/pediatrics/use-of-
j anssen-covid19-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=10).
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 coronavinis 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
3
Date Recue/Date Received 2022-04-06
virus in children aged 1 month and older.
A child is born 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
CD4OL 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/jimmuno1.176.1.165. 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.
4
Date Recue/Date Received 2022-04-06
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.,
ICruglova, 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.
Date Recue/Date Received 2022-04-06
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:1, 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:1, 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:1, 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:1, 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
6
Date Recue/Date Received 2022-04-06
ID NO:3 for induction of specific immunity against the severe acute
respiratory syndrome virus
SARS-CoV-2 in children aged I 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:1, 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:1, 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
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Date Recue/Date Received 2022-04-06
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*1019 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+ (B) T
lymphocytes before
immunization (Day 1) and on Day 14 of the study, after immunization of mice
with the
8
Date Recue/Date Received 2022-04-06
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 SARS-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 lx101
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
( - p<0.0001). 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<0.0001).
Y-axis ¨ titer of antigen-specific IgG antibodies;
X-axis ¨ time, days.
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Date Recue/Date Received 2022-04-06
FIG. 4
illustrates titers of IgG antibodies specific to RBD-domain of SARS-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 2x101
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
( - p<0.0001). 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<0.0001).
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 lx101 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
Date Recue/Date Received 2022-04-06
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
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:1 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 13-
actin promoter
and chimeric intron (chicken p-actin and rabbit fl-globin). It was
experimentally shown
11
Date Recue/Date Received 2022-04-06
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. II 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 1(3 (EF-1a). 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-1 a 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-Ia
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
12
Date Recue/Date Received 2022-04-06
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:1, 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:1, 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:1, SEQ ID NO:2, SEQ ID
NO:31 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:1, 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
13
Date Recue/Date Received 2022-04-06
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:1, 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:1, 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
14
Date Recue/Date Received 2022-04-06
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*101 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.
Date Recue/Date Received 2022-04-06
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-d1E1 plasmid was obtained that comprised the genome of human adenovirus
serotype 26 with deleted El site.
Then, in the obtained pAd26-d1E1 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-d1E 1 -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
16
Date Recue/Date Received 2022-04-06
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:1 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.
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-EF1-S-
CoV2 were obtained that comprised expression cassettes SEQ ID NO:1, SEQ ID
NO:2 or
SEQ ID NO:3, 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-
EF l-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-EFI-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:1, SEQ ID NO:2 or SEQ ID NO:3, respectively.
At the fourth stage, the pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2,
pAd26-only-EF1-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:I, SEQ ID NO:2, SEQ ID NO:3.
ii
Date Recue/Date Received 2022-04-06
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:!, 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:1
(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:1
(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
18
Date Recue/Date Received 2022-04-06
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-
EFI-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 EF I
promoter, the
gene of SARS-CoV-2 virus S protein and polyadenylation signal, SEQ ID NO:3
(Ad26-
EF 1-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-d1E1 plasmid was obtained that comprised the genome of simian
adenovirus
serotype 25 with deleted El site.
19
Date Recue/Date Received 2022-04-06
Then, with the use of standard genetic engineering methods in the constructed
pSim25-d1E1 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 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
pSim25-Ends, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2,
pArms-Sim25-EF1-S-CoV2 were obtained that comprised expression cassettes SEQ
ID
NO:4, SEQ ID NO:2 or SEQ ID NO:3, 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-EF1-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-EF1-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 NO:4, SEQ ID NO:2 or SEQ ID NO:3,
respectively.
At the third stage the pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, pSim25-EF1-
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.
Date Recue/Date Received 2022-04-06
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-EF I-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:
I. 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
21
Date Recue/Date Received 2022-04-06
'
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 EF I promoter, the gene of SARS-CoV-2 virus S protein and
polyadenylation signal, SEQ ID NO:3 (simAd25-EF1-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 NO:3 (simAd25-EF1-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).
