EA048516B1 - ISOLATED RECOMBINANT VIRUS BASED ON INFLUENZA VIRUS FOR INDUCING SPECIFIC IMMUNITY TO INFLUENZA VIRUS AND/OR PREVENTING DISEASES CAUSED BY INFLUENZA VIRUS - Google Patents

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 antibody titer to the influenza virus (variants), a recombinant virus based on the influenza virus for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, a pharmaceutical composition and a vaccine that include the above-mentioned recombinant virus based on the influenza virus, as well as their use for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus.

Description

Field of technology

The present invention relates to the field 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 antibody titer to the influenza virus (variants), a recombinant virus based on the influenza virus for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, a pharmaceutical composition and a vaccine that include the above-mentioned recombinant virus based on the influenza virus, as well as their use for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus.

State of the art

Influenza is an acute viral infection that occurs worldwide. Annual flu epidemics, which can affect up to 20% of the population, typically occur in the fall and winter.

Deaths from respiratory illnesses associated with seasonal influenza can be quite high. In particular, a 2017 study estimated that globally, the annual death toll could be as high as 650,000.

Those at higher risk of developing severe illness after influenza infection include people aged 65 and older, pregnant women, young children, people with weakened immune systems, and people with chronic medical conditions.

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

Annual vaccination is the most effective method of preventing influenza and its severe complications. Because influenza viruses vary greatly, WHO updates recommendations for influenza vaccine composition twice a year: in February for the Northern Hemisphere and in September for the Southern Hemisphere. Recommendations for vaccine composition are based on data on viruses detected and characterized by Member States through surveillance.

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

Patent document RU2556833C2 describes an attenuated cold-adapted strain of the influenza virus a/pr/8/59/m2 (h1n1), intended for obtaining vaccine strains of the influenza virus as an attenuation donor, as well as vaccine strains of the influenza virus a/59/m2/california/66/2211 (h2n2) and a/59/m2/tokyo/67/22111 (h2n2).

Patent document RU2563352C2 describes a strain of influenza virus A/17/Texas/2012/30 (H3N2) for the production of a live intranasal influenza vaccine for adults and children.

Despite the many known vaccine strains of the influenza virus, there is a need to obtain a recombinant virus based on the influenza virus that has improved characteristics, for example, allows for obtaining a higher level of antibodies to the influenza virus when administered to a mammal, that is, to be more effective when used.

In addition, as of the filing date of this application, more than 186 million people have fallen ill with diseases caused by the SARS-CoV2 coronavirus, and more than 4 million people have died from diseases caused by the SARS-CoV-2 coronavirus. Moreover, the COVID-19 pandemic is ongoing as of the filing date of this application. There is an urgent global need for effective means of preventing and treating diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2.

Thus, there is a need to obtain a recombinant virus that will allow the formation of specific T-cell immunity to the coronavirus and specific immunity to the influenza virus, which will allow obtaining a vaccine strain that can be used to prevent diseases caused by the influenza virus and diseases caused by the coronavirus.

Description of the invention

The inventors have developed a nucleic acid that encodes a recombinant polypeptide based on fragments of coronavirus proteins. The inventors have developed a recombinant virus based on the influenza virus that includes the above nucleic acid. The inventors have developed a pharmaceutical composition and a vaccine that include the above recombinant virus based on the influenza virus, as well as various options for using them to induce specific immunity to the influenza virus and/or prevent diseases caused by the influenza virus. The inventors have unexpectedly found that the developed nucleic acid that encodes a recombinant polypeptide based on fragments of coronavirus proteins, when inserted into the neuraminidase (NA) gene or the NS gene of the influenza virus, makes it possible to obtain a recombinant influenza virus that produces a higher level of antibodies to the influenza virus when administered to a mammal than an influenza virus without the above insert. Moreover, the recombinant virus

- 1 048516 influenza, including the above-mentioned nucleic acid, which encodes a recombinant polypeptide based on fragments of coronavirus proteins, allows the formation of 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 an influenza virus, which comprises:

at least 1 fragment from the S glycoprotein of coronavirus with the amino acid sequence SEQ ID NO: 1;

at least 1 fragment from the coronavirus nucleoprotein with the amino acid sequence SEQ ID NO: 3;

and, if necessary, 1 fragment from the membrane protein of coronavirus with the amino acid sequence of SEQ ID NO: 5, where at least 1 fragment from the S glycoprotein of coronavirus 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 membrane protein of coronavirus is SEQ ID NO: 22.

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, 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 comprises

- 2 048516 nucleotide sequence, which is selected from the group comprising nucleotide sequences SEQ ID NO: 59-76.

In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to an influenza virus, comprising fragments from coronavirus proteins and comprising an amino acid sequence 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 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 59-76, which respectively encode the amino acid sequences of SEQ ID NO: 23-40.

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

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

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

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

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

In some embodiments of the invention, the isolated recombinant influenza virus is derived from an H2N2 influenza virus.

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

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

In one aspect, the present invention relates to a pharmaceutical composition for inducing specific immunity to influenza virus and/or preventing diseases caused by influenza virus, comprising any of the above-mentioned 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-mentioned recombinant viruses based on the influenza virus or the above-mentioned composition for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus.

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

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

In some embodiments of the use, the coronavirus is a betacoronavirus.

In some embodiments of the use, the coronavirus is selected from the group consisting of human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARSCoV-2.

In one aspect, the present invention relates to a vaccine for inducing specific immunity to influenza virus and/or preventing diseases caused by influenza virus, which comprises any of the above-mentioned recombinant viruses based on influenza virus in an effective amount.

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

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

In some embodiments, the vaccine coronavirus is selected from the group consisting of human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV2.

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

In one aspect, the present invention relates to a method for inducing specific immunity to an influenza virus and/or preventing diseases caused by an influenza virus, or inducing

- 3 048516 specific immunity to the influenza virus and coronavirus or combined prevention of diseases caused by the influenza virus and diseases caused by coronavirus, including the introduction into the body of mammals of any of the above-mentioned recombinant viruses based on the influenza virus or the above-mentioned composition or the above-mentioned vaccine, in an effective amount.

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

In some 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 the coronavirus is specific T-cell immunity.

Brief description of the drawings

Fig. 1 is a diagram of the cloning of the antigen cassettes of the present invention encoding a transgene (polyepitope cassettes consisting of combinations of fragments of the proteins S glycoprotein (or Spike) (SEQ ID NO: 1), nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, optionally, membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV-2) in the NA gene of the influenza virus.

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

_______________________Description of elements for Fig. 1 -2_______________________

Element Note Enhancer and promoter CMV A regulatory element consisting of an enhancer and a promoter of cytomegalovirus CMV, required for the initiation of transcription of the (+)RNA target insert. Intron Chimeric intronic region of human beta-globin and immunoglobulin heavy chain genes that increases mRNA stability. (-)RNA transcription terminator Transcription termination signal of viral (-)RNA from the mouse RNA polymerase I gene. S’-UTO 5'-untranslated region of the sixth segment of the influenza virus genome, regulating the translation of viral genes Neuraminidase (NA) The complete sequence of influenza virus neuraminidase, which is necessary for the separation of viral particles from the infected cell, as well as for preventing their aggregation and facilitating movement to the target cell. P2A Self-cleaving peptide (22 aa) of porcine teschovirus-1 virus, necessary for separation of the C-terminal transgene from the N-terminal region of neuraminidase during translation. Transgene A polyepitope cassette consisting of combinations of fragments of the proteins S glycoprotein (or Spike) (SEQ ID NO: 1), nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, if necessary, membrane protein (or Membrane) (SEQ ID NO: 5) of beta-corona in and rusa SARS-CoV-2 Stop A stop codon required to terminate mRNA translation.

-4 048516

Protective region of RNA A sequence of 157 3'-terminal nucleotides of the sixth segment of the influenza virus genome, necessary for packaging the segment into a capsid (including re-incorporating the sequence encoding forty-two C-terminal ao. neuraminidase, without their translation). 3*-NTO The 3'-untranslated region of the sixth segment of the influenza virus genome, which serves as a polyadenylation region for termination of transcription of viral genes, as well as stabilization and prevention of mRNA degradation. Promoter for (-)RNA The promoter of the human RNA polymerase I gene, required for the initiation of transcription of (-)RNA of the influenza virus for subsequent assembly of virions. SV40 polyadenylation signal Additional transcription termination signal (+)RNA polyadenylation of SV40 virus. fl origin The origin of single-stranded replication of bacteriophage fl. AmpR Promoter Weak constitutive promoter of the wild-type E. coli beta-lactamase (BLa) gene (also found in transposon Tp2660). Includes a promoter and an operator. Beta-lactamase The β-lactamase gene, which causes resistance to ampicillin. Allows selection of E. coli cell culture. Polyadenylation region The polyadenylation signal that terminates transcription of the β-lactamase gene. Origin of replication The complex high-copy origin of replication pUC/pBR322/ColEl/pMBl is a replication origin that functions in prokaryotic cells.

Fig. 3 is a diagram of the cloning of the antigen cassettes of the present invention encoding polyepitope cassettes consisting of combinations of fragments of the proteins of the S glycoprotein (or Spike) (SEQ ID NO: 1), the nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, optionally, the membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV-2, into the NS gene of the influenza virus.

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

Description of elements for Fig. 3-4

Element Note Enhancer and promoter CMV A regulatory element consisting of an enhancer and a promoter of cytomegalovirus CMV, required for the initiation of transcription of the (+)RNA target insert. Intron Chimeric intronic region of human beta-globin and immunoglobulin heavy chain genes that increases mRNA stability. (-)RNA transcription terminator Viral (-)RNA transcription termination signal from mouse RNA polymerase I gene 5'-NTO 5'-untranslated region of the eighth segment of the influenza virus genome from the donor of attenuation A/Leningrad/134/17/57, regulating the translation of viral genes NS1 1-126 A fragment (1-126 aa) of the modified NS1 protein of influenza virus (UniProt: P69273) from the donor of attenuation A/Leningrad/134/17/57, required for transport, splicing and translation of viral RNA. NS2 (NEP) 1-10 The first fragment (1-10 aa) of the NS2 protein of influenza virus (also known as NEP, UniProt: P69266) from the donor of attenuation A/Leningrad/134/17/57, required for the transport of viral RNA from the cell nucleus. 5' splice site 5' splice site of the influenza virus NS gene required for correct expression of NS2. P2A Self-cleaving peptide (22 aa) of porcine teschovirus-1 virus, necessary for separation of the C-terminal insert from the N-terminal fragment of the NS1 protein (1-126 aa) during translation. Transgene A polyepitope cassette consisting of combinations of fragments of the proteins S glycoprotein (or Spike) (SEQ ID NO: 1), nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, if necessary, membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV-2. Stop A stop codon required to terminate mRNA translation. 3' splice site 3' splice site of the influenza virus NS gene required for correct expression of NS2. NS2 (NEP) 11-121 The second fragment (11-121 aa) of the influenza virus NS2 protein (also known as NEP, UniProt: P69266) from the donor of attenuation

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A/Leningrad/134/17/57, necessary for the transport of viral RNA from the cell nucleus. NS2 (NEP) 1-121 The complete sequence of the influenza virus NS2 protein (also known as NEP, UniProt: P69266) from the attenuation donor A/Leningrad/134/17/57, which is required for the transport of viral RNA from the cell nucleus. Z'-NTO 3'-untranslated region of segment 8 of the influenza virus genome from the donor of attenuation A/Leningrad/134/17/57, which functions as a poly(A) region for termination of transcription of viral genes, as well as stabilization and prevention of mRNA degradation. Promoter for (-)RNA The promoter of the human RNA polymerase I gene, required for the initiation of transcription of (-)RNA of the influenza virus for subsequent assembly of virions. SV40 polyadenylation signal Additional transcription termination signal (+)RNA polyadenylation of SV40 virus fl origin The origin of single-stranded replication of bacteriophage fl. AmpR Promoter Weak constitutive promoter of the beta-lactamase (BLa) gene in wild-type E. coli (also found in transposon Tp2660). Includes a promoter and an operator. Beta-lactamase The β-lactamase gene that causes resistance to ampicillin. Allows selection of E. coli cell cultures Polyadenylation region The polyadenylation signal that terminates transcription of the β-lactamase gene Origin of replication Complex high-copy origin of replication pUC/pBR322/ColEl/pMBl - origin of replication functioning in prokaryotic cells

Fig. 5 is a graph showing the levels of serum IgG antibodies in the blood sera of mice inoculated twice with recombinant influenza viruses containing SARS-CoV-2 coronavirus polyepitope cassette inserts, compared to the viral vector. The optical densities in ELISA are shown as a function of serum dilution. Comparison of AUC data was performed using one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

PBS is phosphate buffered saline.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 6 is a graph showing the levels of serum IgG antibodies in the blood sera of mice inoculated twice with recombinant influenza viruses containing SARS-CoV-2 coronavirus polyepitope cassette inserts, compared to the viral vector. The optical densities in ELISA are shown as a function of serum dilution. Comparison of AUC data was performed using one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

PBS is phosphate buffered saline.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 7 is a graph showing the levels of serum IgG antibodies in the blood sera of mice inoculated twice with recombinant influenza viruses containing SARS-CoV-2 coronavirus polyepitope cassette inserts compared to the viral vector. The area under the curve OD ₄₅₀ values for each animal are indicated. Comparison of AUC data was performed using one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001.

PBS is phosphate buffered saline.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 8 is a graph showing the levels of serum IgG antibodies in the blood sera of mice inoculated twice with recombinant influenza viruses containing inserts

- 7,048,516 SARS-CoV-2 coronavirus polyepitope cassettes compared to the viral vector. The area under the _OD450 curve values for each animal are shown. AUC data were compared using one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001.

PBS is phosphate buffered saline.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 9 is a graph showing the levels of virus-specific IFN-γ-producing memory CD4+ T cells in peripheral blood mononuclear cell samples from COVID-19 convalescents after stimulation with the CoVac-13 vaccine candidate and the H7N9 LAIV viral vector.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 10 is a graph showing the levels of virus-specific IFN-γ-producing memory CD4+ T cells in peripheral blood mononuclear cell samples from COVID-19 convalescents after stimulation with the CoVac-15 vaccine candidate and the H7N9 LAIV viral vector.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 11 is a graph showing the levels of virus-specific IFN-γ-producing memory CD4+ T cells in peripheral blood mononuclear cell samples from COVID-19 convalescents after stimulation with the CoVac-16 vaccine candidate and the H7N9 LAIV viral vector.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

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

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 18 is a graph showing virus-specific IFN-γ-producing CD4+ (top) and CD8+ (bottom) memory T cells in peripheral blood mononuclear cell samples from control donors after stimulation with vaccine candidates and the H7N9 LAIV viral vector.

LAIV H7N9 is a live attenuated H7N9 influenza virus.

Fig. 19 is a comparison of the sequences of glycoprotein S fragments of different coronaviruses.

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Definitions and General Methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. Generally, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is customary in the art, or as described herein.

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

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

In the present description and in the following claims, unless the context otherwise requires, the words include and contain, or variations thereof such as includes, including, contains, or comprising, are to be understood as including the stated whole or group of wholes but not excluding any other whole or group of wholes.

