EP4370681A1

EP4370681A1 - Influenza virus-based isolated recombinant virus

Info

Publication number: EP4370681A1
Application number: EP22842553.4A
Authority: EP
Inventors: Larisa Georgievna Rudenko; Irina Nikolayevna ISAKOVA-SIVAK; Ekaterina Alekseevna STEPANOVA; Viktoriia Arkadienva MATIUSHENKO; Sergei Andreevich NISKANEN; Bogdan Olegovich NETEREBSKII; Anna Konstantinovna VLADIMIROVA; Pavel Andreevich IAKOVLEV; Iakov Iurevich USTIUGOV; Timur Mugdinovich SHEUDZHEN; Aleksandr Nikolaevich Doronin; Tatiana Urevna OSTROUKHOVA; Aleksei Aleksandrovich ALEKSANDROV; Dmitry Valentinovich MOROZOV
Original assignee: Federal State Budgetary Scientific Institution Institute Of Experimental Medicine; Biocad JSC
Current assignee: Federal State Budgetary Scientific Institution Institute Of Experimental Medicine; Biocad JSC
Priority date: 2021-07-16
Filing date: 2022-05-30
Publication date: 2024-05-22
Also published as: TW202306968A; AR126008A1; ECSP24003913A; WO2023287324A1

Abstract

The present invention relates to the fields of biotechnology, immunology, virology, genetics, and molecular biology. More specifically, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to influenza virus (variants), an influenza virus-based recombinant virus for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases, a pharmaceutical composition and a vaccine that include the above influenza virus-based recombinant virus, as well as their use for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases.

Description

Influenza virus-based isolated recombinant virus

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Influenza is an acute viral infection that occurs worldwide. Annual influenza epidemics usually occur during autumn and winter and can infect up to 20% of the population.

Seasonal influenza-related respiratory diseases may cause quite a large mortality. In particular, a 2017 study found that such deaths may reach 650000 each year worldwide.

People most at risk of severe disease following influenza infection include people aged 65 and over, pregnant women, young children, immunocompromised people and people with chronic pathologies.

There are 2 main types of seasonal influenza viruses that cause illness in humans: type A and type B. Influenza A viruses are further classified into subtypes, while influenza B viruses are categorized in lineages. The most common circulating influenza A viruses belong to subtypes A(H1N1) and A(H3N2), and the most common circulating influenza B viruses belong to Yamagata and Victoria lineages.

Annual vaccination is the most effective way to prevent influenza and severe outcomes thereof. Due to the high variability of influenza viruses, WHO updates recommendations for the composition of the influenza vaccine twice a year: in February for the northern hemisphere and in September for the southern hemisphere. The recommendations for the vaccine composition are based on viruses detected and characterized by Member States through surveillance.

Patent document RU92006182 provides an attenuated cold-adapted influenza virus strain A/Leningrad/134/17/57 (H2N2) for producing live intranasal influenza vaccine strains.

Patent document RU2556833C2 provides an attenuated cold-adapted influenza virus strain a/pr/8/59/m2 (hlnl) intended for producing vaccine influenza virus strains as an attenuation donor, as well as vaccine influenza virus strains a/59/m2/califomia/66/2211 (h2n2) and a/59/m2/tokyo/67/22111 (h2n2).

Patent document RU2563352C2 provides an influenza virus strain A/17/Texas/2012/30 (H3N2) for producing live influenza intranasal vaccine for adults and children.

Despite the plurality of known vaccine influenza virus strains, there is a need to produce an influenza virus-based recombinant virus that has improved properties, for example, one that allows to obtain higher levels of antibodies to influenza virus when administered to a mammal, i.e. be more effective in use.

In addition, as of the date of filing of the present application, more than 186 million people have contracted SARS-CoV-2-related diseases, more than 4 million people have died from SARS-CoV-2-related diseases. Furthermore, the COVID-19 pandemic still continues at the date of filing of the application. There is an urgent global need for effective means for the prevention and treatment of diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2.

Thus, there is a need to produce a recombinant virus that will allow to form specific T- cell immunity to coronavirus and specific immunity to influenza virus, thus making it possible to produce a vaccine strain that may be used to prevent influenza virus-related diseases and coronavirus-related diseases.

Description of the invention

The inventors have developed a nucleic acid that encodes a recombinant polypeptide based on coronavirus protein fragments. The inventors have developed an influenza virus-based recombinant virus, which includes the above nucleic acid. The authors of the invention have developed a pharmaceutical composition and a vaccine that include the above influenza virus- based recombinant virus, as well as various variants for their use for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases. The inventors surprisingly found that the developed nucleic acid that encodes the recombinant polypeptide based on coronavirus protein fragments, when embedded in the neuraminidase (NA) gene or the NS gene of influenza virus, makes it possible to produce a recombinant influenza virus that produces higher levels of antibodies to influenza virus when administered to a mammal, as compared to influenza virus without of the above insert. Furthermore, the recombinant influenza virus including the above nucleic acid that encodes the recombinant polypeptide based on coronavirus protein fragments allows to form specific T-cell immunity to coronavirus, when administered to a mammal.

Brief description of the invention In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to influenza virus, which comprises: at least 1 fragment from the coronavirus S glycoprotein with the amino acid sequence of SEQ ID NO: 1; at least 1 fragment from the coronavirus nucleoprotein with the amino acid sequence of SEQ ID NO: 3; and, if necessary, 1 fragment from the coronavirus membrane protein with the amino acid sequence of SEQ ID NO: 5, wherein at least 1 fragment from the coronavirus S glycoprotein is selected from the group of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; at least 1 fragment from the coronavirus nucleoprotein is selected from the group of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; the fragment from the coronavirus membrane protein is SEQ ID NO: 22.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes from 1 to 4 fragments from the coronavirus S glycoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 1 fragment from the coronavirus S glycoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 4 fragments from the coronavirus S glycoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes from 1 to 4 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 1 fragment from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus nucleoprotein. In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 4 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes a fragment of the coronavirus membrane protein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:36.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 35.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 37.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO: 39.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO: 40.

In some embodiments of the invention, the isolated nucleic acid includes a nucleotide sequence that is selected from the group comprising the nucleotide sequences of SEQ ID NO: 59-76.

In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide to increase the titer of antibodies to the influenza virus, including fragments from coronavirus proteins and comprising an amino acid sequence that is selected from the group comprising the amino acid sequences of SEQ ID NO: 23-40. In some embodiments of the invention, the isolated nucleic acid includes a nucleotide sequence that is selected from the group comprising the nucleotide sequences of SEQ ID NO: 59-76 that encode the amino acid sequences of SEQ ID NO: 23-40, respectively.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide from coronavirus, wherein the coronavirus is betacoronavirus.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide from betacoronavirus, wherein the beta-coronavirus is SARS-CoV-2 virus.

In one aspect, the present invention relates to an influenza virus-based isolated recombinant virus for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases, which comprises any of the above nucleic acids.

In some embodiments of the invention, the influenza virus-based isolated recombinant virus is produced from an attenuated influenza virus.

In some embodiments of the invention, the influenza virus-based isolated recombinant virus is produced from a cold-adapted influenza virus.

In some embodiments of the invention, the influenza virus-based isolated recombinant virus is produced from the H2N2 influenza virus.

In some embodiments of the invention, the influenza virus-based isolated recombinant virus is produced from the virus A/Leningrad/134/17/57 (H2N2) or derivatives thereof.

In some embodiments of the invention, the influenza virus-based isolated recombinant virus includes any of the above nucleic acids of the invention, which is present in the neuraminidase (NA) gene or the NS gene of influenza virus.

In one aspect, the present invention relates to a pharmaceutical composition for the induction of specific immunity to influenza virus and/or the prevention of diseases caused by influenza virus, which comprises any of the above recombinant viruses in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention relates to the use of any of the above influenza virus- based recombinant viruses or the above composition for the induction of specific immunity to influenza virus and/or prevention of influenza virus-related diseases.

In some embodiments of the use, any of the above influenza virus-based recombinant viruses or the above composition are used to induce specific immunity to influenza virus and coronavirus and/or to prevent influenza virus-related diseases and coronavirus-related diseases.

In some embodiments of the use, the specific immunity to coronavirus is specific T-cell immunity.

In some embodiments of the use, the coronavirus is betacoronavirus. In some vaccine embodiments of the use, the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

In one aspect, the present invention relates to a vaccine for induction of specific immunity to influenza virus and/or prevention of influenza virus-related diseases, which comprises any of the above influenza virus-based recombinant viruses in an effective amount.

In some embodiments of the invention, the vaccine is used to induce specific immunity to influenza virus and coronavirus and/or to prevent influenza virus-related diseases and coronavirus-related diseases.

In some embodiments of the vaccine, the coronavirus is betacoronavirus.

In some embodiments of the vaccine, the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

In some embodiments of the vaccine, the specific immunity to coronavirus is a specific T-cell immunity.

In one aspect, the present invention relates to a method for induction of specific immunity to influenza virus and/or prevention of influenza virus-related diseases, or induction of specific immunity to influenza virus and coronavirus, or combined prevention of influenza virus-related diseases and coronavirus-related diseases, comprising administering into a mammalian organism any of the above influenza virus-based recombinant viruses or the above composition or the above vaccine, in an effective amount.

In some embodiments of the method, the coronavirus is betacoronavirus.

In some vaccine embodiments of the method, the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

In some embodiments of the method, the specific immunity to coronavirus is specific T- cell immunity.

Brief description of drawings

Figure 1 is a cloning scheme for the subject antigenic cassettes encoding the transgene (polyepitope cassettes consisting of combinations of protein fragments of S (or Spike) glycoprotein (SEQ ID NO: 1), nucleoprotein (SEQ ID NO: 3), and, if necessary, membrane protein (SEQ ID NO: 5) of SARS-CoV-2 betacoronavirus) in the NA gene of influenza virus.

Figure 2 is a scheme of genetic construct based on the influenza virus reverse genetics vector pCIPolISapIT, which genetic construct contains sequences of the sixth segment of the influenza virus genome, including the NA gene, as well as the transgene (polyepitope cassette consisting of combinations of protein fragments of S (or Spike) glycoprotein proteins (SEQ ID NO: 1), nucleoprotein (SEQ ID NO: 3), and, if necessary, membrane protein (SEQ ID NO: 5) of SARS-CoV-2 betacoronavirus) to produce modified influenza virus particles of the vaccine strain.

Description of elements for Figures 1-2.

Figure 3 is a cloning scheme for the subject antigenic cassettes encoding poly epitope cassettes consisting of combinations of protein fragments of S (or Spike) glycoprotein (SEQ ID NO: 1), nucleoprotein (SEQ ID NO: 3), and, if necessary, membrane protein (SEQ ID NO: 5) of SARS-CoV-2 betacoronavirus in the NA gene of influenza virus.

Figure 4 is a scheme of genetic construct based on the influenza virus reverse genetics vector pCIPolISapIT, which genetic construct contains sequences of the eighth segment of the influenza virus genome, including the modified NS gene from the attenuation donor A/Leningrad/134/17/57 or derivatives thereof, as well as the transgene (polyepitope cassette consisting of combinations of protein fragments of S (or Spike) glycoprotein proteins (SEQ ID NO: 1), nucleoprotein (SEQ ID NO: 3), and, if necessary, membrane protein (SEQ ID NO: 5) of SARS-CoV-2 betacoronavirus) to produce modified influenza virus particles of the vaccine strain.