22
Date Recue/Date Received 2022-04-06
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-dIE1 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-
d1E1 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:1 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-EFI-S-CoV2 were obtained that comprised expression cassettes SEQ ID NO:1,
SEQ
ID NO:2 or SEQ ID NO:3, 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-EF1-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-EF1-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:1, SEQ ID NO:2 or SEQ ID NO:3, respectively.
At the fourth stage the pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, pAd5-
too-EF1-S-CoV2 plasmids were hydrolyzed with the specific restriction
endonuclease to
23
Date Recue/Date Received 2022-04-06
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:1), Ad5-CAG-S-CoV2 (comprising the
expression
cassette SEQ ID NO:2), Ad5-EF1-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:1, 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:1, 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:1 (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
24
Date Recue/Date Received 2022-04-06
polyadenylation signal, SEQ ID NO:1 (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 NO:3 (Ad5-EF1-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 NO:3 (Ad5-EF1-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.
Date Recue/Date Received 2022-04-06
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*levirus 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*101 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.
26
Date Recue/Date Received 2022-04-06
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
Buffer composition
Group Magnesium
Sodium Ethanol
No. Tris Sucrose chloride EDTA Polysorbate-
chloride 95% Water
(mg) (mg) hexahydrate (mg) 80 (mg)
(mg) (mg)
(mg)
under
1 0.968 2.19 25 0.102 0.019 0.25 0.0025
0.5 ml
under
2 1.815 2.19 25 0.102 0.019 0.25 0.0025
0.5 ml
under
3 1.21 1.752 25 0.102 0.019 0.25 0.0025
0.5 ml
under
4 1.21 3.285 25 0.102 0.019 0.25 0.0025
0.5 ml
under
1.21 2.19 20 0.102 0.019 0.25 0.0025
0.5 ml
under
6 1.21 2.19 37.5 0.102 0.019 0.25 0.0025
0.5 ml
under
7 1.21 2.19 25 0.0816 0.019 0.25 0.0025
0.5 ml
27
Date Recue/Date Received 2022-04-06
under
8 1.21 2.19 25 0.153 0.019 0.25 0.0025
0.5 ml
under
9 1.21 2.19 25 0.102 0.0152 0.25 0.0025
0.5 ml
under
1.21 2.19 25 0.102 0.0285 0.25 0.0025
0.5 ml
under
11 1.21 2.19 25 0.102 0.019 0.2 0.0025
0.5 ml
under
12 1.21 2.19 25 0.102 0.019 0.375 0.0025
0.5 ml
under
13 1.21 2.19 25 0.102 0.019 0.25 0.002
0.5 ml
under
14 1.21 2.19 25 0.102 0.019 0.25 0.00375
0.5 ml
under
1.21 2.19 25 0.102 0.019 0.25 0.0025
0.5 ml
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. %;
28
Date Recue/Date Received 2022-04-06
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. A);
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:
I. 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*101 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.
29
Date Recue/Date Received 2022-04-06
Table 2 - Composition of Experimental Buffers.
TABLE 2
Buffer composition
Group
Sodium Magnesium
No. Tris Sucrose EDTA Polysorbate-
chloride chloride Water
(mg) (mg) (mg) 80 (mg)
(mg) hexahydrate (mg)
under
1 0.1936 1.403 73.5 0.0204 0.0038 0.05
1 ml
under
2 0.363 1.403 73.5 0.0204 0.0038 0.05
1 ml
under
3 0.242 1.1224 73.5 0.0204 0.0038 0.05
1 ml
under
4 0.242 2.1045 73.5 0.0204 0.0038 0.05
1 ml
under
0.242 1.403 58.8 0.0204 0.0038 0.05
1 ml
under
6 0.242 1.403 110.25 0.0204 0.0038 0.05
1 ml
under
7 0.242 1.403 73.5 0.01632 0.0038 0.05
1 ml
under
8 0.242 1.403 73.5 0.0306 0.0038 0.05
1 ml
under
9 0.242 1.403 73.5 0.0204 0.00304 0.05
1 ml
10 0.242 1.403 73.5 0.0204 0.0057 0.05 under
Date Recue/Date Received 2022-04-06
1 ml
under
11 0.242 1.403 73.5 0.0204 0.0038 0.04
1 ml
under
12 0.242 1.403 73.5 0.0204 0.0038 0.075
1 ml
under
13 0.242 1.403 73.5 0.0204 0.0038 0.05
1 ml
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 BALM line (aged 21-28 days) were used. The
animals
were distributed by groups of 10 and the following agents were administered to
them:
31
Date Recue/Date Received 2022-04-06
=
1) Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 5x109 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 5x109v.p./100u1; in 21 days Ad5-CMV-S-CoV2,
i/n, dose 5x109 v.p./100u1.