Protein (peptide).

As used herein, the terms peptide, polypeptide and protein are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and longer chains, typically referred to in the art as proteins, of which many types exist. Polypeptides include, but are not limited to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules.

The terms nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucleotide sequence, which are used interchangeably in this specification, denote a distinct sequence of nucleotides, modified or unmodified, defining a fragment or section of nucleic acid, containing or not containing non-natural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.

A person skilled in the art has a general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric nucleotides. Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any methods available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, etc., and synthetic methods.

It should also be mentioned here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The sequences of the present invention have been isolated and/or purified, i.e. taken directly or indirectly, for example by copying, while their environment has been at least partially modified. Thus,

- 9 048516 here it should be understood as isolated nucleic acids obtained by genetic recombination, for example, with the help of host cells, or obtained by chemical synthesis.

The term nucleotide sequence includes its complement unless otherwise specified. Thus, a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

Vector.

The term vector, as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Additionally, the term vector, as used herein, means a viral particle capable of transporting a nucleic acid.

Application.

The term prophylaxis or prevention and similar terms 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 specifically controlling or influencing the activity of an 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 herein refers to a state of non-susceptibility to a disease due to the induction of an immune response.

The term disorder means any condition that can be improved by treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to develop the disorder.

Disease is a health condition of an animal where the animal is unable to maintain homeostasis and where, unless the disease is alleviated, the animal's health continues to deteriorate.

The term subject, patient, individual, etc. are used interchangeably herein and refer to any animal susceptible to the methods described herein. In certain non-limiting embodiments, the subject, patient, or individual is a human. The aforementioned subject may be male or female of any age.

A therapeutically effective amount or effective amount is the amount of a therapeutic agent administered that will relieve to some extent one or more symptoms of the disease being treated.

Detailed description of the invention

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

In one aspect, the present invention relates to an isolated nucleic acid encoding a recombinant polypeptide for increasing the titer of antibodies to an influenza virus, which comprises:

at least 1 fragment of the coronavirus S glycoprotein with the amino acid sequence

MF VFL VLLPL VS S QC VNLTTRTQLPP AYTNSFTRGVY YPDKVFRS S VLHSTQDLFLPFF S NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKN1DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLP1GINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGtYQTSNFRVQPTESJVRFPNlTNLCPFGEVFNATRFASVYAWNRKRlSN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV1RGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRD1ADTTDAVRDPQTLE1LDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT NFTISVTTE1LPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQ1YKTPP1KDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP

- 10048516

FAMQMAYRFNG1GVTQNVLYENQKL1ANQFNSA1GK1QDSLSSTASALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLND1LSRLDKVEAEVQ1DRLITGRLQSLQTYVTQQLIRAA EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC KFDEDDSEPVLKGVKLHYT (SEQ ID NO: I);

at least 1 fragment of the coronavirus nucleoprotein with the amino acid sequence

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQ HGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTG PEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEG SRGGSQ AS SRS S SRSRNS SRNSTPGS SRGTSPARMAGNGGD AALLALLLLDRLNQLESKM SGKGQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQEL IRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQV ILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQ SMSSADSTQA (SEQ ID NO: 3), and, if necessary, 1 fragment from the membrane protein of coronavirus with the amino acid sequence

MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPV TLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPEINILL NVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKL GASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ (SEQ ID NO: 5), wherein at least 1 fragment from the S glycoprotein of the coronavirus is selected from the group: DRLNEVA (SEQ ID NO: 7), FIAGLIAIV (SEQ ID NO: 8), APHGVVFLHVTYV (SEQ ID NO: 9), GYQPYRVVVLSF (SEQ ID NO: 10), ALNTLVKQL (SEQ ID NO: 11), VLNDILSRL (SEQ ID NO: 12) or LITGRLQSLQTYV (SEQ ID NO: 13);

at least 1 fragment from the coronavirus nucleoprotein is selected from the group: ALALLLLDRLNQL (SEQ ID NO: 15), LSPRWYFYYL (SEQ ID NO: 16), ALNTPKDHI (SEQ ID NO: 17), QLPQGTTL (SEQ ID NO: 18), RQGTDYKHWP (SEQ ID NO: 19), GMSRIGMEV (SEQ ID NO: 20) or ILLNKHIDA (SEQ ID NO: 21);

The fragment from the coronavirus membrane protein is FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

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

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

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

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

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

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

In some embodiments of the invention, the isolated nucleic acid encodes

- 11 048516 is a lipopeptide 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 a coronavirus nucleoprotein.

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

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.

- 12 048516

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 selected from the group consisting of 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 that includes 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 selected from the group consisting of 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 consisting of 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 selected from the group consisting of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 37.

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

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

In some embodiments, 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 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 59-76.

Any of the above nucleic acids encodes a recombinant polypeptide for increasing the titer of antibodies to the influenza virus. This polypeptide is a polyepitope cassette for a T-cell response to coronavirus proteins. In the application materials, this polypeptide is also called the cassette according to the invention.

The cassettes according to the invention comprise at least one sequence which is selected from the group:

1) a fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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 can be presented in cassettes as part of, for example, the following options:

fragment (1180-1197 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: QKEIDRLNEVAKNLNESL (SEQ ID NO: 95);

fragment (1164-1178 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region involved in the penetration of the virus into the infected cell: EIDRLNEVAKNLNES (SEQ ID NO: 96).

2) Fragment (1220-1228 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8).

This fragment can be presented in cassettes as part of, for example, the following options:

fragment (1199-1213 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): WLGFIAGLIAIVMVT (SEQ ID NO: 97);

fragment (1220-1231 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): FIAGLIAIVMVT (SEQ ID NO: 98);

- 13 048516 fragment (1220-1229 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): FIAGLIAIVM (SEQ ID NO: 99).

3) Fragment (1056-1068 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9).

This fragment can be presented in cassettes as part of, for example, the following options:

fragment (1035-1062 aa) of the S glycoprotein of the SARS-CoV-1 virus, partially coinciding with that of the S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): PQAAPHGVVFLHVTYVPSQERNFTTAPA (SEQ ID NO: 100);

fragment (1056-1078 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): APHGVVFLHVTYVPAQEKNFTTA (SEQ ID NO: 101);

fragment (1056-1073 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): APHGVVFLHVTYVPAQEK (SEQ ID NO: 102);

fragment (1055-1073 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): SAPHGVVFLHVTYVPAQEK (SEQ ID NO: 103);

fragment (1055-1070 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): SAPHGVVFLHVTYVPA (SEQ ID NO: 104).

4) A fragment (504-515 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the 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 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSFELLHAPA (SEQ ID NO: 105).

6) A fragment (504-516 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSFE (SEQ ID NO: 106).

7) A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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 can be presented in cassettes as part of, for example, the following variants: a fragment (946-971 aa) of the above-described glycoprotein S of the SARS-CoV-2 virus (SEQ ID NO: 1), partially from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: GKLQDVVNQNAQALNTLVKQLSSNFG (SEQ ID NO: 107);

fragment (947-970 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 1 region involved in the penetration of the virus into the infected cell: KLQDWNQNAQALNTLVKQLSSNF (SEQ ID NO: 108).

8) Fragment (976-984 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12).

9) Fragment (996-1008 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): LITGRLOSLQTYV (SEQ ID NO: 13).

10) Fragment (996-1004 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): LITGRLQSL (SEQ ID NO: 14).

The cassettes according to the invention comprise at least one sequence which is selected from the group:

1) Fragment (218-230 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15).

This fragment can be presented in cassettes as part of, for example, the following variants: a fragment (216-234 aa) of the nucleoprotein N of the SARS-CoV-1 virus, partially coinciding with that of the nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): GETALALLLLDRLNQLESK (SEQ ID NO: 109);

fragment (214-234 aa) of the nucleoprotein described above N virus SARS-CoV-2 (SEQ ID NO: 3): GGDAALALLLLDRLNQLESKM (SEQ ID NO: 110); fragment (216-235 aa) of the above-described nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): DAALALLLLDRLNQLESKMS (SEQ ID NO: 111); fragment (216-234 aa) of the above-described nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): DAALALLLLDRLNQLESKM (SEQ ID NO: 112); fragment (216-233 aa) of the above-described nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): DAALALLLLDRLNQLESK (SEQ ID NO: 113); 2) Fragment (104-113 aa) of the nucleoprotein described above N virus SARS-CoV-2

(SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16).

This fragment can be presented in cassettes as part of, for example, the following options:

fragment (92-118 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): RRIRGGDGKMKDLSPRWYFYYLGTGPE (SEQ ID NO: 114);

- 14 048516 fragment (92-117 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): RRIRGGDGKMKDLSPRWYFYYLGTGP (SEQ ID NO: 115);

fragment (98-119 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): DGKMKDLSPRWYFYYLGTGPE (SEQ ID NO: 116)

3) Fragment (138-146 aa) of the above-described nucleoprotein (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17).

4) Fragment (160-167 aa) of the nucleoprotein described above (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

5) Fragment (293-302 aa) of the above-described nucleoprotein (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19).

6) Fragment (316-324 aa) of the above-described nucleoprotein (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

virus virus virus virus virus virus

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

N

N

N

N

This fragment can be presented in cassettes as part of, for example, the following variant:

fragment (303-337 aa) of the N nucleoprotein of the SARS-CoV-1 virus, partially coinciding with that of the N nucleoprotein of the SARS-CoV-2 virus (SEQ ID NO: 3): PQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGA (SEQ ID NO: 117).

7) Fragment (351-359 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21)

This fragment can be presented in cassettes as part of, for example, the following options:

fragment (340-365 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): DDKDPNFKDQVILLNKHIDAYKTFPP (SEQ ID NO: 118);

fragment (341-359 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): DKDPNFKDQVILLNKHIDA (SEQ ID NO: 119);

fragment (340-359 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): DDKDPNFKDQVILLNKHIDA (SEQ ID NO: 120).

SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus

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

This fragment can be presented in cassettes as part of, for example, the following options:

fragment (40-75 aa) of the membrane protein M of the SARS-CoV-1 virus, partially coinciding with that of the membrane protein M of the SARS-CoV-2 virus (SEQ ID NO: 5): NRNRFLYIIKLVFLWLLWPVTLACFVLAAVYRINWV (SEQ ID NO: 121);

fragment (42-75 aa) of the above-described membrane protein M (SEQ ID NO: 5): RNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINW (SEQ ID NO: 122);

fragment (50-79 aa) of the above-described membrane protein M (SEQ ID NO: 5): KLIFLWLLWPVTLACFVLAAVYRINWITGG (SEQ ID NO: 123);

fragment (50-77 aa) of the above-described membrane protein M (SEQ ID NO: 5): KLIFLWLLWPVTLACFVLAAVYRINWIT (SEQ ID NO: 124).

SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus

In some variants, the sequences listed above were used as part of specific antigen cassettes:

Cassette 1 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region involved in the entry of the DRLNEVA virus (SEQ ID NO: 7);

Fragment (1220-1228 aa) of the above-described glycoprotein S (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described glycoprotein S (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

into the infected cell:

SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus

Antigen cassette 1 is described by the following sequence:

NRNRFLYIIKLVFLWLLWPVTLACFVLAAVYRINWVGETALALLLLDRLNQLES

KEIDRLNEVAKNLNESWLGFIAGLIAIVMVTPQAAPHGVVFLHVTYVPSQERNFTTAPA (SEQIDNO: 23)

It should be taken into account that the above-mentioned antigen cassette 1 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

- 15 048516

GGSGATNFSLLKQAGDVEENPGPNRNRFLYIIKLVFLWLLWPVTLACFVLAAVYRI

NWVGETALALLLLDRLNQLESKEIDRLNEVAKNLNESWLGFIAGLIAIVMVTPQAAPHG

VVFLHVTYVPSQERNFTTAPA (SEQ ID NO: 41)

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 41 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

AATCGGAACAGGTTTTTGTACATAATAAAGCTTGTTTTTCCTCTGGCTCTTGTG

GCCAGTAACACTTGCTTGTTTTGTGCTTGCTGCTGTCTACAGAATTAATTGGGTGGGT

GAAACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACCAGCTTGAGAGCAAAGA

AATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAATCATGGCTCGGCTTCAT

TGCTGGACTAATTGCCATCGTCATGGTTACACCACAAGCAGCCCCGCATGGTGTTGT

CTTCCTACATGTCACGTATGTGCCATCCCAGGAGGAACTTCACCACAGCGCCAGC

A (SEQ ID NO: 59).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTAATCGGAACAGGTTTTTGTACATAATAAAGCTTGTTTTCCTC

TGGCTCTTGTGGCCAGTAACACTTGCTTGTTTTGTGCTTGCTGCTGTCTACAGAATTA

ATTGGGTGGGTGAAACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACCAGCTTG

AGAGCAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAATCATGG CTCGGCTTCATTGCTGGACTAATTGCCATCGTCATGGTTACACCACAAGCAGCCCCG

CATGGTGTTGTCTTCCTACATGTCACGTATGTGCCATCCCAGGAGAGGAACTTCACC

ACAGCGCCAGCA (SEQ ID NO: 77).

Cassette 2 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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);

Fragment (1220-1228 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (316-324 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

The following sequence describes antigen cassette 2:

IQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVVTILL

CPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGA (SEQ Ш NO: 24).

It should be taken into account that the above-mentioned antigen cassette 2 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK

WPWYVWLGFIAGLIAIVMTILLCPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGA (SEQ Ш NO: 42).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 42 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a particular sequence should

- 16 048516 to be understood as encompassing its complementary chain with its complementary sequence.

In a particular embodiment, the nucleic acid that encodes antigen cassette 2 includes the nucleotide sequence:

ATTCAAAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAAT

CACTCATTGACCTTCAAGAATTGGGAAAATATGAGCAATATATTAAATGGCCTTGGT

ATGTTTGGCTCGGCTTCATTGCTGGACTAATTGCCATCGTCATGGTTACAATCTTGCT TTGTCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTTGGAATGTCACGC

ATTGGCATGGAAGTCACACCTTCGGGAACATGGCTGACTTATCATGGAGCC (SEQ ID

NO: 60).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTATTCAAAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAA

TTTAAATGAATCACTCATTGACCTTCAAGAATTGGGAAAATATGAGCAATATATTAA

ATGGCCTTGGTATGTTTGGCTCGGCTTCATTGCTGGACTAATTGCCATCGTCATGGTT

ACAATCTTGCTTTGTCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTTG

GAATGTCACGCATTGGCATGGAAGTCACACCTTCGGGAACATGGCTGACTTATCATG

GAGCC (SEQ ID NO: 78).

Cassette 3 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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);

Fragment (1220-1228 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (316-324 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

Antigen cassette 3 is described by the following sequence:

PQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIQKEIDRLNEVAKNLNESLID

LQELGKYEQYIKWPWYVWLGFIAGLIAIVVMVTILLC (SEQ ID NO: 25).