Description of elements for Figures 3-4.

Figure 5 is a graph that shows the levels of serum IgG antibodies in the blood sera of mice vaccinated twice with recombinant influenza viruses containing inserts of SARS-CoV-2 polyepitope cassettes, as compared to the viral vector. It indicates optical densities by ELISA as a function of serum dilution. AUC data was compared using the ANOVA one-factor analysis of variance followed by the Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001.

PBS is phosphate-buffer saline.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 6 is a graph that shows the levels of serum IgG antibodies in the blood sera of mice vaccinated twice with recombinant influenza viruses containing inserts of SARS-CoV-2 polyepitope cassettes, as compared to the viral vector. It indicates optical densities by ELISA as a function of serum dilution. AUC data was compared using the ANOVA one-factor analysis of variance followed by the Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001.

PBS is phosphate-buffer saline.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 7 is a graph that shows the levels of serum IgG antibodies in the blood sera of mice vaccinated twice with recombinant influenza viruses containing inserts of SARS-CoV-2 polyepitope cassettes, as compared to the viral vector. It indicates values of area under the curve of OD450 for each animal. AUC data was compared using the ANOVA one-factor analysis of variance followed by the Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001.

PBS is phosphate-buffer saline.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 8 is a graph that shows the levels of serum IgG antibodies in the blood sera of mice vaccinated twice with recombinant influenza viruses containing inserts of SARS-CoV-2 polyepitope cassettes, as compared to the viral vector. It indicates values of area under the curve of OD450 for each animal. AUC data was compared using the ANOVA one-factor analysis of variance followed by the Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001.

PBS is phosphate-buffer saline.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 9 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-13 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 10 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-15 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 11 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-16 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 12 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-17 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 13 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-29 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 14 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-30 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated influenza virus H7N9.

Figure 15 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-31 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated influenza virus H7N9.

Figure 16 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-32 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated influenza virus H7N9.

Figure 17 is a graph that shows the levels of virus-specific IFN-γ-producing CD4+ (left) and CD8+ (right) memory T cells in peripheral blood mononuclear samples from COVID-19 convalescents following stimulation with the CoVac-33 vaccine candidate and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 18 is a graph that shows virus-specific IFN-γ-producing CD4+ (top) and CD8+ (bottom) memory T cells in peripheral blood mononuclear samples from control donors following stimulation with vaccine candidates and the LIV H7N9 viral vector.

LIV H7N9 is live attenuated H7N9 influenza virus.

Figure 19 is a comparison of sequences of fragments of S glycoprotein in various coronaviruses. Definitions and general methods

Unless defined otherwise herein, all technical and scientific terms used in connection with the present invention will have the same meaning as is commonly understood by those skilled in the art.

Furthermore, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the present classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and purification methods are performed according to the manufacturer's guidelines, as is common in the art, or as described herein.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in an animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid or protein can exist substantially in purified form, or can exist in a non- native environment such as, for example, a genetically modified cell.

The terms “naturally occurring,” “native,” or “wild-type” are used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and that has not been intentionally modified by a person in the laboratory, is naturally occurring.

As used in the present description and claims that follow, unless otherwise dictated by the context, the words "include" and "comprise," or variations thereof such as "includes", "including", "comprises," or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Protein (Peptide)

As used in the present description, the terms "peptide", “polypeptide” and “protein” are used interchangeably, and they refer to a compound consisting of amino acid residues that are covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used in the present description, the term refers to both short chains, which also commonly are referred to in the art, for example, as peptides, oligopeptides and oligomers, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present description, mean a precise sequence of nucleotides, modified or not, determining a fragment or a region of a nucleic acid, containing unnatural nucleotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.

One skilled in the art has the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric “nucleotides”. Monomeric nucleotides can be hydrolyzed into nucleosides. As used in the present description, polynucleotides include, as non-limiting examples, all nucleic acid sequences which are obtained by any means available in the art, including, as non-limiting examples, recombinant means, i.e. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means.

It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in a natural state. The sequences of the present invention have been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by copying, their environment having been at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by chemical synthesis should also be mentioned here.

Unless otherwise indicated, the term nucleotide sequence encompasses its complement. Thus, a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

Vector

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Furthermore, the term "vector" herein refers to a viral particle capable of transporting a nucleic acid.

Use

The term "prophylaxis" or "prevention" and the like means slowing down or preventing the onset of symptoms of a disease, disorder or infection. The term "induction of an immune response" as used in the present invention refers to the specific control of or effect on the activity of the immune response and includes activation of an immune response, stimulation of an immune response, enhancement of an immune response.

The term "specific immunity" as used in the present invention refers to a condition of being immune to disease after the induction of an immune response.

The term "disorder" means any condition that would benefit from treatment with the compound of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.

“Disease” is a state of health of an animal where the animal cannot maintain homeostasis, and where if the disease is not ameliorated then the animal's health continues to deteriorate.

The terms “subject,” “patient,” “individual,” and the like are used interchangeably in the present description, and they refer to any animal amenable to the methods described in the present description. In certain non-limiting embodiments, the subject, patient or individual is a human. Said subject may be either male or female, of any age.

"Therapeutically effective amount" or "effective amount" refers to that amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disease being prevented.

Detailed description of the invention

Recombinant polypeptide for increasing the titer of antibodies to influenza virus, and nucleic acid encoding same

In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to influenza virus, which comprises: at least 1 fragment from the coronavirus S glycoprotein with the amino acid sequence

at least 1 fragment from the coronavirus nucleoprotein with the amino acid sequence and, if necessary, 1 fragment from the coronavirus membrane protein with the amino acid sequence

MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPV TL ACF VL AAVYRINWIT GGI AIAM ACL V GLMWL S YFI ASFRLF ARTRSMW SFNPETNILL NVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKL GASQRVAGDSGF AAY SRYRIGNYKLNTDHS S S SDNIALL VQ (SEQ ID NO: 5), wherein the at least 1 fragment from the coronavirus S glycoprotein is selected from the group: the at least 1 fragment from the coronavirus nucleoprotein is selected from the group: the fragment from the coronavirus membrane protein FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 4 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein and 1 fragment from the coronavirus nucleoprotein. In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein, 1 fragment from the coronavirus nucleoprotein and a fragment of the coronavirus membrane protein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein and 3 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 1 fragment from the coronavirus S glycoprotein and 3 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein and 2 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein and 4 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 4 fragments from the coronavirus S glycoprotein and 3 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein and 4 fragments from the coronavirus nucleoprotein.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, as well as 1 fragment from the coronavirus nucleoprotein with the amino acid sequence of SEQ ID NO: 20.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, 1 fragment from the coronavirus nucleoprotein with the amino acid sequence of SEQ ID NO: 15 and a fragment of the coronavirus membrane protein with the amino acid sequence of SEQ ID NO: 22.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, as well as 3 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, as well as 3 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 21.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 1 fragment from the coronavirus S glycoprotein with the amino acid sequence of SEQ ID NO: 10 and 3 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 1 fragment from the coronavirus S glycoprotein with the amino acid sequence of SEQ ID NO: 10 and 3 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 21.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, as well as 2 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 21

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 2 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13 and 4 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 4 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, as well as 3 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that includes 3 fragments from the coronavirus S glycoprotein with the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, as well as 4 fragments from the coronavirus nucleoprotein with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:36. In some embodiments of the invention, the isolated nucleic acid encodes a polypeptide that comprises an amino acid sequence that is selected from the group comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

In some embodiments of the invention, the isolated nucleic acid includes a nucleotide sequence that is selected from the group that comprises the nucleotide sequences of SEQ ID NO: 59-76.

Any of the above nucleic acids encodes a recombinant polypeptide to increase the titer of antibodies to influenza virus. Said polypeptide is a polyepitope cassette for T cell response against coronavirus proteins. In the application materials, said polypeptide is also referred to as a cassette according to the invention.

The cassettes according to the invention include at least one sequence that is selected from the group:

1) a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (1180-1197 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: QKEIDRLNE V AKNLNE SL (SEQ ID NO: 95); a fragment (1164-1178 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: EIDRLNE V AKNLNE S (SEQ ID NO: 96).

2) a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (1199-1213 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): WLGFIAGLIAIVMVT (SEQ ID NO: 97); a fragment (1220-1231 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIVMVT (SEQ ID NO: 98); a fragment (1220-1229 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIVM (SEQ ID NO: 99).

3) a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (1035-1062 aar) of SARS-CoV-1 virus S glycoprotein, partially coinciding with that of SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1):

PQAAPHGVVFLHVTYVPSQERNFTTAPA (SEQ ID NO: 100); a fragment (1056-1078 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVT YVP AQEKNFTT A (SEQ ID NO: 101); a fragment (1056-1073 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYVPAQEK (SEQ ID NO: 102); a fragment (1055-1073 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): S APHGVVFLHVTYVPAQEK (SEQ ID NO: 103); a fragment (1055-1070 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): S APHGVVFLHVT YVP A (SEQ ID NO: 104).

4) a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10). 5) a fragment (504-522 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GY QP YR V VVL SFELLHAP A (SEQ ID NO: 105).

6) a fragment (504-516 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GY QP YR V VVL SFE (SEQ ID NO: 106).

7) a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (946-971 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) partially from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: GKLQDVVNQNAQ ALNTLVKQL S SNF G (SEQ ID NO: 107); a fragment (947-970 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: KLQDVVNQNAQALNTLVKQLSSNF (SEQ ID NO: 108).

8) a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12).

9) a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLOSLOTYV (SEQ ID NO: 13).

10) a fragment (996-1004 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSL (SEQ ID NO: 14).

1) a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (216-234 aar) of the SARS-CoV-1 virus N nucleoprotein, partially coinciding with that of the SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GET AL ALLLLDRLN QLESK (SEQ ID NO: 109); a fragment (214-234 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GGDAALALLLLDRLNQLESKM (SEQ ID NO: 110); a fragment (216-235 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO:

3): DAALALLLLDRLNQLESKMS (SEQ ID NO: 111); a fragment (216-234 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): DAALALLLLDRLNQLESKM (SEQ ID NO: 112); a fragment (216-233 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): DAALALLLLDRLNQLESK (SEQ ID NO: 113);

2) a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (92-118 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): RRIRGGDGKMKDLSPRWYFYYLGTGPE (SEQ ID NO: 114); a fragment (92-117 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): RRIRGGDGKMKDL SPRW YF Y YLGT GP (SEQ ID NO: 115); a fragment (98-119 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): D GKMKDL SPRW YF YYLGT GPE (SEQ ID NO: 116).

3) a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17).

4) a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

5) a fragment (293-302 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19).

6) a fragment (316-324 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

This fragment may be provided in cassettes as part of, for example, the following variant: a fragment (303-337 aar) of the SARS-CoV-1 virus N nucleoprotein, partially coinciding with that of the SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): PQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGA (SEQ ID NO: 117).

7) a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKFUDA (SEQ ID NO: 21).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (340-365 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): DDKDPNFKDQVILLNKHIDAYKTFPP (SEQ ID NO: 118); a fragment (341-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): DKDPNFKDQVILLNKHIDA (SEQ ID NO: 119); a fragment (340-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): DDKDPNFKDQVILLNKHIDA (SEQ ID NO: 120).