3) simAd25-CMV-S-CoV2, i/m, dose 5x109v.p./100u1; in 21 days simAd25-CMV-
S-CoV2, i/m, dose 5x109 v.p./100u1
4) Ad26-null, i/m, dose 5x109v.p./100u1; in 21 days Ad26-null, i/m, dose 5x109
v.p./100u1
5) Ad5-null, i/m, dose 5x109v.p./100u1; in 21 days Ad5-null, i/m, dose 5x109
v.p./100u1
6) simAd25-null, i/m, dose 5x109v.p./100u1; in 21 days simAd25-null, i/m, dose
5x109 v.p./100u1
7) Ad26-CMV-S-CoV2, intranasally (i/n), dose 5x109 v.p./100u1; in 21 days
Ad26-CMV-S-CoV2 i/n, dose 5x109 v.p./100u1.
8) Ad5-CMV-S-CoV2, i/n, dose 5x109 v.p./100u1; in 21 days Ad5-CMV-S-CoV2,
i/n, dose 5x109v.p./100u1.
9) Ad26-CMV-S-CoV2, i/m, dose 5x109v.p./100u1; in 21 days Ad26-CMV-S-
CoV2 i/n, dose 5x109v.p./100u1.
10) Ad5-CMV-S-CoV2, i/m, dose 5x109v.p./100u1; in 21 days Ad5-CMV-S-CoV2,
i/n, dose 5x109v.p./100u1.
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.
32
Date Recue/Date Received 2022-04-06
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 irrununoglobulins 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 tetrarnethylbenzidine (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
Titer of IgG Titer of IgA
Group name
antibodies antibodies
1 Ad26-CMV-S-CoV2 ihn; Ad26-CMV-S-CoV2 162 3
i/m
33
Date Recue/Date Received 2022-04-06
Titer of IgG Titer of IgA
Group name
antibodies antibodies
2 Ad5-CMV-S-CoV2 i/m, Ad5-CMV-S-CoV2 325 4
3 simAd25-CMV-S-CoV2 iim, simAd25-CMV-S- 214 3
CoV2 i/m
4 Ad26-CMV-S-CoV2 i/n; Ad26-CMV-S-CoV2 123 76
i/n
Ad5-CMV-S-CoV2 i/n, Ad5-CMV-S-CoV2 264 81
6 Ad26-CMV-S-CoV2 i/m; Ad26-CMV-S-CoV2 373 283
i/n
7 Ad5-CMV-S-CoV2 i/m, Ad5-CMV-S-CoV2 348 303
8 Ad26-null i/m; Ad26-null i/m; 0 0
9 Ad5-null i/m; Ad5-null i/m; 0 0
simAd25-null i/m; simAd25-null i/m; 0 0
11 Ad26-null i/n; Ad26-null i/n; 0 0
12 Ad5-null i/n; Ad5-null i/n; 0 0
13 Buffer 0 0
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 mucosa] immune response against SARS-CoV-2 virus on the
mucous
34
Date Recue/Date Received 2022-04-06
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.
Date Recue/Date Received 2022-04-06
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
No. Age of animals Titer of IgG antibodies
1 15-18 days 6400
2 28-35 days 5572
3 50-60 days 4222
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 BAL13/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:
36
Date Recue/Date Received 2022-04-06
1. Ad26-CMV-S-CoV2, intramuscularly (i/m), dose 1010 vp/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 ug/m1). 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 AriaIII (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.