It should be taken into account that the above-mentioned antigen cassette 3 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYH

GAIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVVTILLC (SEQ IDNO: 43).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 43 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

In a particular embodiment, the nucleic acid that encodes antigen cassette 3 includes the nucleotide sequence:

CCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTTGGAATGTCACG

CATTGGCATGGAAGTCACACCTTCGGGAACATGGCTGACTTATCATGGAGCCATTCA

AAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCATTG

ACCTTCAAGAATTGGGAAAATATGAGCAATATATTAAATGGCCTTGGTATGTTTGGC TCGGCTTCATTGCTGGACTAATTGCCATCGTCATGGTTACAATCTTGCTTTGT (SEQ Ш NO: 61).

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

- 17 048516

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTT

GGAATGTCACGCATTGGCATGGAAGTCACACCTTCGGGAACATGGCTGACTTATCAT

GGAGCCATTCAAAAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGA

ATCACTCATTGACCTTCAAGAATTGGGAAAATATGAGCAATATATTAAATGGCCTTG

GTATGTTTGGCTCGGCTTCATTGCTGGACTAATTGCCATCGTCATGGTTACAATCTTG

CTTTGT (SEQ ID NO: 79).

Cassette 4 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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);

Fragment (1220-1228 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described N virus nucleoprotein (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M of the virus

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2 (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigen cassette 4 is described by the following sequence:

QKEroRLNEVAKNLNESLroLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTKLIF

LWLLWPVTLACFVLAAVYRINWITDAALALLLLDRLNQLESKMSAPHGWFLHVTYVP

A (SEQ ID NO: 26).

It should be taken into account that the above-mentioned antigen cassette 4 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK

WPWYIWLGFIAGLIAIVVTKLIFLWLLWPVTLACFVLAAVYRINWITDAALALLLLDR

LNQLESKMSAPHGVVFLHVTYVPA (SEQ Ш NO: 44).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 44 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

CAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTTAAATGAATCTC

TCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTAC

ATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAAAGTTAATT TTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACA GAATAAATTGGATCACCGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACC AGCTTGAGAGCAAAATGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATG TCCCTGCA (SEQ Ш NO: 62).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAATTT AAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAAT GGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGA CAAAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGC

- 18 048516

TGCTGTTTACAGAATAAATTGGATCACCGATGCTGCTCTTGCTTTGCTGCTGCTTGAC

AGATTGAACCAGCTTGAGAGCAAAATGTCAGCACCTCATGGTGTAGTCTTCTTGCAT

GTGACTTATGTCCCTGCA (SEQ ID NO: 80).

Cassette 5 contains the following epitope fragments:

Fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region, which is involved in the penetration of the virus into the infected cell:

DRLNEVA (SEQ ID NO: 7);

Fragment (1220-1228 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described N virus nucleoprotein (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M of the virus

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2 (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigen cassette 5 is described by the following sequence:

QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTGGD

AALALLLLDRLNQLESKMSAPHGWFLHVTYVPAQEKKLIFLWLLWPVTLACFVLAAV

YRINWITGG (SEQ ID NO: 27).

It should be taken into account that the above-mentioned antigen cassette 5 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK

WPWYIWLGFIAGLIAIVMVTGGDAALALLLLDRLNQLESKMSAPHGVVFLHVTYVPAQ EKKLIFLWLLWPVTLACFVLAAVYRINWITGG (SEQ Ш NO: 45).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 45 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

In a particular embodiment, the nucleic acid that encodes antigen cassette 5 includes the nucleotide sequence:

CAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTTAAATGAATCTC

TCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTAC ATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAGGCGGTGAT GCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCA GCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAAG TTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGT TTACAGAATAAATTGGATCACCGGTGGA (SEQ ID NO: 63).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAATTT AAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAAT GGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGA CAGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGA GCAAAATGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCAC AAGAAAAGAAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGT GCTTGCTGCTGTTTACAGAATAAATTGGATCACCGGTGGA (SEQ Ш NO: 81).

Cassette 6 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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);

Fragment (1220-1228 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus

- 19 048516 (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M of the SARS-CoV-2 virus (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

Antigen cassette 6 is described by the following sequence:

QKEIDRLNEVAKNLNESLAPHGVVFLHVTYVPAQEKKLIFLWLLWPVTLACFVL

AAVYRINWITDAALALLLLDRLNQLESKMSFIAGLIAIVMVT (SEQ ID NO: 28).

It should be taken into account that the above-mentioned antigen cassette 6 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLAPHGVVFLHVTYVP

AQEKKLIFLWLLWPVTLACFVLAAVYRINWITDAALLALLLLDRLNQLESKMSFIAGLIAI

VMVT (SEQ ID NO: 46).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 46 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

CAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTTAAATGAATCTC

TCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGA

AGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGC

TGTTTACAGAATAAATTGGATCACCGATGCTGCTCTTGCTTTGCTGCTGCTTGACAG

ATTGAACCAGCTTGAGAGCAAAATGTCTTTTATAGCTGGCTTGATTGCCATAGTAAT

GGTGACA (SEQ ID NO: 64).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCCTGGACCTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTT

AAATGAATCTCTCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCA

CAAGAAAAGAAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTG

TGCTTGCTGCTGTTTACAGAATAAATTGGATCACCGATGCTGCTCTTGCTTTGCTGCT

GCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTTTTATAGCTGGCTTGATTGC

CATAGTAATGGTGACA (SEQ ID NO: 82).

Cassette 7 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region involved in the entry of the DRLNEVA virus (SEQ ID NO: 7);

into the infected cell:

Fragment (1220-1228 aa) of the above (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

glycoprotein glycoprotein nucleoprotein

Fragment (53-73 aa) of the membrane protein described above (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

The following sequence describes antigen cassette 7:

virus virus virus virus

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

QKEIDRLNEVAKNLNESLAPHGVVFLHVTYVPAQEKDAALALLLLDRLNQLESK

MSFIAGLIAIVMKLIFLWLLWPVTLACFVLAAVYRINWITGG (SEQ ID NO: 29).

- 20 048516

It should be taken into account that the above-mentioned antigen cassette 7 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLAPHGVVFLHVTYVP

AQEKDAALALLLLDRLNQLESKMSFIAGLIAIVMKLIFLWLLWPVTLACFVLAAVYRIN

WITGG (SEQ ID NO: 47).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 47 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

CAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTTAAATGAATCTC

TCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGG

ATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGT CTTTTATAGCTGGCTTGATTGCCATAGTAATGAAGTTAATTTTCCTCTGGCTGTTATG

GCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGATCACC

GGTGGA (SEQ ID NO: 65).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCCTGGACCTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTT

AAATGAATCTCTCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCA

CAAGAAAAGGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAG

AGCAAAATGTCTTTTATAGCTGGCTTGATTGCCATAGTAATGAAGTTAATTTTCCTCT

GGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAA

TTGGATCACCGGTGGA (SEQ ID NO: 83).

Cassette 8 contains the following epitope fragments:

A fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region involved in the entry of the DRLNEVA virus (SEQ ID NO: 7);

Fragment (1220-1228 aa) of the above-described glycoprotein S (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described glycoprotein S (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

into the infected cell:

SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus

The following sequence describes antigen cassette 8:

QKEIDRLNEVAKNLNESLAPHGVVFLHVTYVPAQEKNFTTAFIAGLIAIVMVTDA

ALALLLLDRLNQLESKKLIFLWLLWPVTLACFVLAAVYR.INWITGG (SEQ ID NO: 30).

It should be taken into account that the above-mentioned antigen cassette 8 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPQKEIDRLNEVAKNLNESLAPHGVVFLHVTYVP

AQEKNFTTAFIAGLIAIVVTDAALLLLDRLNQLESKKLIFLWLLWPVTLACFVLAA VYRINWITGG (SEQ Ш NO: 48).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 48 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to persons skilled in the art to obtain such alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of

- 21 048516 of the present invention.

A reference to a nucleotide sequence includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

CAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTTAAATGAATCTC

TCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGA

ACTTCACAACTGCTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAGATGCTG

CTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAAAGTTAATTT TCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAG AATAAATTGGATCACCGGTGGA (SEQ Ш NO: 66).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCCTGGACCTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGATTT

AAATGAATCTCTCGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCA

CAAGAAAAGAACTTCACAACTGCTTTTATAGCTGGCTTGATTGCCATAGTAATGGTG

ACAGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAA

AAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTG

CTGTTTACAGAATAAATTGGATCACCGGTGGA (SEQ Ш NO: 84).

Cassette 9 contains the following epitope fragments:

Fragment (1184-1190 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 2 region, which is involved in the penetration of the virus into the infected cell:

DRLNEVA (SEQ ID NO: 7);

Fragment (1220-1228 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): FIAGLIAIV (SEQ ID NO: 8);

Fragment (1056-1068 aa) of the above-described S virus glycoprotein (SEQ ID NO: 1): APHGVVFLHVTYV (SEQ ID NO: 9);

Fragment (218-230 aa) of the above-described N virus nucleoprotein (SEQ ID NO: 3): ALALLLLDRLNQL (SEQ ID NO: 15);

Fragment (53-73 aa) of the above-described membrane protein M of the virus (SEQ ID NO: 5): FLWLLWPVTLACFVLAAVYRI (SEQ ID NO: 22).

The following sequence describes antigen cassette 9:

RNRFLYnKLIFLWLLWPVTLACFVLAAVYRINWKEIDRLNEVAKNLNESLIDLQE

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

SARS-CoV-2

LGKYEQYIKWPWYIWLGFIAGLIAIVMVTGGDAALALLLLDRLNQLESKMSAPHGVVF

LHVTYVPA (SEQ ID NO: 31).

It should be taken into account that the above-mentioned antigen cassette 9 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRIN

WKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTGGDAALA

LLLLDRLNQLESKMSAPHGVVFLHVTYVPA (SEQ Ш NO: 49).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 49 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

- 22 048516

AGGAATAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGGCTGTTATGGCC

AGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGAAAGAAAT TGACCGCCTCAATGAGGTTGCCAAATTTAAATGAATCTCTCATCGATCTCCAAGA

ACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTAT AGCTGGCTTGATTGCCATAGTAATGGTGACAGGCGGTGATGCTGCTCTTGCTTTGCT GCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCAGCACCTCATGGTGTAGT CTTCTTGCATGTGACTTATGTCCCTGCA (SEQ ID NO: 67).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTAGGAATAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGG CTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATT GGAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATC GATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTG

GCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAGGCGGTGATGCTGC

TCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCAGCACC

TCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCA (SEQ ID NO: 85).

Cassette 10 contains the following epitope fragments:

A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10);

Fragment (104-113 a.o.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); Fragment (138-146 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); Fragment (160-167 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2

(SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

The following sequence describes antigen cassette 10:

RRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKD

HIGTRNPANNAAIVLQLPQGTTLPKGKLQDVVNQNAQALNTLVKQLSSNFGYQPYRVV

VLSFELLHAPA (SEQ ID NO: 32).

It should be taken into account that the above-mentioned antigen cassette 10 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPY

GANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGKLQDVVNQNAQ

ALNTLVKQLSSNFGYQPYRVVVLSFELLHAPA (SEQ Ш NO: 50).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 50 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

- 23 048516

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT

ATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAG

ACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATT

GGCACCCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACA

ACATTGCCAAAAGGCAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAA

CACGCTTGTTAAACAACTTAGCTCCAATTTTGGTTACCAACCATACAGAGTAGTAGT

ACTTTCTTTTGAACTTCTACATGCACCAGCA (SEQ ID NO: 68).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG

TCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGG

TGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAA

AAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTC

CTCAAGGAACAACATTGCCAAAAGGCAAACTTCAAGATGTGGTCAACCAAAATGCA

CAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTTACCAACCATAC

higher nucleoprotein N virus SARS-CoV-2 higher nucleoprotein N virus SARS-CoV-2 higher nucleoprotein N virus SARS-CoV-2

AGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCA (SEQ Ш NO: 86).

Cassette 11 contains the following epitope fragments:

A fragment (504-515 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10);

Fragment (104-113 aa) of the described (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16);

Fragment (138-146 aa) of the described (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17);

Fragment (160-167 aa) described (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18).

The following sequence describes antigen cassette 11:

RRIRGGDGKMKDLSPRWYFYYLGTGPERSGARSKQRRPQGLPNNTASWFTALT

QHGKEDLGYQPYRVVVLSFETEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYA

EGSRGG (SEQ ID NO: 33).

It should be taken into account that the above-mentioned antigen cassette 11 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPERSGAR

SKQRRPQGLPNNTASWFTALTQHGKEDLGYQPYRVVVLSFETEGALNTPKDHIGTRNP

ANNAAIVLQLPQGTTLPKGFYAEGSRGG (SEQ ID NO: 51).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 51 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

In a particular embodiment, the nucleic acid that encodes antigen cassette 11 includes the nucleotide sequence:

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT

ATTTCTACTACCTAGGAACTGGGCCAGAACGCAGTGGGGCGCGATCAAAACAACGT

CGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACAT

GGCAAGGAAGACCTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAAACT

GAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAA

TGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGC

AGAAGGGAGCAGAGGCGGC (SEQ Ш NO: 69).

In a particular embodiment, the nucleic acid that encodes antigen cassette 11 and self-cleaves

- 24 048516 the peptide being synthesized includes the nucleotide sequence:

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG TCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAACGCAGTGGGGCGCGAT

CAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTC

TCACTCAACATGGCAAGGAAGACCTTGGTTACCAACCATACAGAGTAGTAGTACTTT

CTTTTGAAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAAT

CCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAA

GGCTTCTACGCAGAAGGGAGCAGAGGCGGC (SEQ ID NO: 87).

Cassette 12 contains the following epitope fragments:

A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 1 region involved in the entry of the ALNTLVKQL virus (SEQ ID NO: 11);

Fragment (976-984 aa) of the described (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12);

Fragment (996-1008 aa) described (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13);

Fragment (104-113 aa) of the described (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16);

Fragment (351-359 aa) of the above described above above above into the infected cell:

glycoprotein glycoprotein nucleoprotein nucleoprotein

S virus SARS-CoV-2 S virus SARS-CoV-2 N virus SARS-CoV-2 N virus SARS-CoV-2

(SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

The following sequence describes antigen cassette 12:

RRIRGGDGKMKDLSPRWYFYYLGTGPDKDPNFKDQVILLNKHIDAGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIR AA (SEQ GO NO: 34).

It should be taken into account that the above-mentioned antigen cassette 12 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GGSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPDKDPN

FKDQVILLNKHIDAGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAE

VQIDRLITGRLQSLQTYVTQQLIRAA (SEQ GO NO: 52).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 52 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT ATTTCTACTACCTAGGAACTGGGCCAGGAAAACTTCAAGATGTGGTCAACCAAAAT GCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCA AGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATT GATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTA ATTAGAGCTGCAGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAA GCATATTGACGCA (SEQ GO NO: 70).

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

- 25 048516

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG

TCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGGAAAACTTCAAGATGTGGT

CAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGG

TGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGA

higher nucleoprotein N virus SARS-CoV-2 higher nucleoprotein N virus SARS-CoV-2 higher nucleoprotein N virus SARS-CoV-2

AGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGA

CTCAACAATTAATTAGAGCTGCAGACAAAGATCCAAATTTCAAAGATCAAGTCATTT

TGCTGAATAAGCATATTGACGCA (SEQ ID NO: 88).