The cassettes according to the invention include, if necessary, the sequence of a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

This fragment may be provided in cassettes as part of, for example, the following variants: a fragment (40-75 aar) of the SARS-CoV-1 virus membrane M protein, partially coinciding with that of the SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): NRNRFLYIIKLVFLWLLWPVTLACFVLAAVYRINWV (SEQ ID NO: 121); a fragment (42-75 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): RNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINW (SEQ ID NO: 122); a fragment (50-79 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): KLIFLWLLWPVTLACFVLAAVYRINWITGG (SEQ ID NO: 123); a fragment (50-77 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): KLIFLWLLWPVTLACFVLAAVYRINWIT (SEQ ID NO: 124).

In particular variants, the above sequences were used as part of specific antigenic cassettes:

Cassette 1 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 1 is described by the following sequence: NRNRFL YIIKL VFLWLL WPVTLACFVL AAV YRINWV GET AL ALLLLDRLN QLE S KEIDRLNE V AKNLNES WLGFI AGLI AIVM VTPQ A APHGV VFLH VT YVP S QERNF TT AP A (SEQ ID NO: 23)

Further, it should be noted that above antigenic cassette 1 as part of the vector is fused with a self-cleaving peptide. This Fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPNRNRFL YIIKL VFLWLL WPVTLACFVLAAVYRJ NWVGETALALLLLDRLNQLESKEIDRLNEVAKNLNESWLGFIAGLIAIVMVTPQAAPHG VVFLH VT YVP S QERNF TT AP A (SEQ ID NO: 41)

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 41. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

A reference to a nucleotide sequence encompasses the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

In a particular embodiment, the nucleic acid that encodes the antigenic cassette 1 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 1 and the self-cleaving peptide includes the nucleotide sequence:

Cassette 2 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (316-324 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

Antigenic cassette 2 is described by the following sequence:

Further, it should be noted that above antigenic cassette 2 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 42. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes the antigenic cassette 2 includes the nucleotide sequence: In a particular embodiment, the nucleic acid that encodes the antigenic cassette 2 and a self-cleaving peptide includes the nucleotide sequence:

Cassette 3 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (316-324 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

Antigenic cassette 3 is described by the following sequence:

Further, it should be noted that above antigenic cassette 3 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 43. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

A reference to a nucleotide sequence encompasses the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof. In a particular embodiment, the nucleic acid that encodes antigenic cassette 3 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 3 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 4 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 4 is described by the following sequence:

Further, it should be noted that above antigenic cassette 4 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 44. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 4 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 4 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 5 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 5 is described by the following sequence:

Further, it should be noted that above antigenic cassette 5 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 45. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 5 includes the nucleotide sequence: In a particular embodiment, the nucleic acid that encodes antigenic cassette 5 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 6 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 6 is described by the following sequence:

QKEIDRLNEVAKNLNESLAPHGVVFLHVTYVPAQEKKLIFLWLLWPVTLACFVL AAVYRINWITDAALALLLLDRLNQLESKMSFIAGLIAIVMVT (SEQ ID NO: 28).

Further, it should be noted that above antigenic cassette 6 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLAPHGVVFLHVTYVP AQEKKLIFLWLLWPVTLACFVLAAVYRINWITDAALALLLLDRLNQLESKMSFIAGLIAI VMVT (SEQ ID NO: 46).

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 46. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention. A reference to a nucleotide sequence encompasses the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 6 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 6 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 7 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 7 is described by the following sequence:

Further, it should be noted that above antigenic cassette 7 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 47. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 7 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 7 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 8 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 8 is described by the following sequence:

QKEIDRLNEVAKNLNESLAPHGVVFLHVTYVPAQEKNFTTAFIAGLIAIVMVTDA ALALLLLDRLNQLESKKLIFLWLLWPVTLACFVLAAVYRINWITGG (SEQ ID NO: 30).

Further, it should be noted that above antigenic cassette 8 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence: ( )

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 48. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 8 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 8 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 9 includes the following epitope fragments: a fragment (1184-1190 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: DRLNEVA (SEQ ID NO: 7); a fragment (1220-1228 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8); a fragment (1056-1068 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9); a fragment (218-230 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): AL ALLLLDRLN QL (SEQ ID NO: 15); a fragment (53-73 aar) of the above SARS-CoV-2 virus membrane M protein (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigenic cassette 9 is described by the following sequence:

Further, it should be noted that above antigenic cassette 9 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 49. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 9 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 9 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 10 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

Antigenic cassette 10 is described by the following sequence:

Further, it should be noted that above antigenic cassette 10 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 50. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 10 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 10 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 11 includes the following epitope fragments: a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

Antigenic cassette 11 is described by the following sequence:

Further, it should be noted that above antigenic cassette 11 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 51. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

A reference to a nucleotide sequence encompasses the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof. In a particular embodiment, the nucleic acid that encodes antigenic cassette 11 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 11 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 12 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

Antigenic cassette 12 is described by the following sequence: Further, it should be noted that above antigenic cassette 12 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 52. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 12 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 12 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 13 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

Antigenic cassette 13 is described by the following sequence:

Further, it should be noted that above antigenic cassette 13 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 53. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 13 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 13 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 14 includes the following epitope fragments: a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

Antigenic cassette 14 is described by the following sequence:

Further, it should be noted that above antigenic cassette 14 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 54. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 14 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 14 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 15 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

Antigenic cassette 15 is described by the following sequence:

Further, it should be noted that above antigenic cassette 15 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 55. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 15 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 15 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 16 includes the following epitope fragments: a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21); a fragment (293-302 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19); a fragment (316-324 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

Antigenic cassette 16 is described by the following sequence:

Further, it should be noted that above antigenic cassette 16 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 56. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 16 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 16 and a self- cleaving peptide includes the nucleotide sequence:

Cassette 17 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); a fragment (504-515 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from a region of the receptor-binding domain (RBD) responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (138-146 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); a fragment (160-167 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

Antigenic cassette 17 is described by the following sequence:

Further, it should be noted that above antigenic cassette 17 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 57. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 17 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 17 and a self- cleaving peptide includes the nucleotide sequence: Cassette 18 includes the following epitope fragments: a fragment (958-966 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: ALNTLVKQL (SEQ ID NO: 11); a fragment (976-984 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); a fragment (996-1008 aar) of the above SARS-CoV-2 virus S glycoprotein (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); a fragment (104-113 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); a fragment (351-359 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21); a fragment (293-302 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19); a fragment (316-324 aar) of the above SARS-CoV-2 virus N nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

Antigenic cassette 18 is described by the following sequence:

Further, it should be noted that above antigenic cassette 18 as part of the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the polypeptide with the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 58. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In a particular embodiment, the nucleic acid that encodes antigenic cassette 18 includes the nucleotide sequence:

In a particular embodiment, the nucleic acid that encodes antigenic cassette 18 and a self- cleaving peptide includes the nucleotide sequence:

In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide to increase the titer of antibodies to the influenza virus, comprising an amino acid sequence that is selected from the group comprising the amino acid sequences of SEQ ID NO: 23-40.

In some embodiments of the invention, the isolated nucleic acid includes a nucleotide sequence that is selected from the group comprising the nucleotide sequences of SEQ ID NO: 59-76 that encode the amino acid sequences of SEQ ID NO: 23-40, respectively.

Influenza virus-based recombinant virus for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases

The term "influenza virus-based recombinant virus" (or "influenza virus-based virus-like particle", or "recombinant strain of influenza virus") in the context of the present description refers to influenza virus that includes any of the above nucleic acids that encodes the above recombinant polypeptide for inducing specific immunity to influenza virus and/or preventing influenza virus-related-diseases. Thanks to to the constructive feature thereof, the influenza virus-based recombinant virus is able to produce a higher level of antibodies to influenza virus when administered to a mammal. Furthermore, the recombinant influenza virus including the above nucleic acid encoding a recombinant polypeptide based on coronavirus protein fragments allows to form specific T cell immunity to coronavirus when administered to a mammal, and, accordingly, the given influenza virus-based recombinant virus may be used for the combined prevention of coronavirus-related diseases and influenza virus-related diseases. The feature "influenza virus" refers to a virus from the orthomyxovirus family (i Orthomyxoviridae ), including the monotypic genera Alphainfluenzavinis (previously referred to as type A influenza virus), Betainfluenzavirus (previously referred to as type B influenza virus), Gammainfluenzavints (previously referred to as type C influenza virus) and Deltainfluenzaviriis (previously referred to as type D influenza virus), wherein the division by genus is based on the antigenic properties of the rib onucleo protein complex. Influenza virus is a spherical enveloped virus of 100-120 nm in diameter, the outer layer of which consists of a lipid membrane that exposes the main glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA), and the matrix protein M2 forming ion channels. Under the lipid membrane there is disposed the matrix protein 1 (Ml) that defines the shape of the viral particle. The genome of influenza virus is segmented and characterized by a negative-stranded RNA that is located inside the virion in the form of a ribonucleoprotein complex (RNP), In type A influenza virus, 8 RNA fragments encode at least 12 proteins: HA, Ml, M2, NA, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, PB2. Based on the antigenic properties of HA and NA surface antigens, influenza A viruses are divided into serotype subtypes, for example, H1N1, H1N2, H2N2, H3N2, H5N1, etc.

In some embodiments, the isolated influenza virus-based recombinant virus is vaccine influenza virus strains.

The vaccine strains are characterized in that they contain genes encoding influenza virus internal and non- structural proteins (Ml, M2, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, PB2) from a special laboratory strain A/Leningrad/134/17/57 (H2N2) or derivatives thereof. The A/Leningrad/134/17/57 (H2N2) strain was produced by cold adaptation of the epidemic virus A/Leningrad/134/57 (H2N2), by way of 17-fold passaging of same in embryonated chicken eggs at a temperature lowered to 25 °C [Alexandrova GL.Smorodintsev A. A. Obtaining of an additionally attenuated vaccinating cryophilic influenza strain// Rev Roum Inframicrobiol. - 1965. - V 2 - - p. 179-189.]. As a result of passages, the virus A/Leningrad/134/17/57 (H2N2) acquired a set of mutations in the genes encoding the surface and internal proteins of the virion (Table A), which defined its temperature-sensitive, cold-adapted and attenuated phenotype. We determined the strain's primary nucleotide sequence (GISAID database isolate number the EPI ISL 169836, gene access codes EPI555079 to EPI555086). It was previously shown that the mutant PB2 protein is the critical factor of the attenuated phenotype in the strain A/Leningrad/134/17/57 (H2N2), while the polymerase genes PB1 and PA complement this feature [Klimov A.I., Kiseleva I.V., Alexandrova G.I., Cox N.J. Genes coding for polymerase proteins are essential for attenuation of the cold-adapted A/Leningrad/l 34/17/57 (H2N2) influenza virus, in Options for the Control of Influenza IV. 2001, Okinawa, Japan: Elsevier Science BV.]. We further determined the role of individual mutations in the genome of the strain A/Leningrad/134/17/57 (H2N2) in terms of the main phenotypic feature of the virus - sensitivity to elevated incubation temperatures [Isakova-Sivak I, Chen LM, Matsuoka Y, Voeten JT, Kiseleva I, Heldens JG, den Bosch Hv. Klimov A, Rudenko I,, Cox NJ, DonisRO. Genetic bases of the temperature-sensitive phenotype of a master donor vims used in live attenuated influenza vaccines: A/Leningrad/ 134/ 17/57 (H2N2). Virology. 2,011 Apr 10;412(2):297-305. doi: 10 1016/j.virol 2011.01.004],

Table A. Amino acid differences in genome of Len/17 and Len/134 viruses

In some embodiments of the invention, the influenza virus-based isolated recombinant vims is produced from an attenuated influenza vims.