37
Date Recue/Date Received 2022-04-06
Example 11
Study of immunogenicity of the developed agent on young mice with different
administration methods.
Young BALB/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-EF1-S-CoV2,intramuscularly,dose 109vp/100 ul
6. Ad5-CMV-S-CoV2,intramuscularly,dose 109vp/100 ul
7. Ad5-CAG-S-CoV2,intramuscularly,dose 109vp/100 ul
8. Ad5-EF 1 -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-EF1-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-EF1-S-CoV2,intranasally (i/n),dose 109vp/I00 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-EF1-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-EF1-S-CoV2,intranasally (i/n),dose 109vp/100 ul
38
Date Recue/Date Received 2022-04-06
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:
I. 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.
39
Date Recue/Date Received 2022-04-06
Table 5¨ Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean
Geometrical Value Of The Titer Of Antibodies)
TABLE 5
Group of animals Titer of IgG antibodies
1 Ad26-CMV-S-CoV2, i/m 696
2 Ad26-CAG-S-CoV2, i/m 528
3 Ad26-EF1-S-CoV2 i/m 459
4 Ad5-CMV-S-CoV2, i/m 9701
Ad5-CAG-S-CoV2, i/m 11143
6 Ad5-EF1-S-CoV2, i/m 14703
7 simAd25-CMV-S-CoV2, i/m 3676
8 simAd25-CAG-S-CoV2, i/m 5572
9 simAd25-EF1-S-CoV2, i/m 3676
Ad26-CMV-S-CoV2, i/n 230
11 Ad26-CAG-S-CoV2, i/n 200
12 Ad26-EF1-S-CoV2, i/n 174
Date Recue/Date Received 2022-04-06
Group of animals Titer of IgG antibodies
13 Ad5-CMV-S-CoV2, 606
14 Ad5-CAG-S-CoV2, i/n 459
15 Ad5-EF I -S-CoV2, i/n 459
16 simAd25-CMV-S-CoV2, i/n 459
17 simAd25-CAG-S-CoV2, i/n 400
18 simAd25-EF1-S-CoV2, i/n 303
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, 10'0vp/100 ul,
3) Ad5-CMV-S-CoV2, 5*101 vp/100 ul.
41
Date Recue/Date Received 2022-04-06
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.
42
Date Recue/Date Received 2022-04-06
Table 6¨ Titer Of Igg Antibodies To S Protein In Blood Serum Of Mice (Mean
Geometrical Value Of The Titer Of Antibodies)
TABLE 6
Group of animals Titer of IgG antibodies
1 Ad5-CMV-S-CoV2, 5*109vp/100 ul, 22286
2 Ad5-CMV-S-CoV2, 101 vp/100 ul, 44572
3 Ad5-CMV-S-CoV2, 5*101 vp/100 ul. 117627
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;
43
Date Recue/Date Received 2022-04-06
5) Ad5-CMV-S-CoV2, i/m, 109vp/100 ul; in 3 weeksAd5-CMV-S-CoV2 i/m,
l0svp/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.