Cassette 13 includes the following epitope fragments:

A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10);

Fragment (138-146 aa) of the described (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17);

Fragment (160-167 aa) described (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18);

Fragment (351-359 aa) of the described (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

The following sequence describes antigen cassette 13:

DDKDPNFKDQVILLNKHIDAGKLQDVVNQNAQALNTLVKQLSSNFGTEGALNTP

KDHIGTRNPANNAAIVLQLPQGTTLPKGFYAGYQPYRVVVLSFELLHAPA (SEQ Ш NO: 35).

It should be taken into account that the above-mentioned antigen cassette 13 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLL1<QAGDVEENPGPDDI<DPNFI<DQVILLNI<HIDAGI<LQDVVNQNA

QALNTLVKQLSSNFGTEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAGYQPY

RVVVLSFELLHAPA (SEQ Ш NO: 53).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 53 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

GATGACAAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATA

TTGACGCAGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACG

CTTGTTAAACAACTTAGCTCCAATTTTTGGTACTGAGGGAGCCTTGAATACACCAAAA

GATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCT CAAGGAACAACATTGCCAAAAGGCTTCTACGCAGGTTACCAACCATACAGAGTAGT AGTACTTTCTTTTGAACTTCTACATGCACCAGCA (SEQ ID NO: 71).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG

GAGAACCCCTGGACCTGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTG

AATAAGCATATTGACGCAGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGC TTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTACTGAGGGAGCCTTGAA

TACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCT

ACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGGTTACCAACCAT

ACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCA (SEQ Ш NO: 89).

- 26 048516

Cassette 14 contains the following epitope fragments:

A fragment (504-515 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10);

Fragment (138-146 aa) of the described (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17);

Fragment (160-167 aa) of the described (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18);

Fragment (351-359 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus nucleoprotein N of the SARS-CoV-2 virus above nucleoprotein N of the virus

SARS-CoV-2 (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

Antigen cassette 14 is described by the following sequence:

DDKDPNFKDQVILLNKHIDANGERSGARSKQRRPQGLPNNTASWFTALTQHGKE

DLGYQPYRVVVLSFETEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRG

G (SEQ ID NO: 36).

It should be taken into account that the above-mentioned antigen cassette 14 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPDDKDPNFKDQVILLNKHIDANGERSGARSKQR

RPQGLPNNTASWFTALTQHGKEDLGYQPYRVVVLSFETEGALNTPKDHIGTRNPANNA

AIVLQLPQGTTLPKGFYAEGSRGG (SEQ Ш NO: 54).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 54 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

GATGACAAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATA

TTGACGCAAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGT

TTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGAC

CTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAAACTGAGGGAGCCTTG

AATACACCAAAAGATCACATTGGCACCCCGCAATCCTGCTAACAATGCTGCAATCGT

GCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCA GAGGCGGC (SEQ Ш NO: 72).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCTGGACCTGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTG AATAAGCATATTGACGCAAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCG GCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGG CAAGGAAGACCTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAAACTGA

GGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATG CTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAG AAGGGAGCAGAGGCGGC (SEQ Ш NO: 90).

Cassette 15 contains the following epitope fragments:

A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the heptad repeat 1 region involved in the entry of the ALNTLVKQL virus (SEQ ID NO: 11);

Fragment (976-984 aa) of the above-described glycoprotein S (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12);

Fragment (996-1008 aa) of the above-described glycoprotein S (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13);

Fragment (104-113 aa) of the above-described nucleoprotein N (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16);

into the infected cell:

SARS-CoV-2 virus SARS-CoV-2 virus SARS-CoV-2 virus

- 27 048516

Fragment (351-359 aa) of the above-described nucleoprotein N of the SARS-CoV-2 virus (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21).

The following sequence describes antigen cassette 15:

DDKDPNFKDQVILLNKHIDAYKTFPPDGKMKDLSPRWYFYYLGTGPEKLQDVV

NQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQL IRAA (SEQ ID NO: 37).

It should be taken into account that the above-mentioned antigen cassette 15 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPDDKDPNFKDQVILLNKHIDAYKTFPPDGKMKD

LSPRWYFYYLGTGPEKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAE

VQIDRLITGRLQSLQTYVTQQLIRAA (SEQ Ш NO: 55).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 55 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

GATGACAAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATA TTGACGCATACAAAACATTCCCACCAGACGGTAAAATGAAAGATCTCAGTCCAAGA TGGTATTTCTACTACCTAGGAACTGGGCCAGAAAAACTTCAAGATGTGGTCAACCAA AATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATT TCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAA ATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAA TTAATTAGAGCTGCA (SEQ Ш NO: 73).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCTGGACCTGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTG AATAAGCATATTGACGCATACAAAACATTCCCACCAGACGGTAAAATGAAAGATCT CAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAAAACTTCAAGATGT GGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTT TGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCT GAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGT

GACTCAACAATTAATTAGAGCTGCA (SEQ ID NO: 91). Cassette 16 includes the following epitope fragments: S virus SARS-CoV-2 Fragment (976-984 a.o.) of the described higher glycoprotein (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); Fragment (996-1008 a.a.) of the described higher glycoprotein S virus SARS-CoV-2 (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); Fragment (104-113 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); Fragment (351-359 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21); Fragment (293-302 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19); Fragment (316-324 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20). The following sequence describes the antigen cassette 16: RRLRGGDGKMKDLSPRWYFYYLGTGPERQGTDYKHWPQIAQFAPSASAFFGMS

RIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKSVLNDILS

RLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA (SEQ Ш NO: 38).

It should be taken into account that the above-mentioned antigen cassette 16 in the vector is fused with

- 28 048516 self-cleaving peptide. This fusion polypeptide has the following sequence: GSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPERQGT

DYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNK HIDAYI<TFPPTEPI<I<SVLNDILSRLDI<VEAEVQIDRLITGRLQSLQTYVTQQLIRAA (SEQ IDNO: 56).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 56 may be encoded by a wide variety of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT

ATTTCTACTACCTAGGAACTGGGCCAGAAAGACAAGGAACTGATTACAAACATTGG

CCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATT

GGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATT

GGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGA

CGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGAGTGTTTTAAATGATATCCT TTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCA GACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCA (SEQ ID NO: 74).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG TCCAAGATGGTATTTCTACTACCTAGGAACTGGGGCCAGAAAGACAAGGAACTGATT ACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAA TGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTG CCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATA

AGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGAGTGTTTTA AATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTG ATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCT GCA (SEQIDNO: 92).

Cassette 17 contains the following epitope fragments:

A fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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);

Fragment (976-984 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12);

A fragment (504-515 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (SEQ ID NO: 1) from the region of the receptor-binding domain (RBD), responsible for binding to receptors on the surface

type of infected cells: GYQPYRVVVLSF (SEQ ID NO: 10); glycoprotein S virus SARS-CoV-2 Fragment (996-1008 a.o.) of the described higher (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); Fragment (104-113 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); Fragment (138-146 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): ALNTPKDHI (SEQ ID NO: 17); Fragment (160-167 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): QLPQGTTL (SEQ ID NO: 18). The following sequence describes the antigen cassette 17:

- 29 048516

RRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKD HIGTRNP ANN AAIVLQLPQGTTLPKGALNTL VKQLS SNFGAIS SVLNDILSRLDKVEAEV QIDRLITGRLQSLQTYVTQQLIRAAEIRAGYQPYRVVVLSFELLHAPA (SEQ ID NO: 39).

It should be taken into account that the above-mentioned antigen cassette 17 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPY GANKDGIIWVATEGALNTPKDHIGTRNP ANN AAIVLQLPQGTTLPKGALNTL VKQLSSN FGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRAGYQPYRVVVL SFELLHAPA (SEQ ID NO: 57).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 57 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT ATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAG ACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATT GGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACA ACATTGCCAAAAGGCGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGT GCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAA GTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACT CAACAATTAATTAGAGCTGCAGAAATCAGAGCTGGTTACCAACCATACAGAGTAGT AGTACTTTCTTTTTGAACTTCTACATGCACCAGCA (SEQ Ш NO: 75).

In a particular embodiment, the nucleic acid that encodes antigen cassette 17 and the self-cleaving peptide includes the nucleotide sequence:

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG TCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGG TGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAA AAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTC CTCAAGGAACAACATTGCCAAAAGGCGCTTTAAACACGCTTGTTAAACAACTTAGCT CCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGT TGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGA CATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTGGTTACCAACCAT ACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCA (SEQ Ш NO: 93).

Cassette 18 contains the following epitope fragments:

Fragment (958-966 aa) of the above-described S glycoprotein of the SARS-CoV-2 virus (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); glycoprotein S virus SARS-CoV-2 Fragment (976-984 a.o.) of the described higher (SEQ ID NO: 1): VLNDILSRL (SEQ ID NO: 12); Fragment (996-1008 a.a.) of the described higher glycoprotein S virus SARS-CoV-2 (SEQ ID NO: 1): LITGRLQSLQTYV (SEQ ID NO: 13); Fragment (104-113 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): LSPRWYFYYL (SEQ ID NO: 16); Fragment (351-359 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): ILLNKHIDA (SEQ ID NO: 21); Fragment (293-302 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2 (SEQ ID NO: 3): RQGTDYKHWP (SEQ ID NO: 19); Fragment (316-324 a.a.) of the described higher nucleoprotein N virus SARS-CoV-2

- 30 048516 (SEQ ID NO: 3): GMSRIGMEV (SEQ ID NO: 20).

The following sequence describes antigen cassette 18:

A (SEQ Ш NO: 40).

It should be taken into account that the above-mentioned antigen cassette 18 in the vector is fused with a self-cleaving peptide. This fused polypeptide has the following sequence:

GSGATNFSLLKQAGDVEENPGPRRIRGGDGKMKDLSPRWYFYYLGTGPERQGT DYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNK HIDAYKTFPPTEPKKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEV QIDRLITGRLQSLQTYVTQQLIRAA (SEQ ID NO: 58).

It will be obvious to a person skilled in the art that a polypeptide with the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 58 may be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such 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 includes its complement unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

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

AGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGT ATTTCTACTACCTAGGAACTGGGGCCAGAAAGACAAGGAACTGATTACAAACATTGG CCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATT GGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATT GGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGA CGCATACAAAACATTCCCACCAACAGAGCCTAAAAAACTTCAAGATGTGGTCAACC AAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAA TTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGC AAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAAC AATTAATTAGAGCTGCA (SEQ ID NO: 76).

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

GGAAGTGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAG TCCAAGATGGTATTTCTACTACCTAGGAACTGGGGCCAGAAAGACAAGGAACTGATT ACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAA TGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTG CCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATA AGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAACTTCAAGAT GTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAAT TTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGG CTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATAT GTGACTCAACAATTAATTAGAGCTGCA (SEQ Ш NO: 94).

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

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

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

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

A recombinant virus based on the influenza virus for the induction of specific immunity to the influenza virus and/or the prevention of diseases caused by the influenza virus.

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

The term recombinant influenza virus-based virus (or influenza virus-based virus-like particle, or recombinant influenza virus strain) in the context of the present description refers to an influenza virus that includes any of the above nucleic acids that encode the above recombinant polypeptide, for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus. Due to its design feature, the recombinant influenza virus-based virus is capable of producing a higher level of antibodies to the influenza virus when administered to a mammal. Moreover, the recombinant influenza virus, including the above-mentioned nucleic acid, which encodes a recombinant polypeptide based on fragments of coronavirus proteins, allows the formation of specific T-cell immunity to coronavirus when administered to a mammal, and thus this recombinant virus based on the influenza virus can be used for the combined prevention of diseases caused by the coronavirus virus and diseases caused by the influenza virus.

The influenza virus sign means a virus from the Orthomyxoviridae family, which includes the monotypic genera Alphainfluenzavirus (formerly influenza virus type A), Betainfluenzavirus (formerly influenza virus type B), Gammainfluenzavirus (formerly influenza virus type C) and Deltainfluenzavirus (formerly influenza virus type D), in which the division into genera is determined by the antigenic properties of the ribonucleoprotein complex. The influenza virus is an enveloped spherical virus with a diameter of 100-120 nm, the outer layer of which consists of a lipid membrane on which the main glycoproteins of the virus are exposed - hemagglutinin (HA) and neuraminidase (NA), and the matrix protein M2, which forms ion channels. Matrix protein 1 (M1), which determines the shape of the viral particle, is located under the lipid membrane. The influenza virus genome is segmented and represented by minus-strand RNA, which is located inside the virion as a ribonucleoprotein complex (RNP). Influenza A virus, 8 RNA fragments encode at least 12 proteins: HA, M1, M2, NA, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, PB2. Based on the antigenic properties of the HA and NA surface antigens, influenza A viruses are divided into serosubtypes, such as H1N1, H1N2, H2N2, H3N2, H5N1, etc.

In some embodiments, the isolated recombinant virus based on the influenza virus is a vaccine strain of the influenza virus.

Vaccine strains are characterized by the fact that they contain genes encoding internal and non-structural proteins of the influenza virus (M1, M2, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, РВ2), from a special laboratory strain A/Leningrad/134/17/57 (H2N2) or its derivatives. Strain A/Leningrad/134/17/57 (H2N2) was obtained by the method of cold adaptation of the epidemic virus A/Leningrad/134/57 (H2N2), by its 17-fold passaging in developing chicken embryos at a temperature reduced to 25°C [Alexandrova G.I., Smorodintsev A.A. Obtaining of an additionally attenuated vaccinating cryophilic influenza strain//Rev Roum Inframicrobiol. - 1965. - V. 2. - No. - p. 179-189.]. As a result of passages, the A/Leningrad/134/17/57 (H2N2) virus acquired a set of mutations in the genes encoding the surface and internal proteins of the virion (Table A), which determined its temperature-sensitive, cold-adapted and attenuated phenotype. The primary nucleotide sequence of the strain was determined (isolate number in the GISAID database EPI_ISL_169836, gene access codes EPI555079 to EPI555086). It has been previously shown that the mutant PB2 protein is the main determinant of the attenuated phenotype in the A/Leningrad/134/17/57 (H2N2) strain, and the polymerase genes PB1 and PA complement this trait [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/134/17/57 (H2N2) influenza virus, in Options for the Control of Influenza IV. 2001. Okinawa, Japan: Elsevier Science BV.]. The role of individual mutations in the genome of the A/Leningrad/134/17/57 (H2N2) strain in the manifestation of the main phenotypic feature of the virus - sensitivity to elevated incubation temperatures was also determined [Isakova-Sivak I, Chen LM, Matsuoka Y, Voeten JT, Kiseleva I, Heldens JG, den Bosch Hv, Klimov A, Rudenko L, Cox NJ, Donis RO. Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2). Virology. 2011 Apr 10; 412(2):297-305. doi: 10.1016/j.virol.2011.01.004].