In some embodiments of the invention, the influenza vims-based isolated recombinant vims is produced from a cold-adapted influenza vims.

In some embodiments of the invention, the influenza vims-based isolated recombinant vims is produced from the H2N2 influenza vims.

In some embodiments of the invention, the influenza vims-based isolated recombinant vims is produced from the vims A/Leningrad/134/17/57 (H2N2) or derivatives thereof. In some embodiments of the invention, the influenza virus-based isolated recombinant virus includes any of the above nucleic acids of the invention, which is present in the neuraminidase (NA) gene or the NS gene of influenza virus.

The derivatives of A/Leningrad/134/17/57 (H2N2) virus refer to:

1) influenza A viruses in which the HA and NA surface antigens belong to previously circulating, potentially pandemic, pandemic or seasonal epidemic influenza A viruses of subtypes H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, H5N6, H5N8, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N7, H17N10, H18N11, and the genes of internal and non-structural proteins belong to the A/Leningrad/134/17/57 (H2N2) strain;

2) influenza A viruses in which one or more of the A/Leningrad/134/17/57 (H2N2) genes are replaced with the respective genes of any other type A influenza virus;

3) type A influenza viruses having an amino acid sequence of viral proteins encoded by any nucleotide sequence (with the consideration of the degeneracy of codons for some amino acids) that is identical to A/Leningrad/134/17/57 (H2N2);

4) influenza A viruses of any serotype subtype that comprise amino acid residues in internal and non-structural proteins described for the attenuation donor A/Leningrad/134/17/57 (H2N2) (PB2-Leu478; PB1-Asn265; PB1-Ile591; PA-Pro28; PA-Leu341; Ml-Vall5; Ml- Leul44; NS2-Ilel00), in full or in various combinations;

5) type A influenza viruses, the sequence of internal and non-structural proteins of which (Ml, M2, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, PB2) differs from the A/Leningrad/134/17/57 (H2N2) virus in a number of amino acids;

6) influenza A virus of any serotype subtype comprising modified genes of the A/Leningrad/134/17/57 (H2N2) strain (with the consideration of truncation of the NS1 gene reading frame).

Pharmaceutical composition/vaccine

In some embodiments of the invention, the active substance in the above compositions is present in an effective amount, for example, in a biologically effective amount.

In some embodiments of the invention, the active substance in the above compositions is present in an effective amount, for example, in a therapeutically effective amount.

In particular embodiments, the present invention relates to a pharmaceutical composition comprising the influenza virus-based recombinant virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid carrier. For other methods of administration, the carrier may be either solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate- buffered saline solution. For inhalation administration, the carrier is respirable, and preferably is in a solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives that are common for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

"Pharmaceutical composition" means a composition comprising the above influenza virus-based recombinant virus of the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, auxiliary, distributing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery controllers, the choice and proportions of which depend on the type and route of administration and dosage. The pharmaceutical compositions of the present invention and methods of preparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements. The composition may comprise a buffer composition, tonicity agents, stabilizers and solubilizers.

"Pharmaceutically acceptable" means a material that does not have biological or other negative side effects, for example, the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, in transduction of a cell ex vivo or in administration in vivo of the influenza virus-based recombinant virus of the invention directly to a subject.

The term "excipient" is used herein to describe any ingredient other than the above ingredients of the invention. These are substances of inorganic or organic nature which are used in the pharmaceutical production/manufacturing in order to give drug products the necessary physicochemical properties.

"Stabilizer" refers to an excipient or a mixture of two or more excipients that provide the physical and/or chemical stability of the active agent.

The term "buffer", "buffer composition", "buffering agent" refers to a solution, which is capable of resisting changes in pH by the action of its acid-base conjugate components, which allows the influenza virus-based vector product to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range from 4.0 to 8.0. Examples of buffers used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions. The pharmaceutical composition is "stable" if the active agent retains physical stability and/or chemical stability and/or biological activity thereof during the specified shelf life at storage temperature, for example, of 2-8 °C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.

A pharmaceutical composition according to the invention may be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready formulation. The term "single unit dose" as used herein refers to discrete quantity of a pharmaceutical composition containing a predetermined quantity of an active ingredient. The quantity of the active ingredient typically equals the dose of the active ingredient to be administered in a subject, or a convenient portion of such dose, for example, half or a third of such dose.

Coronavirus refers to viruses that are members of the coronavirus family (Coronaviridae).

The family includes the alphacoronavirus genus and the betacoronavirus genus.

The alphacoronavirus genus includes:

1) human coronavirus 229E, causing ARVI;

2) human coronavirus NL63, causing ARVI.

The betacoronavirus genus includes:

1) human coronavirus OC43, causing ARVI;

2) human coronavirus HKU1, causing ARVI;

3) MERS-CoV;

4) SARS-CoV;

5) SARS-CoV-2.

The above pharmaceutical composition or vaccine may be used to prevent diseases related to any of the above coronaviruses.

In some embodiments of the vaccine, the coronavirus is betacoronavirus.

In some embodiments of the vaccine, the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2. In some embodiments of the vaccine, the specific immunity to coronavirus is a specific T-cell immunity.

The term "vaccine" refers to an immunogenic composition comprising an antigen derived from a pathogen that is used to induce an immune response against a pathogen that confers protective immunity (e.g., immunity that protects a subject from an infection caused by a pathogen and/or reduces the severity of a disease or a condition caused by an infection as a result of a pathogen). Protective immunity may include the production of antibodies and/or cell- mediated response.

Depending on the context, the term "vaccine" may also refer to an antigen suspension or solution that is administered to a vertebrate to develop protective immunity.

In some embodiments of the invention, the vaccine includes an influenza virus-based recombinant virus that is preferably present in a biologically effective amount.

In some embodiments of the invention, the vaccine includes an influenza virus-based recombinant virus that is preferably present in a therapeutically effective amount.

All definitions and explanations relating to the pharmaceutical composition shall also apply to the vaccine.

Use

Any of the above influenza virus-based recombinant viruses or the above composition may be used to induce specific immunity to influenza virus and coronavirus and/or prevent influenza virus-related diseases and coronavirus-related diseases, wherein the coronavirus refers to viruses from the coronavirus family (Coronaviridae), in particular, alphacoronaviruses that include human coronavirus 229E or human coronavirus NL63, and betacoronaviruses that include human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV, SARS- CoV-2.

In some embodiments of the method, the coronavirus is betacoronavirus.

Any method for administering the influenza virus-based recombinant virus, which is recognized in the art, may be suitably used for the above influenza virus-based recombinant virus of the present invention.

Exemplary modes of administration include topical application, intranasal, inhalation, transmucosal, transdermal, enteral (e.g. oral, rectal), parenteral (e.g. intravenous, subcutaneous, intradermal, intramuscular) administrations, as well as direct tissue or organ injections.

The preferred method of administration of the above influenza virus-based recombinant virus of the present invention is intranasal administration.

Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for the preparation of solution or suspensions in liquid prior to injection, or as emulsions. Alternatively, one may administer the above influenza virus-based recombinant virus of the present invention in a local rather than systemic manner, for example in a depot or sustained-release formulation.

The influenza virus-based recombinant virus of the present invention is administered into an organism in an effective amount.

In some embodiments, the influenza virus-based recombinant virus of the present invention is preferably administered into an organism in a biologically effective amount.

In some embodiments, the influenza virus-based recombinant virus of the present invention is preferably administered into an organism in a therapeutically effective amount. Dosages of the above influenza virus-based recombinant virus of the invention will depend on the mode of administration, the particular viral vector, and they can be determined in a routine manner. Exemplary doses to achieve the therapeutic effect are viral titers of at least about 10⁵, 10⁶, 10⁷, 10⁸ infectious units or more, preferably from 10⁷ to 10⁸ infectious units.

Any of the above influenza virus-based recombinant viruses or the above composition may be used to induce specific immunity to viruses that are members of the coronavirus family (Coronaviridae), which are selected from the group: human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-

CoV or SARS-CoV-2. The versatility of the above recombinant virus according to the invention, as a means for prevention of any coronavirus from the above group, is achieved due to the following.

The antigenic cassettes of the present invention which were employed to induce the cellular immune response against SARS-COV-2 used at least 102 fragments of amino acid sequences coinciding in SARS-CoV-1 and SARS-COV-2 proteins registered in the IEDB database (iedb.org) as immunogenic ones: GY QPYRVWL, QPYRVVVLSF, PYRVVVLSF,

LQDVVNQNAQALNTL, DWNQNAQALNTLVKQL, AQALNTLVK,

Q ALNTL VKQL S SNF GAI, ALNTLVKQL, LNTLVKQLSSNFGAI, S SNF GAIS S VLNDIL, AISSVLNDILSRLDKVE, S VLNDIL SR, VLNDILSRL, ILSRLDKVEAEVQIDRL, RLDKVEAEV, E AE V QIDRLIT GRLQ SL, AEVQIDRLIT, AEVQIDRLI, VQIDRLITGR, IDRLITGRLQSLQTY, RLITGRLQSLQTYVTQQ, LIT GRLQ SL, TGRLQSLQTYVTQQL, GRLQSLQTY, RLQSLQTYV, LQSLQTYVTQQLIRA, SLQTYVTQQLIRAAEIR, LQTYVTQQLIRAAEI, QTYVTQQLIRAAEIR, APHGVVFLHV, GVVFLHVTY, VVFLHVTYV, EIDRLNEVAKNLNESLIDLQELGKYEQY,

EIDRLNEVAKNLNESLIDLQELGKYEQY, RLNEVAKNL, EVAKNLNESLIDLQELG, NLNESLIDL, SLIDLQELGK, LIDLQELGKY, QELGKYEQYI, YEQYIKWPWY, WLGFIAGLIAIVMVT, LGFIAGLIAIVMVTI, FIAGLIAIV, GLIAIVMVTI, LSPRWYFYY, SPRWYFYYL, ATEGALNTPK, ALNTPKDHI, VLQLPQGTTLPKGFY, VLQLPQGTTL, LQLPQGTTL, QLPQGTTLPKGFYAEGSR, QLPQGTTLPKGFYAEGSRGGSQ, QLPQGTTLPKGFYAE, QLPQGTTLPK, LPQGTTLPKG, GTTLPK, GTTLPKGFY, ALALLLLDR, LALLLLDRL, LLLLDRLNQ, LLLLDRLNQL, LLLDRLNQL, IRQGTDYKHWPQIAQFA, QGTDYKHW, KHWPQIAQFAPSASAFF,

AQFAPSASAFFGMSR, AQFAPSASA, AQFAPSASAFFGMSRIGM,

QFAPSASAFFGMSRIGM, APSASAFFGM, SASAFFGMSR, ASAFFGMSR,

AFF GMSRIGMEVTPSGTW, FF GM SRIGME VTP S GTW, GMSRIGMEV, MSRIGMEVTPSGTWL, GMEVTPSGTWL, MEVTPSGTWL, VTPSGTWLTY, TPSGTWLTY, ILLNKHID, ILLNKHIDA, LLNKHIDAYKTFPPTEPK,

LNKHIDAYKTFPPTEPK, NKHIDAYKTFPPTEP, TFPPT, KTFPPTEPKK, KTFPPTEPK, TFPPTEPK, FLWLLWPVT, FLWLLWPVTLACFVL, FLWLLWPVTL, LWLLWPVTL, WLLWPVTLA, LWPVTLACF, WPVTLACFVL, TLACFVLAA, TLACFVLAAV, FVLAAVYRF

In some cases, epitopes overlapped each other to form more extended immunogenic regions (accordinly, also coinciding in SARS-COV-1 and SARS-COV-2): LQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTY VTQQLIRAAEIR, EIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWY,

GYQPYRVVVLSF, WLGFIAGLIAIVMVTI,

IRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTY,

VLQLPQGTTLPKGFYAEGSRGGSQ, ILLNKHID AYKTFPPTEPKK, ALALLLLDRLNQL, ATEGALNTPKDHI, LSPRWYFYYL, FLWLLWPVTLACFVLAAVYRI.