44
Date Recue/Date Received 2022-04-06
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
Group of animals Titer of IgG
antibodies
1 Ad26-CMV-S-CoV2 i/m, Ad26-CMV-S-CoV2 i/m 11143
2 Ad26-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2 i/m, 29407
3 Ad26-CMV-S-CoV2, i/n, Ad5-CMV-S-CoV2 22286
4 Ad26-CMV-S-CoV2, simAd5-CMV-S-CoV2 i/m, 14703
Ad5-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2 i/m, 16890
6 Ad5-CMV-S-CoV2, i/m, Ad26-CMV-S-CoV2 i/m, 33779
7 Ad5-CMV-S-CoV2, i/n, Ad26-CMV-S-CoV2 i/m, 25600
Date Recue/Date Received 2022-04-06
Group of animals Titer of IgG
antibodies
8 Ad5-CMV-S-CoV2, i/m, sirnAd5-CMV-S-CoV2 i/m, 14703
9 simAd5-CMV-S-CoV2, i/m, simAd5-CMV-S-CoV2 i/m, 12800
simAd5-CMV-S-CoV2, i/m, Ad5-CMV-S-CoV2 i/m, 16890
11 simAd5-CMV-S-CoV2, Ad26-CMV-S-CoV2, i/m 14703
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
46
Date Recue/Date Received 2022-04-06
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
Group 1 Group 2
Total
(dose 1/10) .. (dose 1/5)
N = 100 P-
Description N=50 N=50
value
N % N % N %
Any PT 37 74.0 36 72.0 73 73.0 0.822
General disorders and reactions at the place of administration (78 AE
episodes)
Pain at place of injection 15 30.0 11 22.0 26
26.0 0.362
Tenderness (1) 7 14.0 7 14.0 14 14.0
1.000
Hyperthermia 5 10.0 3
6.0 8 8.0 0.715
Influenza like illness 3 6.0 5 10.0 8 8.0 0.715
Erythema at place of injection 4 8.0 0 0.0 4 4.0 0.117
Itching at place of injection 1 2.0 0 0.0 1 1.0 1.000
Local temperature increase at place of
1 2.0 0 0.0 1 1.0 1.000
administration
Shivering 1 2.0 0
0.0 1 1.0 1.000
Edema at place of injection 1 2.0 0 0.0 1 1.0 1.000
Pyrexia 0 0.0 1
2.0 1 1.0 1.000
47
Date Recue/Date Received 2022-04-06
Total in the organ system 29 58.0 23 46.0 52 52.0
0.230
Laboratory and instrumental data (76 AE episodes)
Reduction of neutrophil count 17 34.0 18 36.0 35 35.0
0.834
Positive result of urine analysis for
8 16.0 5 10.0 13 13.0 0.372
erythrocytes
Increase of blood bilirubin level 3 6.0 2 4.0 5 5.0
1.000
Presence of protein in urine 1 2.0 2 4.0 3 3.0 1.000
Reduction of hemoglobin level 1 2,0 2 4.0 3 3.0 1.000
Presence of leukocytes in urine 1 2.0 1 2.0 2 2.0 1.000
Increase of blood alkaline phosphatase 1 2.0 1 2.0 2 2.0
1.000
Reduction of lymphocyte count 1 2.0 1 2.0 2 2.0 1.000
Reduction of platelet count 1 2.0 1 2.0 2 2.0 1.000
Test for bacteria (2) 0 0.0 1 2.0 1 1.0 1.000
Presence of glucose in urine 1 2.0 0 0.0 1 1.0 1.000
Presence of casts in urine 0 0.0 1 2.0 1 1.0 1.000
Increase of erythrocyte sedimentation
1 2.0 0 0.0 1 1.0 1.000
rate
Increase of blood lactate
0 0.0 1 2.0 1 1.0 1.000
dehydrogenase level
Increase of blood cholesterol 1 2.0 0 0.0 1 1.0 1.000
Increase of leukocyte count 0 0.0 1 2.0 1 1.0 1.000
Increase of erythrocyte count 1 2.0 0 0.0 1 1.0 1.000
48
Date Recue/Date Received 2022-04-06
Total in the organ system 25 50.0 23 46.0 48 48.0
0.689
Nervous system disorders (18 AE episodes)
Headache 5 10.0 4 8.0 9 9.0 1.000
Somnolence 4 8.0 1 2.0 5 5.0 0.362
Dizziness 1 2.0 1 2.0 2 2.0 1.000
Total in the organ system 9 18.0 5 10.0 14 14.0
0.249
Musculoskeletal and connective tissue disorders (9 AE episodes)
Pain in extremity 0 0.0 3 6.0 3 3.0 0.242
Myalgia 2 4.0 1 2.0 3 3.0 1.000
Arthralgia 0 0.0 1 2.0 1 1.0 1.000
Total in the organ system 2 4.0 5 10.0 7 7.0
0.436
Gastrointestinal disorders (5 AE episodes)