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Table A

Amino acid differences in the genome of Len/17 and Len/134 viruses

Protein Position Len/134 Len/17 Amino acid Codon Amino acid Codon RV2 478 Vai GTA Leu TTA RV1 265 Lys AAG Asn AAT 591 Vai GTT lie ATT RA 28 Leu CTG Pro CCG 341 Vai GTA Leu TTA NA1 181 Asn AAT Thr ACT 197 Vai GTT lie ATT 225 Met ATG lie ATA NA2 18 He ATT Vai GTT NA 366 He ATS Thr ACC Ml 15 lie ATS Vai GTC 144 Phe TTT Leu TTG NS2 100 Met ATG lie ATA

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

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

In some embodiments of the invention, the isolated recombinant influenza virus is derived from an H2N2 influenza virus.

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

In some embodiments of the invention, the isolated recombinant virus based on the influenza virus comprises any of the above nucleic acids of the invention, which is located in the neuraminidase (NA) gene or the NS gene of the influenza virus. Derivatives of the A/Leningrad/134/17/57 (H2N2) virus are understood to mean:

1) influenza A viruses in which the surface antigens HA and NA belong to previously circulating, potentially pandemic, pandemic or seasonal epidemic influenza A viruses of the 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 strain A/Leningrad/134/17/57 (H2N2);

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

3) influenza A viruses that have an amino acid sequence of viral proteins identical to A/Leningrad/134/17/57 (H2N2), encoded by any nucleotide sequence (the degeneracy of codons for a number of amino acids is taken into account);

4) influenza A viruses of any serosubtype containing amino acid residues in internal and nonstructural 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-Ile100), in full or in various combinations;

5) influenza A viruses, the sequence of internal and non-structural proteins of which (M1, M2, NP, NS1, NS2, PA, PA-X, PB1, PB1-F2, PB2) differs from the A/Leningrad/134/17/57 (H2N2) virus by a number of amino acids;

6) influenza A virus of any serosubtype containing modified genes of the A/Leningrad/134/17/57 (H2N2) strain (shortening of the reading frame of the NS1 gene is taken into account).

Pharmaceutical composition/vaccine.

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

- 33 048516

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

In specific embodiments, the present invention relates to a pharmaceutical composition comprising a recombinant virus based on the influenza virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. The carrier for injections is usually liquid. The carrier for other routes of administration can be either solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For administration by inhalation, the carrier is respirable and is preferably in solid or liquid dispersed form. As an injection medium, it is preferable to use water containing additives conventional for injection solutions, such as stabilizing agents, salts or saline solutions and/or buffers.

The pharmaceutical composition means a composition comprising the above-mentioned recombinant virus based on the influenza virus of the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as fillers, solvents, diluents, carriers, auxiliary, distributing, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, regulators of prolonged delivery, the choice and ratio of which depends on the nature and method of administration and dosage. The pharmaceutical compositions of the present invention and the methods for their manufacture will be undoubtedly obvious to those skilled in the art. The manufacture of pharmaceutical compositions should preferably comply with the requirements of GMP (good manufacturing practice). The composition may include a buffer composition, tonic agents, stabilizers and solubilizers.

A pharmaceutically acceptable material is one that does not have biological or other contraindications, for example, the material can be administered to a subject without any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, for ex vivo cell transduction or for in vivo administration of a recombinant virus based on the influenza virus of the invention directly to a subject.

The term excipient or auxiliary substance is used in this document to describe any component different from those previously described in this invention. These are substances of inorganic or organic origin used in the process of production, manufacturing of medicinal products to impart the necessary physicochemical properties to them.

A stabilizer is an auxiliary substance or a mixture of two or more auxiliary substances that ensure physical and/or chemical stability of the active agent.

The term buffer, buffer composition, buffer agent means a solution capable of maintaining a pH value due to the interaction of acidic and alkaline components included in its composition, which enables the preparation of the influenza virus-based vector to exhibit resistance to changes in pH. In general, the pH values of the pharmaceutical composition from 4.0 to 8.0 are preferable. For example, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions can be used as buffer agents, but are not limited to them.

A pharmaceutical composition is stable if the active agent retains its physical stability and/or chemical stability and/or biological activity during the stated shelf life at a storage temperature of, for example, 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The storage period is selected based on the results of stability studies under accelerated and natural storage.

The pharmaceutical composition of the present invention may be manufactured, packaged, or commercially sold in a single unit dose or multiple single unit doses in a finished dosage form. As used herein, the term unit dose means a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The quantity of the active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

In one aspect, the present invention relates to a vaccine for inducing specific immunity to influenza virus and/or preventing diseases caused by influenza virus, which comprises any of the above-mentioned recombinant viruses based on influenza virus in an effective amount.

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

Coronavirus refers to viruses from the coronavirus family (Coronaviridae).

- 34 048516

This family includes the genus Alphacoronavirus and the genus Betacoronavirus.

The genus Alphacoronavirus includes:

1) human coronavirus 229E - causes acute respiratory viral infections;

2) human coronavirus NL63 - causes acute respiratory viral infections. The genus betacoronaviruses (Betacoronavirus) includes:

1) human coronavirus OC43 - causes acute respiratory viral infections;

2) human coronavirus HKU1 - causes acute respiratory viral infections;

3) MERS-CoV;

4) SARS-CoV;

5) SARS-CoV-2.

The above pharmaceutical composition or vaccine can be used for the prevention of diseases caused by any of the above coronaviruses.

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

In some embodiments, the vaccine coronavirus is selected from the group consisting of human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV2.

In some embodiments of the vaccine, the specific immunity to the coronavirus is 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 the pathogen that provides protective immunity (e.g., immunity that protects a subject from infection by the pathogen and/or reduces the severity of a disease or condition caused by infection due to the pathogen). Protective immunity may include the formation of antibodies and/or a cell-mediated response.

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

In some embodiments of the invention, the vaccine comprises a recombinant virus based on an influenza virus, which is preferably contained in a biologically effective amount.

In some embodiments of the invention, the vaccine comprises a recombinant virus based on an influenza virus, which is preferably contained in a therapeutically effective amount.

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

Application

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant viruses based on the influenza virus or the above-mentioned composition for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus.

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

Any of the above recombinant viruses based on the influenza virus or the above composition can be used to induce specific immunity to the influenza virus and coronavirus and/or prevent diseases caused by the influenza virus and diseases caused by coronavirus, where coronavirus means viruses from the coronavirus family (Coronaviridae), in particular alpha-coronaviruses, which include human coronavirus 229E or human coronavirus NL63, and beta-coronaviruses, which include human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV, SARS-CoV-2.

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

In some embodiments of the use, the coronavirus is a betacoronavirus.

In some embodiments of the use, the coronavirus is selected from the group consisting of human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARSCoV-2.

In one aspect, the present invention relates to a method for inducing specific immunity to an influenza virus and/or preventing diseases caused by an influenza virus, or inducing specific immunity to an influenza virus and a coronavirus, or combined prevention of diseases caused by an influenza virus and diseases caused by a coronavirus, comprising administering to a mammalian body any of the above-mentioned recombinant viruses based on an influenza virus or the above-mentioned composition or the above-mentioned vaccine, in an effective amount.

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

- 35 048516

In some 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 the coronavirus is specific T-cell immunity.

Any method for administering a recombinant influenza virus-based virus accepted in the art can be suitably used for the above-mentioned recombinant influenza virus-based virus of the present invention.

Examples of routes of administration include topical, intranasal, inhalational, transmucosal, transdermal, enteral (e.g., oral, rectal), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular) administration, and injection directly into tissue or into an organ.

The preferred method of administering the above-mentioned recombinant virus based on the influenza virus according to the present invention is intranasal administration.

Injectable preparations can be prepared in conventional dosage forms: as liquid solutions or suspensions, solid forms suitable for preparing solutions or suspensions in liquid before injection, or as emulsions. Alternatively, the above-mentioned recombinant virus based on the influenza virus of the present invention can be administered locally rather than systemically, for example, as a depot or in a sustained-release composition.

The recombinant virus based on the influenza virus according to the present invention is introduced into the body in an effective amount.

In some embodiments, the recombinant influenza virus of the present invention is preferably administered to the body in a biologically effective amount.

In some embodiments, the recombinant influenza virus-based virus of the present invention is preferably administered to the body in a therapeutically effective amount.

The dosages of the above recombinant virus based on the influenza virus of the present invention will depend on the route of administration, the specific viral vector and can be determined by routine methods. Exemplary doses for achieving a therapeutic effect are viral titers of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ infectious units or more, preferably from 10 ⁷ to 10 ⁸ infectious units.

Any of the above recombinant viruses based on the influenza virus or the above composition can be used to induce specific immunity to viruses from 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 preventing any coronavirus from the above group, is achieved due to the following.

The antigen cassettes of the present invention used to induce a cellular immune response against SARS-CoV-2 used at least 102 amino acid sequence fragments that match the SARS-CoV-1 and SARS-CoV-2 proteins registered in the IEDB database (iedb.org) as immunogenic:

- 36 048516

GYQPYRVVVL, QPYRVVVLSF, PYRVVVLSF, LQDVVNQNAQALNTL, DVVNQNAQALNTLVKQL, AQALNTLVK, QALNTLVKQLSSNFGAI, ALNTLVKQL, LNTLVKQLSSNFGAI, SSNFGAISSVLNDIL, AISSVLNDILSRLDKVE, SVLNDILSR, VLNDILSRL, ILSRLDKVEAEVQIDRL, RLDKVEAEV, EAEVQIDRLITGRLQSL, AEVQIDRLIT, AEVQIDRLI, VQIDRLITGR, IDRLITGRLQSLQTY, RLITGRLQSLQTYVTQQ, LITGRLQSL, 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, AFFGMSRIGMEVTPSGTW, FFGMSRIGMEVTPSGTW, GMSRIGMEV,

MSRIGMEVTPSGTWL, GMEVTPSGTWL, MEVTPSGTWL, VTPSGTWLTY, TPSGTWLTY, ILLNKHID, ILLNKHIDA, LLNKHIDAYKTFPPTEPK, LNKHIDAYKTFPPTEPK, NKHIDAYKTFPPTEP, TFPPT, KTFPPTEPKK, KTFPPTEPK, TFPPTEPK, FLWLLWPVT, FLWLLWPVTLACFVL, FLWLLWPVTL, LWLLWPVTL, WLLWPVTLA, LWPVTLACF, WPVTLACFVL, TLACFVLAA, TLACFVLAAV, FVLAAVYRI.

In a number of cases, epitopes overlapped each other to form longer immunogenic regions (respectively, also coinciding in SARS-CoV-1 and SARS-CoV-2):

LQDVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTY VTQQLIRAAEIR, EIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWY, GYQPYRVVVLSF, WLGFIAGLIAIVMVTI, IRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTY, VLQLPQGTTLPKGFYAEGSRGGSQ, ILLNKHIDAYKTFPPTEPKK, ALALLLLDRLNQL, ATEGALNTPKDHI, LSPRWYFYYL, FLWLLWPVTLACFVLAAVYRI.

In most of these regions, one can see consensus hydrophobic motifs characteristic of antigenic peptides bound by proteins of the major histocompatibility complex (MHC) of different classes, for example

FLWLLWPVTLACFVLAAVY. WLGFIAGLIAIVMVTI, APHGVVFLHVTYV,

F APSASAFFGM, FGAISSVL, ALALLLL (hydrophobic residues are underlined). Due to this characteristic feature, some fragments of coronavirus proteins containing the most universal motifs are recognized by several allelic variants of HLA. For example, the LSPRWYFYY epitope binds to at least seven allelic forms of HLA (http://www.iedb.org/epitope/39576). And for the FIAGLIAIV epitope, binding to all variants of HLA group A2, including the most common in the world population HLA-A*02:01 (http://www.iedb.org/epitope/16156) has been shown.

Among the previously selected sequences used in this invention, at least 10 antigenic fragments were found that have significant homology with the sequences of other pandemic and epidemic strains of coronaviruses, including those causing seasonal acute respiratory viral infections (beta-coronaviruses HKU1 and OC43, alpha-coronaviruses 229E and NL63).

Most of the conserved regions used in the invention belong to the C-terminal region of glycoprotein S. They have the highest immunogenicity in healthy people due to their greater similarity to the sequences of coronaviruses that cause seasonal ARVI: HKU1, OC43, 229E and NL63 [Braun, J, 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].

Thus, the ILSRLDKVEAEVQIDRLITGRL fragment of the S glycoprotein of SARS-CoV-2 is 81.8% identical to a similar fragment of the betacoronaviruses HKU1 and OC43, which cause acute respiratory infections

- 37 048516 human; section VVNQNAQ ALNTLVKQL - by 81.2%, and section AEVQIDRLITGR - by 83.3%:

SARS-CoV-2

LQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ L

SARS-CoV-1

LQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ L MERS

VQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQ L HKU1

IQSWNANAQALNSLLQQLFNKFGAISSSLQEILSRLDNLEAQVQIDRLINGRLTALNAYVSQQ L OC43

IQAWNANAEALNNLLQQLSNRFGAISASLQEILSRLDALEAEAQIDRLINGRLTALNAYVSQQ

L 229E IQDWNQQGNSLNHLTSQLRQNFQAIS S SIQAIYDRLDTIQADQQVDRLITGRLAALNVFVSHT L NL63

IQDWNQQGSALNHLTSQLRHNFQAISNSIQAIYDRLDSIQADQQVDRLITGRLALNAFVSQV L

The general sequence (948-1012 aa of the S glycoprotein of the SARS-CoV-2 coronavirus) includes the following fragments used in the invention:

ALNTLVKQL (IEDB ID: 2801), VLNDILSRL (IEDB ID: 69657), RLDKVEAEV (IEDB ID: 54507),

LITGRLQSL (IEDB ID: 36724), RLQSLQTYV (IEDB ID: 54725).

For all of the listed fragments, binding to the most common allelic variant in the world population, HLA-A*02:01, is described, which is characterized by interaction with peptides that have structures listed in The Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/143).

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

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

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

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

All key residues of these consensus sequences that act as a hydrophobic anchor for binding to HLA (in the ninth, second and, in one case, first position) are found in all corresponding epitopes (with the exception of RLDKVEAEV of all considered coronaries)

viruses (here and below, residues that match SARS-CoV-2 are highlighted in underline, key residues for binding to HLA are in bold): SARS-CoV-2 ALNTLVKQL VLNDILSRL RLDKVEAEV LITGRLQSL RLQSLQTYV SARS-CoV-1 ALNTLVKQL VLNDILSRL RLDKVEAEV LITGRLQSL RLQSLQTYV MERS ALSKLASEL SIGDIIQRL RLDVLEQDA LINGRLTTL RLTTLNAFV HKU1 ALNSLLQQL SLQEILSRL RLDNLEAQV LINGRLTAL RLTALNAYV OC43 ALNNLLQQL SLQEILSRL RLDALEAEA LINGRLTAL RLTALNAYV 229E SLNHLTSQL SIQAIYDRL LITTGRLAAL RLAALNVFV NL63 ALNHLTSQL SIQAIYDRL LITTGRLAAL RLAALNAFV

According to the IEDB, the remaining amino acid variations in the sequences of different strains either do not have a noticeable effect on binding to the corresponding HLA, or further enhance the interaction with it (for example, for HLA-A*02:01, the replacement of the fourth lysine residue in RLDKVEAEV

- 38 048516 in SARS-CoV-2 to an asparagine residue in HKU1 and an alanine residue in OC43; or a substitution of the seventh lysine residue in ALNTLVKQL in SARS-CoV-2 to a serine or glutamine residue in the remaining strains). Detailed binding efficiency maps for each amino acid residue at each position of the antigen are available on the IEDB website: HLA-A*02:01 (www.iedb.org/motif_output/HLA-A-02:01-9.html). HLA-A*02:02 (www.iedb.org/motif_output/HLA-A-02:02-9.html). HLA-A*02:03 (www.iedb.org/motif_output/HLA-A-02:03-

9.html).