Most of these regions exhibit consensus hydrophobic motifs that are typical of antigenic peptides bound by the major histocompatibility complex (MHC) proteins of different classes, for example, FLWLLWPVTLACFVL AAVY. WLGFIAGLIAIVMVTI. APHGVVFLHVTYV. FAPSASAFFGM, FGAISSVL, ALALLLL (hydrophobic residues are underlined). Due to this characteristic feature, some fragments of coronavirus proteins comprising the most versatile motifs are recognized by several HLA allelic variants. For example, the epitope LSPRWYFYY binds to at least seven HLA allelic forms (http://www.iedb.org/epitope/39576). In the meantime, the epitope FIAGLIAIV was shown to bind to all HLA-A2 variants, including the one that is most common in the world population, HLA-A*02:01 (http://www.iedb.org/epitope/16156).

Among the previously selected sequences used in the present invention, at least 10 antigenic fragments were found that have significant homology with those of other pandemic and epidemic strains of coronaviruses, including those causing seasonal ARVI (betacoronaviruses HKU1 and OC43, alphacoronaviruses 229E andNL63).

Most of the conserved sites used in the invention relate to the C-terminal region of S glycoprotein. They have the greatest immunogenicity in healthy people due to their greater similarity to the sequences of coronaviruses that cause seasonal ARVI: HKU1, OC43, 229E and NL63 [Braun, T, Loyal, L., Frentsch, M. et al. SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature 587, 270-274 (2020). https://doi.org/10.1038/s41586-020- 2598-9],

Accordingly, the fragment IL SRLDK VE AE V QIDRLIT GRL of the SARS-CoV-2 S glycoprotein coincides by 81.8% with the respective fragment of betacoronaviruses HKU1 and OC43 that cause acute respiratory infections in humans; the region WNQNAQALNTLVKQL coincides by 81.2%, and the region AEVQIDRLITGR coincides by 83.3%:

The common sequence (948-1012 aar of SARS-CoV-2 S glycoprotein) includes the following fragments used in the present invention: ALNTLVKQL (IEDB ID: 2801), VLNDILSRL (IEDB ID: 69657), RLDKVEAEV (IEDB ID: 54507), LITGRLQSL (IEDB ID: 36724), RLQSLQTYV (IEDB ID: 54725).

For all the given fragments, binding to the most common allelic variant in the world population, HLA-A*02:01, has been described, which binding is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB)

(http s . //www i edb . or g/mh c/ 143 ) .

For the epitopes RLDKVEAEV, LITGRLQSL, RLQSLQTYV, binding to the allelic variant HLA-A*02:02 has also been described (whereas for LITGRLQSL, binding to all HLA- A*02 has been described), which binding is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/200).

Finally, binding to allelic variants HLA-A*02:03, HLA-A*02:06 and HLA-A*68:02 has also been described for epitopes RLDKVEAEV and RLQSLQTYV.

HLA-A*02:03 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/201). HLA-A*02:06 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (htps://www.iedb.org/mhc/204)_·

HLA-A*68:02 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://wrww.iedb.org/mhc/231).

All key residues of these consensus sequences, acting as a "hydrophobic anchor" for binding to HLA (at position 9, 2, and, in one case, 1), are found in all respective epitopes (with the exception of RLDKVEAEV) of all coronaviruses in question (hereinafter the residues that coincide with SARS-CoV-2 are underlined, key residues for binding to HLA are in bold):

According to the IEDB, the remaining amino acid variations of the sequences of different strains either do not have a noticeable effect on binding to the respective HLA, or further enhance interaction with same (for example, for HLA-A*02:01, the substitution of lysine residue 4 in RLDKVEAEV in SARS-CoV-2 for an asparagine residue in HKU 1 and alanine residue in OC43; or the substitution of lysine residue 7 in ALNTLVKQL in SARS-CoV-2 for a serine or glutamine residue in the other strains). Detailed binding efficiency maps for each amino acid residue at each antigen position are available on the IEDB website: HLA-A*02:01

(www . iedb . or g/motif ou tpu t/HL A- A- 02 :Ql-9.html), HLA-A*02:02 (www .iedb . org/m otif_ output/HI A- A -02.02-9.html), HLA-A*02:03 (vyww.iedb.org/motif output/HLA~A-Q2:Q3-9 html), HLA-A*02:06 (www.iedb.Org/motif_output/HLA-A-02:06-9.htmi), HLA-A*68:02 (wvyw.iedb.org/motif output/HLA-A-68:02-9.html).

Another conserved C-terminal region of S glycoprotein used in the present invention is the sequence KNLNESLIDLQELGKYEQYIKWPWYIWL (1191-1218 aar in SARS-CoV-2):

This region includes, inter alia, the fragments NLNESLIDL (IEDB ID: 44814) and YEQYIKWPWY (IEDB ID: 73751) used in the invention. For NLNESLIDL, binding to HLA- A*02:01 has been described (see the consensus sequence of binding antigen above), for YEQYIKWPWY binding to HLA-B* 18:01, HLA-B*40:01, HLA-B *44: 02, HLA-B *44: 03 and HLA-B*45:01 has been described, but the consensus sequences of antigen have been described only for HLA-B *44: 03 and HLA-B *45:01.

HLA-B*44:03 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/34).

HLA-B*45:01 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/36).

All key residues of these consensus sequences acting as a "hydrophobic anchor" for binding to HLA are either found in all respective epitopes of all coronaviruses under consideration (with the exception of MERS), or coincide with those of SARS-CoV-2:

According to the IEDB, the remaining amino acid variations in the sequences of different strains do not have a noticeable effect on binding to the respective HLA (ww w i edb . org/moti f ontpnt/HI, A- A-02 : 01 -9. him I ; www i edb . org/motl f__output/HL A -B-44 : 03 - IQ.html; www. iedb . org/motif output/HL A-B-45:01-10, html).

Also, the C-terminal region of S glycoprotein contains the fragments APHGVVFLHVTYV (1056-1068 aar in SARS-CoV-2) and FIAGLIAIV (1220-1228 aar in

SARS-CoV-2). used in the invention.

For the immunogenic sequences VVFLHVTYV (IEDB ID: 71663) and FIAGLIAIV (IEDB ID: 16156), binding to HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:06, HLA-A*68:02 has been described (see above for the consensus sequences of binding antigens). The both fragments are less conserved among most coronaviruses, but retain homology to individual strains (VVFLHVTYV SARS-CoV-2 to a sequence of alphacoronavirus 229E; FIAGLIAIV to a sequence of MERS):

According to the IEDB, insignificant amino acid variations in the sequences of different strains do not have a noticeable effect on binding to the respective HLA (see above for antigen binding efficiency maps).

Among the sequences of the SARS-CoV-2 coronavirus nucleoprotein used in the invention, the fragment 104-118 aar (LSPRWYFYYLGTGPE) is the most conserved: the coincidence with the sequences of betacoronaviruses HKU1 and OC43 reaches 92.8%, and that with the sequences of MERS reaches 86.7%:

The common sequence includes, inter alia, the antigenic fragment SPRWYFYYL (IEDB ID: 60242), for which binding to HLA-B has been described: HLA-B*07:02, HLA-B*51:01, HLA-B*53:01, HLA-B*54:01. The consensus sequences of binding antigen have been described only for HLA-B*07:02 and HLA-B*51:01:

HLA-B*07:02 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/251).

HLA-B*51:01 is characterized by interaction with peptides that have structures specified in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/40).

The key residues acting as a "hydrophobic anchor" for binding to HLA (at positions 2 and 9) remain unchanged in the sequences of all coronaviruses in question (SARS-CoV-2, SARS- CoV-1, HKU1, OC43, 229E, NL63), whereas the other substitutions do not have a significant effect on binding, except for a proline residue at position 1 for NL63, which hinders binding to HLA-B *07: 02 (https .//www . i edb . or g/moti f ou tpu t/HI, A - B-07 : 02-9. him I ; https://www.iedb.org/motir output/HLA-B-51 :Ql-9.html):

Some of the fragments used in the invention (ALNTLVKQL, VLNDILSRL, FIAGLIAIV) were shown to be highly conserved between a wide range of known coronaviruses with up to 100% homology in 21 strains of bat, pangolin and civet coronaviruses [Prakash S, Srivastava R, Coulon PG, et al. Genome-Wide B Cell, CD4+, and CD8+ T Cell Epitopes That Are Highly Conserved between Human and Animal Coronaviruses, Identified from SARS-CoV- 2 as Targets for Preemptive Pan-Coronavirus Vaccines. J Immunol. 2021;206(ll):2566-2582. doi: 10.4049/jimmunol.2001438. Supplemental 1], making it possible to expect the formation of memory cells against new cases of zoonotic coronavirus pandemics in the future. Figure 19 shows a comparison between sequences of S glycoprotein fragments of various coronaviruses and indicates the percentage of identity.

It is hard to accurately assess the effectiveness of immune system activation against a wide range of coronaviruses because of the assessment of identity of antigen processing for sequences of proteins of different coronaviruses in view of a large number of individual factors, such as the different probability of immunoproteasome formation with different proteolytic activity depending on the intensity of the interferon response to the virus [Neefjes, J., Jongsma, M., Paul, P. et al. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11, 823-836 (2011). https://doi.org/10.1038/nri30841 [Seifert U, Bialy LP, Ebstein F, et al. Immunoproteasomes preserve protein homeostasis upon interferon- induced oxidative stress. Cell. 2010;142(4):613-624. doi: 10.1016/j. cell.2010.07.036], as well as poor knowledge of this matter. Nevertheless, in most cases, the sequences flanking the key immunogenic fragments of the coronaviruses used in the present invention are also extremely identical to each other.

Thus, the use of a vaccine containing antigenic cassettes of the present invention may form a stable cellular immunity to SARS-CoV-2 and SARS-CoV-1, equally, as well as to all region-specific variants of SARS-CoV-2 known at the time of publication of the invention, including the "UK" strain SARS-CoV-2 B.l.1.7, "Brazilian" strain SARS-CoV-2 P.1, "South African" strain SARS-CoV-2 B.1.351 and "Indian" strain SARS-CoV-2 B.1.617.2 (variants). For example, it is known that activated T cells that recognize the epitope RLQSLQTYV are observed in more than 70% of patients who have been exposed to COVID-19 in the past [Shomuradova AS, Vagida MS, Sheetikov SA, et al. SARS-CoV-2 Epitopes Are Recognized by a Public and Diverse Repertoire of Human T Cell Receptors. Immunity. 2020;53(6):1245- 1257. e5. doi:10.1016/j.immuni.2020.11.004] [Liao, M., Liu, Y., Yuan, J. et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nat Med 26, 842-844 (2020). https://doi org/10.1038/s41591 "020-0901-9], which epitope is part of the antigenic cassettes of the present invention.