Stomachache 0 0.0 3 6.0 3 3.0 0.242
Vomiting 1 2.0 1 2.0 2 2.0 1.000
Dryness in mouth 1 2.0 0 0.0 1 1.0 1.000
Total in the organ system 2 4.0 3 6.0 5 5.0 1.000
Respiratory, thoracic and mediastinal disorders (5 AE episodes)
Nasal congestion 3 6.0 0 0.0 3 3.0 0.242
Oropharyngeal pain 0 0.0 1 2.0 1 1.0 1.000
Rhinorrhea 1 2.0 0 0.0 1 1.0 1.000
Total in the organ system 3 6.0 1 2.0 4 4.0
0.617
49
Date Recue/Date Received 2022-04-06
Infections and invasions (3 AE episodes)
Rotavirus infection 1 2.0 0 0.0 1 1.0 1.000
Furunculus (3) 1 2.0 0 0.0 1 1.0 1.000
Enterovirus infection 0 0.0 1 2.0 1 1.0 1.000
Total in the organ system 2 4.0 1 2.0 3 3.0 1.000
Vascular disorders (3 AE episodes)
Vaginal hemorrhage (4) 1 2.0 0 0.0 1 1.0 1.000
Haemorrhoids 1 2.0 0 0.0
1 1.0 1.000
Hyperemia 1 2,0 0 0.0
1 1.0 1.000
Total in the organ system 3 6.0 0 0.0 3 3.0 0.242
Metabolic disturbances (1 AE episode)
Loss of appetite 1 2.0 0 0.0 1 1.0 1.000
Skin and subcutaneous tissue disorders (1 AE episode)
Rash 0 0.0 1 2.0 1 1.0 1.000
Blood and lymphatic system disorders (1 AE episode)
Leukopenia 0 0.0 1 2.0
1 1.0 1.000
Ear and labyrinth disorders (1 AE episode)
Earache 1 2.0 0 0.0 1 1.0 1.000
Renal and urinary disorders (1 AE episode)
Po I lakiuria 0 0.0 1 2.0 1 1.0 1.000
Psychiatric disorders (1 AE episode)
Date Recue/Date Received 2022-04-06
Panic attack 0 0.0 1 2.0 1 1.0 1.000
Traumas, intoxications and procedure complications (1 AE episode)
Food poisoning 1 2.0 0 0.0 1 1.0 1.000
Notes:
The results are given in the form: the number of subjects in whom adverse
events (AE) were
recorded, percentage from safety population in the given group.
1
Hypersensitivity at place of injection. PT "Tenderness" corresponds to PT
"Tenderness
[10043224]".
2 PT "Test for bacteria" corresponds to LLT "Presence of bacteria in urine
[10060857]".
3 PT
"Furunculus" corresponds to LLT "Ear furunculus [10017556]", AE ¨ "Left ear
auricle furunculus".
4 PT
"Vaginal hemmorhage" corresponds to LLT "Bloody vaginal discharge {100498511".
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
51
Date Recue/Date Received 2022-04-06
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 lx10I 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
52
Date Recue/Date Received 2022-04-06
6
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
wave length of 450 nm.
The titer of IgG was detelinined as maximum serum dilution at which 0D450
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
(1x10' 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 (2x101 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 1/5th 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 inununobiological agent is capable of inducing formation of high-
level post-
vaccination humoral immunity.
53
Date Recue/Date Received 2022-04-06
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 lx101 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 2x10' virus particles/1 ml, 48 people.
Cell immunity level was determined by assessing the quantity of proliferating
CD4+ and
CD8+ 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 I gimp. 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 Arian (BD Biosciences, USA). The resultant percentage of proliferating
cells in each
54
Date Recue/Date Received 2022-04-06
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.
Date Recue/Date Received 2022-04-06