HLA-A*02:06 (www.iedb.org/motif_output/HLA-A-02:06-9.html).

HLA-A*68:02 (www.iedb.org/motif_output/HLA-A-68:02-9.html).

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

SARS-CoV-2

SARS-CoV-1

MERS

HKU1

OC43

229E

NL63

KNLNESLIDLQELGKYEQYIKWPWYIWL KNLNESLIDLQELGKYEQYIKWPWYIWL KALNESYIDLKELGNYTYYNKWPWYIWL KSLNSSFINLKEIGTYEMYVKWPWYIWL KVLNQSYINLKDIGTYEYYVKWPWYVWL DNINSTLVDLKWLNRVETYIKWPWWVWL DQINSTYVDLKLLNRFENYIKWPWWVWL

This section also includes the fragments used in the invention.

NLNESLIDL (IEDB ID: 44814) and YEQYIKWPWY (IEDB ID: 73751).

For NLNESLIDL, binding to HLA-A*02:01 has been described (for the consensus sequence of the bound antigen, see above), for YEQYIKWPWY - to HLA-B*18:01, HLA-B*40:01, HLA-B*44:02, HLA-B*44:03 and HLA-B*45:01, but consensus antigen sequences 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 listed in the Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/34).

HLA-B*45:01 is characterized by interaction with peptides that have the structures indicated in

The Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/36).

All key residues of these consensus sequences that act as a hydrophobic anchor for binding to HLA are either found in all corresponding epitopes of all considered coronaviruses (except MERS) or coincide with those of SARS-CoV-2:

sars-cov-2

SARS-C0V-1

MERS

HKU1

OS43

229E

NL63

NLNESLIDL YEQYIKWPWY

NLNESLIDL YEQYIKWPWY

ALNESYIDL

SLNSSFINL YEMYVKWPWY

VLNQSYINL YEYYVKWPWY NINSTLVDL VETYIKWPWW QINS TYVDL FENYIKWPWW

According to the IEDB, other amino acid variations in the sequences of different strains do not have a significant effect on binding to the corresponding HLA (www.iedb.org/motif_output/HLAA-02:01-9.html; www.iedb.org/motif_output/HLA-B-44:03-10.html; www.iedb.org/motif_output/HLA-B45:01-10.html).

Also in the C-terminal region of the S glycoprotein are the fragments APHGVVFLHVTYV (1056-1068 aa in SARS-CoV-2) and FIAGLIAIV (1220-1228 aa 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^^^, HLA-A*02:06, HLA-A*68:02 has been described (for the consensus sequences of the bound antigens, see above).

Both fragments have low conservation among most coronaviruses, but retain homology with individual strains (VVFLHVTYV SARS-CoV-2 with the alphacoronavirus 229E sequence; FIAGLIAIV - with the MERS sequence):

SARS-CoV-2 VVFLHVTYV FIAGLIAIV

SARS-CoV-1 VVFLHVTYV FIAGLIAIV

MERS FIAGLVALA

229E VAFLPVEYV

According to the IEDB, minor amino acid sequence variations between strains do not significantly affect binding to the corresponding HLA (see above for antigen binding efficiency maps).

Of the SARS-CoV2 coronavirus nucleoprotein sequences used in the invention, the most conservative is the fragment 104-118 aa (LSPRWYFYYLGTGPE): the coincidence with the sequences of betacoronaviruses HKU1 and OC43 reaches 92.8%, with the sequences of MERS 86.7%:

- 39 048516

SARS-CoV-2 LSPRWYFYYLGTGPE

SARS-CoV-1 LSPRWYFYYLGTGPE

MERS LAPRWYFYYTGTGPE

HKU1 LLPRWYFYYLGTGPY

OS 4 3 LLPRWYFYYLGTGPH

2 9E LSPKLHFYYLGTGPH

NL63LPPKVHFYYLGTGPH

The general sequence also includes the antigen fragment SPRWYFYYL (IEDB U: 60242), for which binding to HLA group B is described: HLA-B*O7:O2, HLAB*51:01, HLA-B*53:01, HLA-B*54:01. Consensus sequences of the binding antigen are 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 listed 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 listed in The Immune Epitope Database (IEDB) (https://www.iedb.org/mhc/40).

The key residues that act as a hydrophobic anchor for binding to HLA (at positions two and nine) remain unchanged in the sequences of all the coronaviruses under consideration (SARS-CoV-2, SARS-CoV-1, HKU1, OC43, 229E, NL63), and the remaining substitutions do not have a significant effect on binding, except for the proline residue at position one for NL63, which hinders binding to HLA-B*07:02 (https://www.iedb.ore/motif_output/HLA-B-07:02-9.html;

https://www.iedb.org/motif_output/HLA-B-51:01-9.html): SARS-CoV-2 SPRWYFYYL SARS-CoV-1 SPRWYFYYL HKU1 LPRWYFYYL

OS43 LPRWYFYYL

229E SPKLHFYYL

NL63 PPKVHFYYL

Some of the fragments used in the invention (ALNTLVKQL, VLNDILSRL, FIAGLIAIV) showed the highest level of conservation among a wide range of known coronaviruses: up to 100% homology among 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 PanCoronavirus Vaccines. J Immunol. 2021; 206(11):2566-2582. doi:10.4049/jimmunol.2001438. Supplementary 1], which allows us to expect the formation of memory cells against new cases of zoonotic coronavirus pandemics in the future. Fig. 19 shows a comparison of the sequences of glycoprotein S fragments of different coronaviruses and the percentage of identity is indicated.

Accurate assessment of the efficiency of immune system activation against a wide range of coronaviruses is complicated by the similarity of antigen processing for protein sequences of different coronaviruses due to a large number of individual factors, such as different probabilities of formation of immunoproteasomes with different proteolytic activities 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 the poor study of this issue. Nevertheless, in most cases, the sequences flanking the key immunogenic fragments of the coronaviruses used in this invention are also extremely similar to each other.

Thus, the use of a vaccine containing the antigen cassettes of the present invention can form stable cellular immunity to SARS-CoV-2 and SARS-CoV-1 equally, as well as to all regional variants of SARS-CoV-2 known at the time of publication of the invention, including the British strain of SARS-CoV-2 B.1.1.7, the Brazilian strain of SARS-CoV-2 P.1, the South African strain of SARS-CoV-2 B.1.351 and the Indian strain of SARS-CoV-2 B.1.617.2 (variants). For example, it is known that more than 70% of those who have recovered from COVID-19 have activated T-lymphocytes that recognize the RLQSLQTYV epitope [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 is part of the antigen cassettes of the present invention.

In addition, for all the above-described antigen fragments used in the present invention, not only was a high degree of similarity shown with similar fragments of other strains of the coronavirus family in interaction with HLA of different groups, but also experimentally confirmed

- 40 048516 induction of T-cell immunity with subsequent activation of the interferon response is expected, which gives reason to assume the possibility of forming cellular immunity also against the potentially pandemic strain of MERS and strains causing seasonal acute respiratory viral infections (beta-coronaviruses HKU1 and OC43, alpha-coronaviruses 229E and NL63).

The above calculations are supported 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] shows a cross-reactive cellular immune response to SARS-CoV-2 and betacoronaviruses causing human acute respiratory viral infections: OC43 and HKU1. In particular, the vast majority of T lymphocytes recognizing epitopes from the DLSPRWYFYYL fragment of SARS-CoV-2 used in the invention showed a pronounced interferon response to the QLLSPRWYFYYL peptide from the 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/s41586021-03594-0] describes antibodies from a universal vaccine that successfully neutralized four different coronaviruses. This article indicates that testing the new nanoparticle vaccine on macaques showed that the antibodies produced after vaccination can effectively neutralize not only SARS-CoV-2, but also two closely related bat coronaviruses and SARS-1. The authors of this article believe that such a vaccine can protect not only against SARS-CoV-2, but also against other potential infections that can be transmitted to humans from animals, since in all cases the antibodies neutralized not only SARS-CoV-2 (including the British variant B.1.1.7, South African B.1.351 and Brazilian P.1), but also SARS-CoV-1 and two bat coronaviruses.

Examples

For a better understanding of the invention, the following examples are given. These examples are given for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example for the purpose of avoiding ambiguity, it will be readily apparent to those skilled in the art, based on the teachings disclosed in this invention, that certain changes and modifications may be made without departing from the spirit and scope of the accompanying embodiments of the invention.

Materials and general methods.

Recombinant DNA methods.

DNA manipulations followed standard procedures described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions. Briefly, plasmid DNA for further manipulations was produced in E. coli cells grown under selective pressure with antibiotics to ensure that plasmids were not lost in the cell population. Plasmid DNA was isolated from the cells using commercial kits, measured for concentration, and used for cloning by restriction endonuclease digestion or PCR amplification methods. DNA fragments were ligated together using ligases and transformed into bacterial cells for clone selection and further development. All resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing.

Gene synthesis.

The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments of 300 to 1000 bp in length, flanked by unique restriction sites, were assembled by annealing the oligonucleotides on each other, followed by PCR amplification from the outer primers. The result was a mixture of fragments, including the desired one. The fragments were cloned into intermediate vectors by restriction sites, after which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

Determination of DNA sequences.

DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data processing were performed using SnapGene 4.2 and higher for sequence generation, mapping, analysis, annotation, and illustration.

General description of the assembly and cloning of genetic constructs carrying recombinant segments of the genome of the vaccine strain of influenza virus based on the attenuated strain A/Leningrad/134/17/57, encoding antigen 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 service regulatory sequences, such as signal peptides, linkers, functional domains of other proteins, self-cleaving peptides, etc., were obtained by de novo assembly from individual oligonucleotide fragments of about 100 nucleotides in length, by renaturing them on each other, followed by

- 41 048516

PCR amplification from extreme primers. To optimize the assembly process and stability of the final secondary structure, the software developed by Biocad CJSC was used. During the synthesis of transgene sequences, the necessary SapI restriction sites or overlaps (sequence sections identical for the insert and vector) 20-40 nucleotides long were added to the 5'- and 3'-ends of the molecule for further molecular cloning by ligase-free methods (SLIC, Gibson method).

The obtained synthetic sequences were introduced into the pCIPolISapIT vector for reverse genetics of the influenza virus, containing sequences of one or another segment of the influenza virus genome, using various methods:

1. Cloning using restriction and ligation.

2. The method of one-step molecular cloning using T4 polymerase SLIC (sequence and ligase independent cloning), described everywhere, including in Sambrook J. et al. Molecular cloning: A laboratory manual, and optimized at ZAO Biocad.

3. Gibson's method.

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

One of the following sequences was used as stop codons to terminate translation of the antigen cassette: TAATGATAA, TAAATGATTAA.

When using the described recombinant plasmids of the invention, a pronounced induction of both branches of the immune response is achieved while maintaining the functionality of the vector, primarily its ability to replicate and repeatedly present target SARS-CoV-2 antigens to the immune system.

Genetic construct schemes were obtained using SnapGene and SnapGene Viewer software (https://www.snapgene.com).

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

The nucleotide sequences of the present invention encoding polyepitope antigen cassettes consisting of combinations of fragments of the proteins S glycoprotein (or Spike) (SEQ ID NO: 1), nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, if necessary, membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV-2, described in the corresponding section of the detailed description of the invention (specific variants are indicated in SEQ ID NOs: 59-94), used to induce a cellular immune response, were obtained by the de novo synthesis described above. During the synthesis of the transgene sequences, the necessary SapI restriction sites were added to the 5'- and 3'-ends of the molecule.

The obtained sequences were introduced by the restriction and ligation method described above into the universal vector for reverse genetics of the influenza virus pCIPolISapIT_NA-AnhuiN9, containing sequences of the sixth segment of the influenza virus genome, including the NA gene (N9), encoding the ninth type neuraminidase of the A/Anhiu/1/2013 (H7N9) strain, after the last 3'-terminal codon of the protein-coding part of the gene (ctc) with preservation of the open reading frame for joint translation with the proteins of the modified influenza virus, before the protective 3'-untranslated region of 157 nucleotides (Fig. 1).

Thus, during translation, a full-length neuraminidase is formed, which is separated from the hybrid polypeptide chain by the self-cleaving peptide P2A, which allows it to perform its functions in full, as well as an antigen cassette containing several target epitopes of SARSCoV-2, which is subsequently cleaved in the cell's proteasomes into specified epitopes, which are then presented on the cell surface in a complex with MHC class I proteins.

The resulting genetic constructs range in size from 6333 to 6577 bp and differ only in the unique transgene sequences indicated above, but are otherwise completely identical. As an example, Fig. 2 shows a map of a vector with a total size of 6577 bp encoding a 597 bp insert as a transgene.

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

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

The nucleotide sequences of the present invention encoding the receptor binding domain (RBD) of the Spike protein of the SARS-CoV-2 coronavirus, its derivatives or polyepitope antigen cassettes consisting of combinations of fragments of the proteins of the S glycoprotein (or Spike) (SEQ ID NO: 1), the nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, if necessary, the membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV-2, described in the relevant paragraphs of detail

- 42 048516 of the present invention description (specific variants are indicated in SEQ ID NO: 59-94), used for inducing an immune response, were obtained by the above-described de novo synthesis. During the synthesis of transgene sequences, the necessary overlaps (sequence sections identical for the insert and vector) of 30-40 nucleotides in length were added to the 5'- and 3'-ends of the molecule for further molecular cloning by ligase-free methods.

The obtained sequences were introduced by the Gibson method described above into the universal vector for reverse genetics of the influenza virus pCIPolISapIT_NS-Len17, containing the sequence of the eighth segment of the influenza virus genome, including the modified NS gene from the attenuation donor A/Leningrad/134/17/57.

Taking into account the complex structure and regulation of alternative splicing of the NS gene, as well as the importance of NS1 and NS2 proteins (NEP) for the proper functioning of the influenza virus, the following scheme can be used for this cloning:

The target sequences are inserted into the first reading frame, 3' from the 126th codon of the NS1 protein-coding part (aag), without changing the regulatory sequences of the NS gene that control its splicing (Fig. 3).

In any case, three separate polypeptide chains are formed during translation: a truncated version of the NS1 protein (1-126 aa), which fully performs the functions necessary to maintain the replicative activity of the recombinant influenza virus that forms the basis of the invention; a full-length NS2 protein; as well as a target insert sequence encoding a combination of fragments of the S glycoprotein (or Spike) (SEQ ID NO: 1), nucleoprotein (or Nucleoprotein) (SEQ ID NO: 3), and, if necessary, membrane protein (or Membrane) (SEQ ID NO: 5) of the betacoronavirus SARS-CoV2.