In addition, all of the above antigenic fragments used in the present invention were shown to have not only a high degree of identity to corresponding fragments of other strains of the coronavirus family in interaction with HLA of different groups, but also experimentally confirmed induction of T cell immunity followed by activation of the interferon response, which fact suggests that cellular immunity may also be formed against potentially pandemic MERS strain and strains causing seasonal acute respiratory viral infections (betacoronaviruses HKU1 and OC43, alphacoronaviruses 229E and NL63).

The above calculations are confirmed by the latest scientific publications in leading scientific journals. For example, the article [Katja G. Schmidt et al., SARS-CoV-2-Seronegative Subjects Target CTL Epitopes in the SARS-CoV-2 Nucleoprotein Cross-Reactive to Common Cold Coronaviruses; Front Immunol. 2021; 12: 627568; doi: 10.3389/fimmu.2021.627568] demonstrates a cross-reactive cellular immune response to SARS-CoV-2 and human ARVI- related betacoronaviruses: OC43 and HKU1. In particular, the vast majority of T cells recognizing epitopes from the SARS-CoV-2 fragment DLSPRWYFYYL used in the invention showed a pronounced interferon response to the peptide QLLSPRWYFYYL from OC43 and HKU1 proteins. Also, the article [Kevin O. Saunders ET ALL., Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses, Nature, 2021, 1-24, https://doi.org/10.1038/s41586-021- 03594-0] describes universal vaccine antibodies that have successfully neutralized four different coronaviruses. According to this article, in tests on macaques, the novel nanoparticle vaccine showed that the antibodies generated following vaccination can effectively neutralize not only SARS-CoV-2, but also two SARS-CoV-2-like bat coronaviruses and SARS-1. The authors of the given article believe that the subject vaccine can protect not only from SARS-CoV-2, but also from other potential infections that can pass to humans from animals, since, in all cases, the antibodies neutralized not only SARS-CoV-2 (including the "UK" variant B.l.1.7, "South African" variant B.1.351 and "Brazilian" variant P.l), but also SARS-CoV-1 and two bat coronaviruses.

Examples The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

Materials and general methods

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer protocols. Briefly, plasmid DNA was produced for further manipulation in E. coli cells grown under selective antibiotic pressure so that the plasmids were not lost in the cell population. We isolated the plasmid DNA from cells using commercial kits, measured the concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. The DNA fragments were ligated to each other using ligases and transformed into bacterial cells for the selection of clones and further production. All resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing.

Gene synthesis

Desired gene segments were prepared from oligonucleotides made by chemical synthesis. Gene segments of 300 to 1000 bp long, which were flanked by unique restriction sites, were collected by renaturing oligonucleotides on top of each other, followed by PCR amplification from border primers. As a result, a mixture of fragments was produced, including the desired one. The fragments were cloned at restriction sites into intermediate vectors, following which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

DNA sequence determination

DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data was processed in SnapGene Viewer 4.2 or higher for sequence creation, mapping, analysis, annotation and illustration.

General description of assembly and cloning of genetic constructs bearing recombinant genome segments of the influenza virus vaccine strain based on the attenuated A/Leningrad/134/17/57 strain, which encode the antigenic cassettes related to the invention. All nucleotide sequences used in the invention, encoding certain immunogenic fragments of SARS-CoV-2 virus proteins, as well as various auxiliary regulatory sequences, such as signal peptides, linkers, functional domains of other proteins, self-cleaving peptides, etc., were produced by assembling de novo from individual oligonucleotide fragments of about 100 nucleotides long by renaturing them on top of each other, followed by PCR amplification from extreme primers. To optimize the assembly process and for the stability of the final secondary structure, software developed by JSC "Biocad" was employed. When synthesising the transgenes sequences, the desired Sapl restriction sites or overlaps (sequence regions identical for insertion and vector) of 20-40 nucleotide long were added to the 5’ and 3’ ends of the molecule for further molecular cloning by ligase-free methods (SLIC, Gibson method).

The resulting synthetic sequences were introduced into the influenza virus reverse genetics vector pCIPolISapIT comprising sequences of a particular segment of the influenza virus genome, by various methods:

1. Cloning by restriction and ligation.

2. One-step molecular cloning SLIC (sequence and ligase independent cloning) utilizing T4 polymerase, described ubiquitously, including in "Sambrook J. et al. Molecular cloning: A laboratory manual", and optimized in JSC "Biocad".

3. Gibson method.

DNA sequences of the cloned fragments were confirmed by sequencing vectors by the Sanger method.

One of the following sequences: TAATGATAA, TAAATGATTAA was used as stop codons for terminating the translation of the antigenic cassette.

The disclosed recombinant plasmids of the invention enable pronounced induction of both branches of the immune response while maintaining the functionality of the vector — primarily maintaining the ability thereof to replicate and repeatedly present the target SARS- CoV-2 antigens to the immune system.

The schemes of genetic constructs were produced using SnapGene and SnapGene Viewer software (https://www.snapgene.com).

Example 1. Assembly of genetic constructs bearing the recombinant sixth segment of the genome of the A/Anhiu/1/2013 (H7N9) influenza virus strain, said genetic constructs encoding the modified NA gene and antigenic cassettes.

The nucleotide sequences of the present invention encoding polyepitope antigenic cassettes consisting of combinations of protein fragments of S (or Spike) glycoprotein (SEQ ID NO: 1), those of nucleoprotein (SEQ ID NO: 3), and, if necessary, those of membrane protein (SEQ ID NO: 5), those of SARS-CoV-2 betacoronavirus, which are described in the relevant paragraphs of the detailed description of the invention (specific variants are provided in SEQ ID NO: 59-94) and used to induce a cellular immune response, were produced by de novo synthesis described above. During the synthesis of transgene sequences, the desired Sapl restriction sites were added to the 5’ and 3’ ends of the molecule.

The resulting sequences were introduced by the restriction and ligation method described above into a universal vector for influenza virus reverse genetics pCIPolISapIT_NA-AnhuiN9 comprising sequences of the sixth segment of the influenza virus genome, including the NA(N9) gene encoding type-9 neuraminidase of the A/Anhiu/1/2013 (H7N9) strain, after the last 3’-end codon of the protein-coding portion of the gene (etc) while maintaining an open reading frame for co-translation with proteins of the modified influenza virus, before the protective 3’- untranslated region of 157 nucleotides (Figure 1).

Thus, a full-length neuraminidase is formed upon translation, which is separated from the hybrid polypeptide chain due to the self-cleaving peptide P2A, thus making said neuraminidase fully functional, as well as an antigenic cassette is formed upon translation comprising several target SARS-CoV-2 epitopes, said cassette being further cleaved in cell proteasomes into desired epitopes, which are then presented on the surface cells in complex with class 1 MHC proteins.

The resulting genetic constructs have a length from 6333 to 6577 bp and differ only in unique sequences of transgenes specified above, and are otherwise completely identical. By way of example, Figure 2 shows a map for the vector having a total length of 6577 bp, which encodes an insert having a length of 597 bp as a transgene.

The final vector comprises all elements required for expression of influenza virus native genes and target transgenes, as well as for assembly and desired functioning of the recombinant vaccine strain.

Example 2. Assembly of genetic constructs bearing the recombinant eighth segment of the genome of an attenuated A/Leningrad/134/17/57 influenza virus strain, said genetic constructs encoding the modified NS gene and antigenic cassettes.

The nucleotide sequences of the present invention encoding the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, its derivatives or polyepitope antigenic cassettes consisting of combinations of protein fragments of S (or Spike) glycoprotein (SEQ ID NO: 1), those of nucleoprotein (SEQ ID NO: 3), and, if necessary, those of membrane protein (SEQ ID NO: 5), those of SARS-CoV-2 betacoronavirus, which are described in the relevant paragraphs of the detailed description of the invention (specific variants are provided in SEQ ID NO: 59-94) and used to induce an immune response, were produced by de novo synthesis described above. When synthesizing the transgenes sequences, the desired overlaps (sequence regions identical for insertion and vector) of 30-40 nucleotide long were added to the 5’ and 3’ ends of the molecule for further molecular cloning by ligase-free methods.

The resulting sequences were introduced by the Gibson method described above into the universal vector for influenza virus reverse genetics pCIPolISapIT_NS-Lenl7 comprising a sequence of the eighth segment of the influenza virus genome, including the modified NS gene from the attenuation donor A/Leningrad/134/17/57.

In view of the complex structure and regulation of alternative splicing of the NS gene, as well as the importance of NS1 and NS2 (NEP) proteins for influenza virus to fully function, the following scheme can be used for the cloning:

The target sequences are inserted into the first reading frame, at the 3’ end from the 126th codon of the protein-encoding NS1 portion (aag), while keeping the regulatory sequences of the NS gene, which control the gene's splicing, unchanged (figure 3).

In any case, the translation results in three separate polypeptide chains: a truncated variant of the NS1 protein (1-126 aar), which variant is fully functional in terms of maintaining the replicative activity of the recombinant influenza virus making up the basis of the invention; a full-length NS2 protein; as well as a target insert sequence encoding the combination of protein fragments of S (or Spike) glycoprotein (SEQ ID NO: 1), nucleoprotein (SEQ ID NO: 3), and, if necessary, membrane protein (SEQ ID NO: 5) of the SARS-CoV-2 betacoronavirus.

Activation of the immune system using the described recombinant plasmids of the invention is achieved in various fashions, depending on the regulatory elements and functional domains described in the section "Detailed description of the invention", and includes, inter alia: presentation of immunogenic epitopes on the membrane of infected cells in combination with MHC proteins of the both classes.

The resulting genetic constructs have a length from 5656 to 6286 bp and, within the bounds of a single cloning scheme, differ only in unique sequences of transgenes specified above, and are otherwise completely identical. By way of examples, a map for the vector having a total length of 5899 bp is shown, generated according to the above cloning scheme, respectively; encoding an insert of 597 long as a transgene (Figure 4).

The final vectors comprise all elements required for expression of influenza virus native genes and target transgenes, as well as for assembly and desired functioning of the recombinant vaccine strain.

Example 3. Assembly of vaccine candidates based on attenuated influenza virus.

Recombinant influenza viruses bearing in their genome the immunogenic sites of the novel SARS-CoV-2 coronavirus were produced on the basis of reverse genetics system developed for the attenuation donor A/Leningrad/134/17/57 (Isakova- Sivak, I. ET AL., "Characterization of reverse genetics-derived cold-adapted master donor virus A/Leningrad/134/17/57 (H2N2) and reassortants with H5N1 surface genes in a mouse model.", 2014, Clin Vaccine Immunol 21(5): 722-731 u Isakova- Sivak, I. ET AL., "Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2).", 2011, Virology 412(2): 297-305). Vaccine candidates were produced by electroporation of the approved Vero (WHO) cell line. This example shows the production of viable recombinant influenza viruses bearing various polyepitope coronavirus cassettes encoded in one of three influenza virus genes.

The following materials were used in the experiments:

• Plasmids (vector for influenza virus reverse genetics), with an insert encoding the genes of the attenuation donor of the live influenza vaccine A/Leningrad/134/17/57 (H2N2) (Table 1).

Table 1

• Plasmids encoding genes of influenza virus A/ Anhui/1/2013 (H7N9) (Table 2).

Table 2

• Plasmids encoding genes of influenza virus A/Guangdong- Maonan/ S WL 1536/2019 (H1N1) (Table 3). Table 3

• Plasmids encoding modified genes of influenza virus A/Leningrad/134/17/57 (H2N2) (Table 4).