Activation of the immune system using the described recombinant plasmids of the invention is achieved in various ways, depending on the regulatory elements and functional domains used, described in the Detailed Description of the Invention section, and includes, among other things: presentation of immunogenic epitopes on the membrane of infected cells in complex with MHC proteins of both classes.

The resulting genetic constructs range in size from 5656 to 6286 bp and within the same cloning scheme differ only in the unique transgene sequences indicated above, but are otherwise completely identical. As an example, a vector map with a total size of 5899 bp, created according to the above cloning scheme, is given, respectively; encoding a 597-sized insert as a transgene (Fig. 4).

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

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

Recombinant influenza viruses carrying immunogenic regions of the novel coronavirus SARS-CoV-2 in their genome were obtained using the reverse genetics system developed for the attenuation donor virus 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 and 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 certified Vero cell line (WHO). This example shows the production of viable recombinant influenza viruses carrying different coronavirus polyepitope cassettes encoded in one of the three influenza virus genes.

The following materials were used in the experiments:

Plasmids (vector for reverse genetics of influenza virus) 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

Gene Plasmid RV2 pCIPolISapIT_Len_PB2 RV1 pCIPolISapITLenPB 1 RA pCIPolISapITLenPA NP pCIPolISapITLenNP M pCIPolISapITLenM NS pCIPolISapITLenNS

- 43 048516

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

_____________________________Table 2______________________

Gene Plasmid ON pCIPolISapITAnhuiHA NA pCIPolISapITAnhuiNA

Plasmids encoding genes of influenza virus A/Guangdong-Maonan/SWL1536/2019 (H1N1) (Table 3).

_____________________________Table 3______________________________

Gene Plasmid ON pCIPolISapITGDMHA NA pCIPolISapITGDMNA

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

Table 4

Gene Insert Plasmid NS Cassette 1 pCIPolISapITNS-126-Len17_cassette# 1 _CTL_MNS S S NS Cassette 2 pCIPolISapITNS-l 26-Len 17_cassette#2-orig_CTL-CD4BLSN NS Cassette 3 pCIPolISapIT_NS-126-Lenl7_cassette#3-reverse_CTLCD4-BLNS NS Cassette 4 pCIPolISapIT_NS-l 26-Len 17_cassette_4_CTLBLSSMNS NS Cassette 6 pCIPolISapITNS-126-Len 17_cassette_6_CTL_S SMNS NS Cassette 7 pCIPolISapITNS-126-Len 17_cassette_7_CTL_S SNSM NS Cassette 9 pCIPolISapIT_NS-l 26-Len 17_cassette#9_CTLBL_MSSNS NS Cassette 10 pCIPolISapITNS-126-Len 17_cassette# 1OCTLBL_NNNSS NS Cassette 14 pCIPolISapIT_NA-AnhuiN9_cassette#14_CTL-CD4BL_NNSNN NS Cassette 15 pCIPolISapITNS-126-Len 17_cassette# 15CTL-CD4BL_15_NNSSS NS Cassette 18 pCIPolISapIT_NS-126-Lenl7_cassette#18

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

- 44 048516

Table 5

Gene Insert Plasmid NA Cassette 1 pCIPolISapIT_NA-AnhuiN9_cassette# 1_CTL_MNS S S NA Cassette 2 pCIPolISapIT_NA-AnhuiN9_cassette#2-orig_CTL-CD4BL_SN NA Cassette 3 pCIPolISapIT_NA-AnhuiN9_cassette#3-reverse_CTL- CD4-BLNS NA Cassette 5 pCIPolISapIT_NA-AnhuiN9_cassette#5 NA Cassette 8 pCIPolISapIT_NA-AnhuiN9_cassette#8 NA Cassette 15 pCIPolISapIT_NA-AnhuiN9_cassette#15 NA Cassette 16 pCIPolISapIT_NA-AnhuiN9_cassette#16_CTL-CD4BL_NNNSS NA Cassette 17 pCIPolISapIT_NA-AnhuiN9_cassette# 17CTL- BL_17_NNNSSSS NA Cassette 18 pCIPolISapIT_NA-AnhuiN9_cassette#18_CTL-CD4BL_18_NNNNSSS

Vero cell line;

serum-free nutrient medium for cell culture;

cell dissociation reagent Antibiotic-antimycotic solution;

cell growth supplement;

trypsin;

bottle for adherent cell cultures;

Dulbecco's phosphate saline solution;

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 inverting the vial and store at -20. To prepare 10 ml of 3 M NaOAc solution, dissolve 4.08 g of NaOAc-3H ₂ O in 8.38 ml of H2O until completely dissolved;

transfection system;

a set of reagents for carrying out the electroporation procedure;

10-11 day old developing chicken embryos (SPF);

96-well round-bottomed plates for immunological reactions;

filter with pore size of 0.2 microns.

Preparation of Vero cell line. Vero cell line was removed from cryostorage in advance. For this purpose, cells removed from cryostorage were washed from dimethyl sulfoxide in 10 ml of heated medium A, centrifuged at 1500 rpm 20°C for 15 min, then the cell pellet was introduced into a T-75 culture flask and culture medium A was added, the cells were incubated in a thermostat at 37°C and 5% CO2. After the cell monolayer reached 95-100% confluency, the further protocol was started.

Preparation of Vero cells for transfection. Medium B was prepared without the addition of antibiotics, the medium was filtered through a filter with a pore diameter of 0.2 μm. Medium A was removed from the flask with cells, the flask was washed twice with a warm DPBS solution and the cells were removed from the substrate with Accutase (1 ml per T75 flask) by incubating the flask for 1 min at 37°C in a CO2 incubator. % of the cells were transferred to a new N150 flask (Nunc), 100 ml of medium B were added, and the flask was incubated for 16-18 h at 37°C in a CO2 incubator.

The next day, the cells were removed from the substrate using Accutase, following the same protocol. 10 ml of medium B heated to 37°C were added, the cells in the medium were transferred to a sterile test tube and centrifuged for 15 min at 1500 rpm at 20°C.

Preparation of plasmids for obtaining influenza virus (assembly and concentration). On the eve of transfection, plasmids encoding 8 influenza virus genes were mixed in 1 Eppendorf tube at a rate of 2 μg of each plasmid, then 120 μl of sodium acetate buffer were added (in the case of low plasmid concentration, the volume of buffer was calculated at a rate of 2.5 volumes of pDNA solution). Mixed on a vortex for 5 s, then incubated at -20 for 16-18 h, then proceeded to the concentration protocol.

For concentration, Eppendorfs with plasmids were centrifuged at 4°C, 13.2x10 ³ rpm for 15 min.

- 45 048516 immediately after centrifugation, the supernatant was completely collected without touching the sediment. If there was no sediment, the protocol was interrupted and started again from the moment of plasmid assembly.

120 μl of 70% ethanol were added to the test tubes, gently affecting the sediment to wash it from the wall of the test tube, without resuspending it with a pipette. Then centrifugation was carried out at 4°C, 13.2x103 rpm for 5 min, after which the supernatant was completely collected without touching the sediment. Then the sediment was dried at room temperature for 15 min in test tubes with open lids to remove residual ethanol.

The pellet was resuspended in 8 µl TE buffer and the transfection protocol was started.

Transfection of Vero cells with plasmids encoding 8 influenza virus genes. Before transfection, the dispenser, Neon cuvettes, and electroporation apparatus were placed in a laminar flow hood and irradiated with UV to ensure sterility of manipulations. 3 ml of heated medium B were added to a 6-well cell culture plate. 3 ml of buffer E2 were poured into a Neon transfection cuvette and placed in an electroporation apparatus (Neon Pipette Station). After centrifugation of the cells, medium B was removed and the cells were carefully resuspended in buffer R at a rate of 110 μl of suspension per 1 virus.

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

After electroporation, the plate was placed in a _CO2 incubator at 37°C for 6 hours to allow cells to attach. After 6 hours, medium B was removed, the cells were washed twice with warm DPBS solution and 3 ml of medium C were added. Then the plate was incubated at 33°C in a CO2 incubator for 64-72 hours.

Infection of developing chicken embryos (ECEs) with the material after transfection. 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 technique. The remains of transfected cells in the nutrient medium were transferred to a vial and stored at -70°C. Embryos were incubated at 33°C in a thermostat.

Virus detection in hemagglutination reaction. After 72 h, the embryos were cooled for 3 h at +4°C and the shells were opened to collect chorioallantoin fluid (CAF). 50 μl of PBS were added to 7 rows of a 96-well round-bottomed plate for immunological reactions, 100 μl of CAF from each embryo to the upper row. The titration was carried out with two-fold dilutions of 50 μl in 7 rows, after which 50 μl of a suspension of 1% chicken erythrocytes were added to each well of the plate. The presence of the virus was recorded after 20-30 min by the presence of erythrocyte agglutination in the wells. The titer of the virus by hemagglutination was determined by the last dilution of the virus in which erythrocyte agglutination was observed.

Blind passage. If no erythrocyte agglutination was observed in any well, 0.3 ml of HAF from each embryo of passage E1 was transferred to a new embryo using a syringe to allow for possible virus accumulation if the amount was insufficient for detection. Embryos were incubated for 72 h at 33°C. A total of up to 3 consecutive blind passages were performed after each transfection.

Accumulation and cloning of the virus in the erythrocyte agglutination. If erythrocyte agglutination was detected, the HAF was collected, clarified by low-speed centrifugation (3000 rpm, 15 min), aliquoted into 1 ml vials and stored at -70°C. Then, the virus was cloned in the erythrocyte agglutination by the limiting dilution method; the HAF was sterilely collected from individual clones into individual vials. Then, based on one of the clones that did not contain mutations, the working stock of the virus was accumulated by titration in the erythrocyte agglutination.

Control of virus assembly by PCR and sequencing. To control virus assembly in the first passage, in which the presence of the virus was detected by the hemagglutination method, RNA was isolated from 140 μl of the HAF to check for the presence of a specific insertion in the gene(s) by the PCR method with specific primers. Analysis of individual clones was performed by PCR followed by Sanger sequencing of the gene sequence containing the insert. The working stock was also analyzed by sequencing.

Table 6 lists the vaccine candidates that were assembled, with transfection dates and assembly results. The last column of the table lists the date of receipt of the working stock, as well as those candidates that were discontinued early for various reasons.

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Table 6

Results of assembly of vaccine candidates

Vaccine candidate Virus genes PB2, — PB1, PA, HA NA NS NP, M With Vac-1 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+KacceTa 1 — NS126Lenl7+ cassette CoVac-2 Lenl7 H7-Anhui N9-Anhui 2 — NS126Lenl7+ cassette With Vac-3 Lenl7 H7-Anhui N9-Anhui 3 Hl- Nl- NS126Lenl7+ cassette With Vac-4 Lenl7 Guangdong- Guangdong- 1 Maonan Maonan 1 Hl- Nl- NS126Lenl7+ cassette CoVac-5 Lenl7 Guangdong- Guangdong- Maonan Maonan z Hl- Nl- NS126Lenl7+ cassette With Vac-6 Lenl7 Guangdong- Guangdong- Λ Maonan Maonan J — N9-Anhui+ CoVac-7 Lenl7 H7-Anhui cassette 1 Lenl7 N9-Anhui+ CoVac-8 Lenl7 H7-Anhui cassette 2 Lenl7 — N9-Anhui+ Co Vac-9 Lenl7 H7-Anhui cassette 3 Lenl7 — N9-Anhui+ NS126Lenl7+ cassette Co Vac-10 Lenl7 H7-Anhui cassette 1 2 N9-Anhui+ NS126Lenl7+ cassette CoVac-11 Lenl7 H7-Anhui cassette 1 3 N9-Anhui+ NS126Lenl7+ cassette CoVac-12 Lenl7 H7-Anhui cassette 3 1 NS126Lenl7+ cassette CoVac-13 Lenl7 H7-Anhui N9-Anhui 6

- 47 048516

CoVac-14 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 7 CoVac-15 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 9 CoVac-16 Lenl7 H7-Anhui N9-Anhui+ cassette 4 Lenl7 CoVac-17 Lenl7 H7-Anhui N9-Anhui+ cassette 5 Lenl7 CoVac-18 Lenl7 H7-Anhui N9-Anhui+ cassette 8 Lenl7 CoVac-25 Lenl7 H7-Anhui N9-Anhui+ cassette 4 NS126Lenl7+ cassette 6 CoVac-26 Lenl7 H7-Anhui N9-Anhui+ cassette 16 Lenl7 CoVac-27 Lenl7 H7-Anhui N9-Anhui+ cassette 17 Lenl7 CoVac-28 Lenl7 H7-Anhui N9-Anhui+ cassette 18 Lenl7 CoVac-29 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 10 Co Vac-30 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 15 Co Vac-31 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 16 Co Vac-3 2 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 17 Co Vac-33 Lenl7 H7-Anhui N9-Anhui NS126Lenl7+ cassette 18 CoVac-42 Lenl7 H7-Anhui N9-Anhui+ cassette 18 Lenl7

Using the specified design of constructs, the assembly of chimeric influenza viruses was successful. Cassettes of this size, integrated into these regions of the influenza virus genes, do not interfere with the assembly of viable influenza virus.

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

Vaccine viruses were accumulated in developing chicken embryos (ECE). The infectious activity of the viruses was determined in ECE by the limiting dilution method, calculated using the Reed & Muench method and expressed as 50% embryonic infectious doses (EID ₅₀ ). The immunogenic properties of the H7N9 LAIV vaccine candidates carrying polyepitope T-cell cassettes from the SARS-CoV-2 virus against the influenza virus were assessed in experiments on HLA-A2.1 RANDOM TRANSGENIC transgenic mice (Taconic, USA). Two independent experiments were carried out with the vaccine viruses listed in Table 7. In the first experiment, the immunogenicity of four recombinant influenza viruses with inserts of SARS-CoV-2 fragments was studied in comparison with the vector control - the H7N9 LAIV strain [PMID: 30903496]. Mice that received a placebo preparation (saline, PBS) served as a negative control. In the second experiment, the immunogenicity of one recombinant strain was studied in comparison with the vector control placebo group. Each group included 6 mice aged 6-10 weeks. Animals were immunized intranasally with vaccine viruses at a dose of 106 EID ₅₀ , twice, with an interval of 21 days, in a volume of 50 μl/mouse under light inhalation anesthesia with isoflurane.