Table 4

• Plasmids encoding modified genes of influenza virus A/Anhui/01/2013 (H7N9) (Table 5).

Table 5

• Vero cell line

• Serum-free cell culture medium

• Cell dissociation reagent Antibiotic-antimycotic solution

• Cell growth supplement

• Trypsin

• Adhesive cell culture vial

• Dulbecco's phosphate buffered saline

• 6-well cell culture plate

• Ethanol 70%

• Tris-EDTA buffer

• Sodium-acetate buffer containing 3 M NaOAc (pH 4.8). To prepare the buffer, mix 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1 ml of sterile water. Mix by turning the bottle over and store at -20. To prepare 10 ml of 3 M NaOAc solution, dissolve 4.08 g of Na0Ac*3H20 in 8.38 ml of H20 until completely dissolved

• Transfection system

• Reagent kit for electroporation procedure

• 10-11 -day-old developing chicken embryos (SPF) • 96-well U-bottom plates for immunological reactions

• 0.2μ filter

Preparation of the Vero cell line. The Vero cell line was removed from the cryogenic storage in advance. To this end, the cells extracted from the cryogenic storage were washed from dimethyl sulfoxide in 10 ml of heated medium A, centrifuged at 1500 rpm 20 °C for 15 minutes, following which the cell precipitate was introduced into a T-75 culture vial, and culture medium A was added, the cells were incubated in a thermostat at 37 °C and 5% CO2. After the cell monolayer achieved 95-100% confluence, the further protocol was started.

Preparation of Vero cells for transfection. Medium b was prepared, without addition of antibiotic, the medium was filtered using a 0.2m filter. Medium A was removed from the vial with cells, the vial was washed twice with a warm DPBS solution and the cells were removed from the substrate with accutase (1 ml per T-75 vial); to this end, the vial was incubated at 37 °C in a CO 2 incubator for 1 minute. ¾ cells were transferred to another N150 vial (Nunc), 100 ml of medium was added, the mixture was incubated for 16-18 hours at 37 °C in a CO2 incubator.

The next day, the cells were removed from the substrate with accutase in accordance with the same protocol. 10 ml of medium B heated to 37 °C was added, cells were transferred in the medium to a sterile tube and centrifuged for 15 minutes at 1500 rpm at 20 °C.

Preparation of plasmids for producing influenza virus (assembly and concentration). The day before transfection, plasmids encoding 8 influenza virus genes were mixed in 1 Eppendorf tube, at the rate of 2 μg of each plasmid, 120 μg of sodium acetate buffer was then added (in the case of a low concentration of plasmids, the buffer volume was based on 2.5 volumes of pDNA solution). The mixture was vortexed for 5 seconds, following which it was incubated at -20 for 16-18 hours, and then we proceeded to the concentration protocol.

For concentration, eppendorphs with plasmids were centrifuged at 4 °C, 13.2x10 ³ rpm for 15 minutes; immediately after centrifugation, the supernatant was completely removed without touching the precipitate. If there was no precipitate, the protocol was interrupted and restarted from the point of plasmid assembly.

120 ml of 70% ethanol was added to each tube, the precipitate was gently handled to wash it from the tube wall, without resuspending with a pipette. Then the mixture was centrifuged at 4 °C, 13.2x10 ³ rpm for 5 minutes, following which the supernatant was completely removed without touching the precipitate. The precipitate was then dried at room temperature for 15 minutes in uncapped test tubes to remove ethanol residues.

The precipitate was resuspended in 8 mΐ of TE buffer, and then we proceeded to the transfection protocol. Transfection of Vero cells by plasmids encoding 8 influenza virus genes. Before transfection, the dispenser, Neon cuvettes, and electroporation apparatus were placed in a laminar-flow cabinet and irradiated with UV to ensure sterile manipulations. 3 ml of heated medium B was added to each well of a 6-well cell culture plate. 3 ml of E2 buffer was poured into the Neon transfection cuvette which was then placed in an electroporation apparatus (Neon Pipette Station). After cell centrifugation, medium B was removed and the cells were carefully resuspended in R buffer at the rate of 110 mΐ of suspension per virus.

110 mΐ of Vero cell suspension in R buffer was added to Eppendorf tube with plasmid DNA (8 mΐ). The cell suspension with a plasmid solution was carefully stirred using the Neon dispenser with a disposable transfection tip, the suspension was then collected into the tip and the Neon dispenser was placed in the electroporation apparatus with a prepared cuvette. Electroporation was performed according to the following protocol: 1150 V, 20ms, 2 pulses. The suspension was then pipetted over the prepared culture dish (plate well) with medium B.

After electroporation, the plate was placed in a CO2 incubator at 37 °C for 6 hours for cell attachment, after 6 hours, medium B was removed, the cells were washed twice with a warm DPBS solution and 3 ml of medium C was added, the plate was then incubated at 33 °C in a CO 2 incubator for 64-72 hours.

Infection of embryonated chicken eggs (ECEs) with material after transfection. The transfected Vero cells were removed from the plate using a cell scraper. The cells were resuspended in the medium. 10-11-day-old chicken embryos were infected with undiluted medium containing transfected cells in a volume of 0.2 ml/embryo according to the standard procedure. The remaining transfected cells in the culture medium were transferred to a vial and stored at -70 °C. The embryos were incubated at 33 °C in a thermostat.

Detection of the virus in hemagglutination reaction. After 72 hours, the embryos were cooled for 3 hours at a temperature of +4 °C, and the shell was opened to remove chorioallantoic fluid (CAF). 7 rows of a 96-well El-bottom immunological reaction plate were loaded with 50 mΐ of PBS, and 100 mΐ of CAF from each embryo was added to the top row. We titrated 50 mΐ volumes by doubling dilutions in 7 rows, following which 50 mΐ of 1% chicken erythrocyte suspension was added to each plate well. The virus was detected after 20-30 minutes by red cell agglutination in the wells. The virus titer based on hemagglutination was determined by the last virus dilution that exhibited red cell agglutination.

Blind passage. If none of the wells exhibited erythrocyte agglutination, 0.3 ml of CAF from each embryo of passage El was transferred with a syringe to a new embryo for possible accumulation of the virus when the amount was insufficient for detection. The embryos were incubated for 72 hours at 33 °C. In total, up to 3 consecutive blind passages were performed after each transfection.

Accumulation and cloning of the virus in ECEs. If red cell agglutination was detected, the CAF was collected, clarified by low-speed centrifugation (3000 rpm, 15 min), aliquoted into 1 ml vials and stored at -70 °C. Further, the virus was cloned in ECEs by limiting dilution, CAF was sterically collected from individual clones into separate vials. Further, on the basis of one of the clones that did not contain mutations, the working virus stock was accumulated by titration in ECEs.

Control of virus assembly by PCR and sequencing. To control the assembly of the virus in the first passage that showed the presence of the virus by hemagglutination, RNA was isolated from 140 pi of CAF to confirm the presence in the virus a specific insert into the gene (genes) by specific primer-PCR. Individual clones was analyzed by PCR, followed by Sanger sequencing of the sequence of the gene containing the insert. The working stock was also analyzed by sequencing.

Table 6 shows a list of vaccine candidates that were assembled, with transfection dates and assembly results. The last column of the table contains data on the date of receipt of the working stock, as well as data on candidates that discontinued the study early for various reasons.

Table 6. Vaccine candidate assembly results.

By way of using the specified construct design, the assembly of chimeric influenza viruses was successful. Cassettes of the given size embedded in the given regions of influenza virus genes do not interfere with the assembly of a viable influenza virus.

Example 4. Induction of humoral immune response to influenza virus with intranasal immunization of mice

Vaccine viruses were accumulated in embryonated chicken eggs (ECEs). Virus infectivity was determined in ECEs by limiting dilution, calculated by the Reed&Muench method and expressed in 50% embryo infectious doses (EID 50). The immunogenic properties of LIV H7N9 vaccine candidates bearing polyepitope T cell cassettes fromSARS-CoV-2 against influenza virus were evaluated in experiments on HLA-A2.1 RANDOM TRANSGENIC mice (Taconic, USA). We carried out two independent experiments with the vaccine viruses as specified in Table 7. The first experiment studied the immunogenicity of four recombinant influenza viruses with SARS-CoV-2 fragment inserts as compared to a vector control, the LIV H7N9 strain [PMID: 30903496], Mice that received a placebo (saline solution, PBS) were used as a negative control. The second experiment studied the immunogenicity of one recombinant strain as compared to a vector control, a placebo group. Each group included 6 mice aged 6-10 weeks. The animals were immunized intranasally with vaccine viruses at a dose of 10 ⁶ ED 50, twice, with an interval of 21 days, in a volume of 50 mΐ/mouse under light inhalation anesthesia with isoflurane.

Blood serums of mice were collected 10 days after the second immunization and the levels of antibodies to influenza were evaluated in an enzyme immunoassay (ELISA). The ELISA antigen was a whole LIV H7N9 virus, purified by sucrose density gradient, at a concentration of 50 ng/well. The antigen plates were washed three times with a PBS solution supplemented with 0.05% Tween 20 (PBS-T), following which non-specific binding was blocked with a 1% BSA solution in PBS. Two-fold dilutions of serums in PBS were introduced into plate wells in 50 mΐ volumes, starting with a dilution of 1:100. After 1-hour incubation at 37 °C, the plates were washed 3 times with PBS-T, loaded with 50 mΐ of secondary anti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma-Aldrich A9044, USA), diluted at 1:10000. After a 30-minute incubation, the plates were washed 4 times with PBS-T solution, dried and loaded with 50 ml of TMB substrate (1-Step™ Ultra TMB-ELISA Substrate Solution, Thermo, USA). The plates were incubated in a dark place for 5-10 minutes, then the reaction was stopped by 25 mΐ of 1M sulfuric acid. The results were read with XMark Microplate spectrophotometer (BIORAD) at a wavelength of 450 nm. The experiment determined the levels of IgG antibodies produced by the optical density parameter Area Under the Curve, AUC, which was calculated according to the trapezoid rule for each serum separately. Visualization of the results and statistical processing were carried out using GraphPad Prism 5. The statistical difference between the groups was determined by the ANOVA one-factor analysis of variance followed by the Tukey's multiple comparison test.

Figures 5-8 show the results of the assessment of IgG levels of antibodies to whole influenza virus. All vaccine variants induced high levels of anti-H7N9 IgG antibodies, significantly differing from the PBS control group (p<0.0001). Furthermore, recombinant viruses comprising polyepitope cassettes from SARS-CoV-2 were more immunogenic as compared to the control LIV H7N9 strain used as a viral vector. These data indicate increased immunogenic properties of the live influenza vaccine against influenza virus with embedding polyepitope T cell cassettes of the novel coronavirus into the genome of the vaccine strain, regardless of which portion of the influenza virus genome has been modified (the NA gene or the NS1 gene).

Table 7. Recombinant influenza virus vaccine strains studied in mice

Example 5. Evaluation of levels of virus-specific memory T cells in in vitro assay on peripheral blood mononuclears from volunteers with in vivo COVID-19 history.

This experiment is designed to confirm the correct processing of poly epitope T cell cassettes of SARS-CoV-2 embedded in the genome of influenza vaccine virus by human immune cells. Since COVID-19 convalescents have a pool of memory T cells for various epitopes of the novel coronavirus, in vitro incubation of PBMCs of these donors with vaccine candidates may give an idea of the ability of recombinant viruses to express embedded T cell epitopes and, accordingly, stimulate the proliferation of SARS-CoV-2-specific T cells. A prerequisite for setting up the experiment is the parallel stimulation of mononuclear cells by the recombinant virus under study and the LIV conventional strain used as a viral vector. The difference in levels of T cell subpopulations following such stimulation will show the activation of T cells to the embedded T cell epitopes of the coronavirus. Two independent experiments were conducted involving recombinant influenza viruses and a vector control (Table 8). The viruses were accumulated in ECEs and purified on sucrose density gradient to purify the virus- containing liquid from chicken proteins. The infectious titer of the purified influenza virus in ECEs was determined by tenfold dilutions of the virus in 3-5 repeats according to the Reed & Meunch method [1938] and expressed in 50% embryo infectious doses per ml (EIDWml).

Experiment 1 involved 13 volunteers, experiment 2 involved 17 volunteers. The demographic data of the subjects are shown in Table 9. COVID-19 in the subject's medical history was confirmed by a positive result in a viral RNA smear assay (real-time RT-PCR) and/or by SARS-CoV-2-specific IgG antibodies present in blood sera (anti-SARS-CoV-2 RBD ELISA). The experiment was approved at the meeting of the Local Ethics Committee of the Federal State Budgetary Scientific Institution "Institute of Experimental Medicine" (extract from the minutes of the LEC meeting No. 2/20 dated April 7, 2020), and all volunteers signed an informed consent for the study. Furthermore, PBMC samples from volunteers who had never contracted SARS-CoV-2 (absence of antibodies to the coronavirus confirmed by ELISA) were used as a negative control.

To obtain a PBMC culture, whole blood from COVID-19 convalescents was collected into EDTA vacuum tubes, then mixed with Hank's solution in a ratio of 1:1 and isolated by centrifugation using Ficoll density gradient. An isolated fraction of mononuclear cells was resuspended in RPMI-1640 medium containing antimycotic antibiotic, 10 mM HEPES, b- mercaptoethanol and 20 U Roncoleukin, and the resulting suspension at a concentration of 4x 10⁷ cells/ml was used for stimulation by the test viruses (50 mΐ/well cell suspension or 2><10⁶ cells per sample). To stimulate the cells, purified influenza viruses, as well as phorbol and ionomycin as positive controls, were used. Vaccine candidates were added to plate wells with lymphocytes in a 50 mΐ volume at a multiplicity of infection of 3 (amount of infectious units per cell), following which the cells were incubated in a medium containing 30% fetal bovine serum for 16- 18 hours at 37 °C and 5% C02. Intracellular staining of cytokines induced by antigenic stimulation was carried out in several steps of: stopping of cell protein transport, staining of surface antigens, fixation and permeabilization of cells, staining of intracellular antigens, i.e. cytokines. To stop protein transport, Brefeldin A at a dilution of 1:1000 was used, following which the cells were further incubated for 5 hours at 37 °C and 5% C02, and then surface antigens were stained with antibodies to CD4, CD8, CD3, CD45RA, CCR7 markers, as well as with the Zombie Aqua reagent to identify live cells. For fixation and permeabilization of cells, a Fix/Perm buffer was added, and then fixed and permeabilized cells were stained with anti- cytokine antibodies to IFN-g. After washing the stained cells with PermWash buffer, the cells were fixed in CytoLast buffer and stored in the dark at 4 °C until analysed on a flow cytometer. The samples were examined using the Navios flow cytometer (Beckman Coulter). FACS files were processed in FlowJo software packages. Percentages of virus-specific memory T cells were obtained by subtracting the proportion of cells with spontaneous interferon production (unstimulated peripheral blood mononuclear cell) from the levels of T cells detected when cells were stimulated by the appropriate virus. Statistical processing of the results was carried out in the GraphPad Prizm 7.0 software by nonparametric analysis using the Wilcoxon signed-rank test.

Figures 9-17 show data illustrating the levels of IFN-γ-producing cytotoxic and/or helper memory T cells with stimulation of peripheral blood mononuclear cells of COVID-convalescents by vaccine candidates, as compared to stimulation by a control vaccine strain - the viral vector. The recombinant CoVac-13, CoVac-15, CoVac-16 viruses were shown to have a significant increase in the subpopulation of CD4+ effector memory T cells (CD3+CD4+CCR7-), as compared to the viral vector (Figures 9-11). The recombinant CoVac-17, CoVac-29, CoVac-30, CoVac-31, CoVac-32 and CoVac-30 viruses were shown to have a significant increase in both the subpopulation of T helper cells (CD4+) and the subpopulation of cytotoxic (CD8+) effector memory T cells, as compared to the viral vector (Figures 12-17).

Stimulation of mononuclear cells from two blood donors who had not previously contracted with SARS-CoV-2 with all the recombinant viruses studied did not lead to a significant increase in the secretion of IFN-g by helper or cytotoxic memory T cells, as compared to stimulation by the LIV H7N9 viral vector (Figure 18), which fact confirms the specificity of the method used to evaluate human immune cell processing of poly epitope SARS-CoV-2 cassettes embedded in the genome of influenza virus.

Table 8. Recombinant vaccine influenza virus strains studied in in vitro assay on peripheral blood mononuclear cells from COVID-19 convalescents

Table 9. Volunteer demographics

Claims

Claims:

1. An isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to influenza virus, which comprises: at least 1 fragment from the coronavirus S glycoprotein with the amino acid sequence of SEQ ID NO: 1; at least 1 fragment from the coronavirus nucleoprotein with the amino acid sequence of SEQ ID NO: 3; and, if necessary, 1 fragment from the coronavirus membrane protein with the amino acid sequence of SEQ ID NO: 5, wherein at least 1 fragment from the coronavirus S glycoprotein is selected from the group: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; at least 1 fragment from the coronavirus nucleoprotein is selected from the group: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; the fragment from the coronavirus membrane protein is SEQ ID NO: 22.

2. The isolated nucleic acid according to claim 1, wherein the encoded polypeptide comprises from 1 to 4 fragments from the coronavirus S glycoprotein.

3. The isolated nucleic acid according to claim 2, wherein the encoded polypeptide comprises 1 fragment from the coronavirus S glycoprotein; or wherein the encoded polypeptide comprises 2 fragments from the coronavirus S glycoprotein; or wherein the encoded polypeptide comprises 3 fragments from the coronavirus S glycoprotein; or wherein the encoded polypeptide comprises 4 fragments from the coronavirus S glycoprotein.

4. The isolated nucleic acid according to claim 1, wherein the encoded polypeptide comprises from 1 to 4 fragments from the coronavirus nucleoprotein.

5. The isolated nucleic acid according to claim 4, wherein the encoded polypeptide comprises 1 fragment from the coronavirus nucleoprotein; or wherein the encoded polypeptide comprises 2 fragments from the coronavirus nucleoprotein; or wherein the encoded polypeptide comprises 3 fragments from the coronavirus nucleoprotein; or wherein the encoded polypeptide comprises 4 fragments from the coronavirus nucleoprotein.

6. The isolated nucleic acid according to claim 1, wherein the encoded polypeptide comprises a fragment of the coronavirus membrane protein.

7. The isolated nucleic acid according to claim 1, wherein the encoded polypeptide comprises an amino acid sequence that is selected from a group that comprises the amino acid sequence of SEQ ID NO:33 or SEQ ID NO: 36; or wherein the encoded polypeptide comprises an amino acid sequence that is selected from a group that comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25; or wherein the encoded polypeptide comprises an amino acid sequence that is selected from a group that comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 35. wherein the encoded polypeptide comprises an amino acid sequence that is selected from a group that comprises the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 37; or wherein the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 38; or wherein the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 39; or wherein the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 40.

8. The isolated nucleic acid according to any of claims 1 and 6, wherein the encoded polypeptide comprises an amino acid sequence that is selected from a group that comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

9. The isolated nucleic acid according to claim 1, which comprises a nucleotide sequence that is selected from a group that includes the nucleotide sequences of SEQ ID NO: 59-76.

10. The isolated nucleic acid according to claim 1, wherein the coronavirus is betacoronavirus.

11. The isolated nucleic acid according to claim 10, wherein the betacoronavirus is SARS-CoV-2 virus.

12. The isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to influenza virus, comprising fragments from coronavirus proteins and comprising an amino acid sequence that is selected from a group that comprises the amino acid sequences of SEQ ID NO: 23-40.

13. The isolated nucleic acid according to claim 12, which comprises a nucleotide sequence that is selected from a group that comprises the nucleotide sequences of SEQ ID NO: 59-76, which encode the amino acid sequences of SEQ ID NO: 23-40, respectively.

14. The isolated nucleic acid according to claim 12, wherein the coronavirus is betacoronavirus.

15. The isolated nucleic acid according to claim 14, wherein the betacoronavirus is SARS-CoV-2 virus.

16. The influenza virus-based isolated recombinant virus for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases, which comprises the nucleic acid according to any of claims 1-15.

17. The influenza virus-based isolated recombinant virus according to claim 16, wherein the influenza virus-based isolated recombinant virus is produced from attenuated influenza virus.

18. The influenza virus-based isolated recombinant virus according to claim 16, wherein the influenza virus-based isolated recombinant virus is produced from H2N2 influenza virus.

19. The influenza virus-based isolated recombinant virus according to claim 16, wherein the influenza virus-based isolated recombinant virus is produced from the virus A/Leningrad/134/17/57 (H2N2) or derivatives thereof.

20. The influenza virus-based isolated recombinant virus according to claim 16, wherein the nucleic acid according to any of claims 1-15 is present in the neuraminidase (NA) gene or the NS gene of influenza virus.

21. A pharmaceutical composition for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases, comprising the influenza virus-based recombinant virus according to any of claims 16-20 in combination with one or more pharmaceutically acceptable excipients.

22. Use of the influenza virus-based recombinant virus according to any of claims 16-20 or the pharmaceutical composition according to claim 21 for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases.

23. The use according to claim 22 for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases and coronavirus-related diseases.

24. The use according to claim 23, wherein the specific immunity to coronavirus is specific T cell immunity.

25. The use according to claim 23, wherein the coronavirus is betacoronavirus.

26. The use according to claim 23, wherein the coronavirus is selected from a group that comprises human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

27. A vaccine for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases, including the influenza virus-based recombinant virus according to any of claims 16-20 in an effective amount.

28. The vaccine according to claim 27 for inducing specific immunity to influenza virus and/or preventing influenza virus-related diseases and coronavirus-related diseases.

29. The vaccine according to claim 28, wherein the coronavirus is betacoronavirus.

30. The vaccine according to claim 28, wherein the coronavirus is selected from a group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

31. The vaccine according to claim 28, wherein the specific immunity to coronavirus is specific T cell immunity.

32. A method for induction of specific immunity to influenza virus and/or prevention of influenza virus-related diseases, or induction of specific immunity to influenza virus and coronavirus, or combined prevention of influenza virus-related diseases and coronavirus-related diseases, including administering the influenza virus-based recombinant virus according to any of claims 16-20, the pharmaceutical composition according to claim 21 or the vaccine according to claim 27, in an effective amount.

33. The method according to claim 32, wherein the coronavirus is betacoronavirus.

34. The method according to claim 32, wherein the coronavirus is selected from a group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2.

35. The method according to claim 32, wherein the specific immunity to coronavirus is specific T cell immunity.