Mouse serum was collected 10 days after the second immunization and the levels of influenza antibodies were assessed using enzyme-linked immunosorbent assay (ELISA). The antigen used in the ELISA was the whole LAIV H7N9 influenza virus purified on a sucrose density gradient at a concentration of 50

- 48 048516 ng/well. The antigen plates were washed three times with PBS solution containing 0.05% Tween 20 (PBS-T), after which non-specific binding was blocked with 1% BSA solution in PBS. Two-fold dilutions of sera in PBS were added to the wells of the plates in a volume of 50 μl, starting with a dilution of 1:100. After incubation for 1 h at 37°C, the plates were washed 3 times with PBS-T, 50 μl of secondary antibodies against mouse IgG conjugated with horseradish peroxidase (Sigma-Aldrich A9044, USA), diluted 1:10000, were added. After 30-minute incubation, the plates were washed 4 times with PBS-T solution, dried and 50 μl of TMB substrate (1-Step™ Ultra TMB-ELISA Substrate Solution, Thermo, USA) were added. The plates were incubated in a dark place for 5-10 min, then the reaction was stopped with 25 μl of 1 M sulfuric acid. The results were read on an xMark Microplate spectrophotometer (BIORAD) at a wavelength of 450 nm. As a result of the experiment, the levels of produced IgG antibodies were determined by the Area Under the Curve (AUC) parameter, which was calculated using the trapezoid rule separately for each serum. Visualization of the obtained results and statistical processing were performed using GraphPad Prism 5. Statistical differences between groups were determined by one-way ANOVA analysis of variance followed by multiple comparisons according to Tukey's criterion.

Figures 5–8 show the results of assessing the levels of IgG antibodies to the whole influenza virus. All vaccine variants induced high levels of anti-H7N9 IgG antibodies, significantly different from the PBS control group (p<0.0001). In addition, recombinant viruses containing polyepitope cassettes from the SARS-CoV-2 virus were more immunogenic compared to the control H7N9 LAIV vaccine strain used as a viral vector. These data indicate that the immunogenic properties of the live influenza vaccine against the influenza virus are enhanced by the incorporation of polyepitope T-cell cassettes of the new coronavirus into the vaccine strain genome, regardless of which region of the influenza virus genome was modified (the NA gene or the NS1 gene).

Table 7

Recombinant influenza virus vaccine strains studied in mice

Conditional designation Description of the vaccine strain strain Experiment #1 With Vac-13 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 6 in the NS1 gene, shortened to 126 aa. CoVac-16 Reassortant strain 6:2 subtype H7N9 with insertion of cassette 4 in the N9-NA gene CoVac-17 Reassortant strain 6:2 subtype H7N9 with insertion of cassette 5 in the N9-NA gene CoVac-18 LAIV H7N9 Experiment #2 6:2 reassortant strain of subtype H7N9 with insertion of cassette 8 in the N9-NA gene Live influenza vaccine strain A/17/Anui/2013/62 (H7N9) obtained by reverse genetics methods [PMID: 30903496] CoVac-27 Reassortant strain 6:2 subtype H7N9 with insertion of cassette 17 in the N9-NA gene H7N9 GI virus Live influenza vaccine strain A/17/Anui/2013/62 (H7N9) obtained by reverse genetics methods [PMID: 30903496]

Example 5. Evaluation of virus-specific memory T cell levels in an in vitro test on peripheral blood mononuclear cells from volunteers who had COVID-19 in vivo.

This experiment is designed to confirm the correct processing of SARS-CoV-2 polyepitope T-cell cassettes integrated into the influenza vaccine virus genome by human immune cells. Since COVID-19 convalescents have a pool of memory T cells to various epitopes of the new coronavirus, in vitro incubation of PBMC from these donors with vaccine candidates can provide an idea of the ability of recombinant viruses to express integrated T-cell epitopes and, accordingly, stimulate the proliferation of SARS-CoV-2-specific T cells. A prerequisite for setting up the experiment is parallel stimulation of mononuclear cells with the studied recombinant virus and the classical LAIV strain used as a viral vector. The difference in the levels of T cell subpopulations after such stimulation will allow us to judge the activation of T cells to the integrated T-cell epitopes of the coronavirus. Two independent

- 49,048,516 experiments with recombinant influenza viruses and vector control (Table 8). The viruses were accumulated in ECE and purified on a sucrose density gradient to clear the virus-containing fluid from chicken proteins. The infectious titer of the purified influenza virus was determined in ECE by tenfold dilutions of the virus in 3-5 replicates according to the method of Reed & Meunch [1938] and expressed as 50% embryonic infectious doses per ml (EID ₅₀ /ml).

Experiment 1 included 13 volunteers, and Experiment 2 included 17 volunteers. The demographic data of the participants are presented in Table 9. A history of COVID-19 was confirmed by a positive test for viral RNA in swabs (real-time RT-PCR) and/or the presence of SARS-CoV-2-specific IgG antibodies in blood serum (ELISA to the RBD protein SARSCoV-2). The experiment was approved by the Local Ethics Committee of the Institute of Experimental Medicine (extract from the minutes of the LEC meeting No. 2/20 dated 07.04.2020), and all volunteers signed informed consent to the study. In addition, PBMC samples from volunteers who had never been in contact with the SARS-CoV-2 virus (confirmed absence of antibodies to the coronavirus in ELISA) were used as a negative control.

To obtain a PBMC culture, whole blood from COVID-19 convalescents was collected in EDTA vacuum tubes, mixed with Hanks' solution at a 1:1 ratio, and isolated by Ficoll density gradient centrifugation. The isolated mononuclear cell fraction was resuspended in RPMI-1640 medium containing an antibiotic-antimycotic, 10 mM HEPES, P-mercaptoethanol, and 20 U Roncoleukin, and the resulting suspension at a concentration of 4x107 cells/ml was used to stimulate with the studied viruses (50 μl/well of cell suspension or 2x106 cells per sample). Purified influenza viruses were used to stimulate the cells, as well as phorbol and ionomycin as a positive control. Vaccine candidates were added to the wells of the lymphocyte plate in a volume of 50 μl with a multiplicity of infection of 3 (the number of infectious units per cell), after which the cells were incubated in a medium containing 30% fetal bovine serum for 16-18 h at 37°C and 5% CO ₂ . Intracellular staining of cytokines induced by antigen stimulation was carried out in several stages: stopping protein transport of the cell, staining of surface antigens, fixation and permeabilization of the cells, staining of intracellular antigens - cytokines. To stop protein transport, Brefeldin A was used at a dilution of 1:1000, after which the cells were incubated for an additional 5 h at 37°C and 5% CO ₂ and then surface antigens were stained with antibodies to the markers CD4, CD8, CD3, CD45RA, CCR7, as well as with the Zombie Aqua reagent to detect live cells. Fix/Perm buffer was added for cell fixation and permeabilization, and then the fixed and permeabilized cells were stained with anti-cytokine antibodies to IFN-γ. After washing the stained cells with PermWash buffer, the cells were fixed in CytoLast buffer and stored in the dark at 4°C until flow cytometry analysis. Samples were analyzed using a Navios flow cytometer (Beckman Coulter). FACS files were processed using FlowJo software. Data on the % content of virus-specific memory T cells were obtained by subtracting the proportion of cells with spontaneous interferon production (unstimulated MGF) from the T cell levels detected upon cell stimulation with the corresponding virus. Statistical processing of the results was performed in GraphPad Prizm 7.0 software using nonparametric analysis with the Wilcoxon T-test.

Figures 9-17 show data illustrating the levels of IFN-γ-producing cytotoxic and/or helper memory T cells upon stimulation of peripheral blood mononuclear cells from COVID convalescents with vaccine candidates, compared to stimulation with a control vaccine strain - a viral vector. For recombinant viruses CoVac-13, CoVac-15, CoVac-16, a significant increase in the subpopulation of effector memory CD4+ T cells (CD3+CD4+CCR7 ^- ) was shown compared to the viral vector (Figures 9-11). For the recombinant viruses CoVac-17, CoVac-29, CoVac30, CoVac-31, CoVac-32 and CoVac-30, a significant increase in both the T helper (CD4+) subset and the cytotoxic (CD8+) subset of effector memory T cells was shown compared to the viral vector (Fig. 12-17).

Stimulation of mononuclear cells from two blood donors who had not previously encountered SARS-CoV-2 with all the recombinant viruses studied did not lead to a significant increase in IFN-γ secretion by helper or cytotoxic memory T cells, compared with stimulation with the H7N9 LAIV viral vector (Fig. 18), which confirms the specificity of the method used to assess the processing of SARS-CoV-2 polyepitope cassettes integrated into the influenza virus genome by human immune cells.

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Table 8

Recombinant vaccine strains of influenza virus studied in vitro on peri-monocule cells

pharmaceutical blood of COVID-19 convalescents Strain symbol Experiment 1 Description of the vaccine strain With Vac-13 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 6 in the NS1 gene, shortened to 126 aa. CoVac-15 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 9 in the NS1 gene, shortened to 126 aa. CoVac-16 Reassortant strain 6:2 subtype H7N9 with insertion of cassette 4 in the N9-NA gene CoVac-17 LAIV H7N9 Experiment 2 6:2 reassortant strain of subtype H7N9 with insertion of cassette 5 in the N9-NA gene Live influenza vaccine strain A/17/Anui/2013/62 (H7N9) obtained by reverse genetics methods [PMID: 30903496] CoVac-29 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 10 in reneNS1, shortened to 126 a.a. With Vac-30 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 15 in reneNS1, shortened to 126 a.a. With Vac-31 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 16 in reneNS1, shortened to 126 a.a. CoVac-32 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 17 in reHeNSl shortened to 126 a.a. CoVac-33 A 6:2 reassortant strain of the H7N9 subtype with an insertion of cassette 18 in reHeNSl shortened to 126 a.a. H7N9 GI virus Live influenza vaccine strain A/17/Anui/2013/62 (H7N9) obtained by reverse genetics methods [PMID: 30903496]

Table 9

Demographic data of volunteers

Volunteer No. floor Age, years COVID-19 confirmed Severity of the disease The period of blood sampling after the disease PCR IFA Experiment 1 1 M 36 + + Light 1 month 2 AND 28 - + Light 1 month 3 AND 44 + + Heavy 1 month 4 AND 47 + + Heavy 1 month 5 M 42 - + Light 2 months 6 AND 34 - + Light 2 months 7 M 40 + + Heavy 2 months 8 M 29 + + Light 2 months 9 M 30 + + Light 1 month 10 AND 33 + + Light 1 month AND AND 39 - + Light 3 weeks 12 M 34 - + Light 2 months 13 AND 43 - + Light 2 months Experiment 2 1 and 18 - + Light 1 month 2 and 44 - + Light 1 month

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3 M 42 - + Light 2 months 4 M 45 + + Light 3 months 5 and 34 - + Light 2 months 6 M 41 - + Light 2 months 7 M 40 + + Heavy 2 months 8 and 44 + + Heavy 6 months 9 M 36 + + Light 6 months 10 and 49 + + Heavy 6 months 11 and 47 + + Heavy 6 months 12 and 33 + + Light 6 months 13 M 46 + + Light 6 months 14 M 34 + + Light 6 months 15 M 39 + + Light 6 months 16 and 37 + + Light 6 months 17 M 31 + + Light 6 months

Claims (29)

1. An isolated nucleic acid for obtaining a recombinant influenza virus that produces a higher level of antibodies to the influenza virus when administered to a mammal, encoding a recombinant polypeptide from epitope fragments of coronavirus proteins that are selected from the group: i) a) fragments from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; and b) a fragment from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which is SEQ ID NO: 15; and c) a fragment from a membrane protein of coronavirus with the amino acid sequence of SEQ ID NO: 5, which is SEQ ID NO: 22; or ii) a) fragments from a glycoprotein S of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 7 and SEQ ID NO: 8; and b) a fragment from a coronavirus nucleoprotein with the amino acid sequence of SEQ ID NO: 3, which is SEQ ID NO: 20; or iii) a) a fragment from a coronavirus S glycoprotein with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 10 and SEQ ID NO: 11; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; or iv) a) a fragment from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which is SEQ ID NO: 10; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; or v) a) fragments from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 16 and SEQ ID NO: 21; or vi) a) fragments from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 10 and SEQ ID NO: 11; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 21; or vii) a) a fragment from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 10; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 21; or viii) a) fragments from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 12 and SEQ ID NO: 13; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; or ix) a) fragments from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and b) fragments from the coronavirus nucleoprotein with the amino acid sequence SEQ ID - 52 048516 NO: 3, which are SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; or x) a) a fragment from the S glycoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 1, which are SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and b) fragments from the nucleoprotein of coronavirus with the amino acid sequence of SEQ ID NO: 3, which are SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. 2. The isolated nucleic acid of claim 1, wherein the encoded polypeptide comprises an amino acid sequence selected from the group comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. 3. The isolated nucleic acid according to claim 1, which comprises a nucleotide sequence that is selected from the group that includes the nucleotide sequences of SEQ ID NO: 59-76. 4. The isolated nucleic acid of claim 1, wherein the coronavirus is a beta-coronavirus. 5. The isolated nucleic acid of claim 4, wherein the betacoronavirus is the SARSCoV-2 virus. 6. An isolated nucleic acid for obtaining a recombinant influenza virus that produces a higher level of antibodies to the influenza virus when administered to a mammal, encoding a recombinant polypeptide from epitope fragments of coronavirus proteins, containing an amino acid sequence selected from the group comprising the amino acid sequences of SEQ ID NO: 23-40. 7. The isolated nucleic acid of claim 6, which comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 59-76, which respectively encode the amino acid sequences of SEQ ID NO: 23-40. 8. The isolated nucleic acid of claim 6, wherein the coronavirus is a beta-coronavirus. 9. The isolated nucleic acid of claim 8, wherein the betacoronavirus is the SARSCoV-2 virus. 10. An isolated recombinant virus based on the influenza virus for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, which contains a nucleic acid according to any one of claims 1 to 9. 11. An isolated recombinant virus based on an influenza virus according to claim 10, wherein the isolated recombinant virus based on an influenza virus is obtained from an attenuated influenza virus. 12. An isolated recombinant virus based on an influenza virus according to claim 10, wherein the isolated recombinant virus based on an influenza virus is obtained from an H2N2 influenza virus. 13. An isolated recombinant virus based on the influenza virus according to claim 10, wherein the isolated recombinant virus based on the influenza virus is obtained from the A/Leningrad/134/17/57 (H2N2) virus or its derivatives. 14. An isolated recombinant virus based on the influenza virus according to claim 10, wherein the nucleic acid according to any of claims 1-9 is located in the neuraminidase (NA) gene or the NS gene of the influenza virus. 15. A pharmaceutical composition for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, containing a recombinant virus based on the influenza virus according to any of claims 10-14 in combination with one or more pharmaceutically acceptable excipients. 16. The use of a recombinant virus based on the influenza virus according to any of paragraphs 10-14 or a pharmaceutical composition according to paragraph 15 for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus. 17. Use according to item 16 for inducing specific immunity to the influenza virus and coronavirus and/or preventing diseases caused by the influenza virus and diseases caused by coronavirus. 18. The use according to claim 17, wherein the specific immunity to coronavirus is specific T-cell immunity. 19. The use according to claim 17, wherein the coronavirus is a beta-coronavirus. 20. The use according to item 17, wherein the coronavirus is selected from the group that contains human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2. 21. A vaccine for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, comprising a recombinant virus based on the influenza virus according to any of paragraphs 10-14 in an effective amount. 22. The vaccine according to item 21 for inducing specific immunity to the influenza virus and coronavirus and/or preventing diseases caused by the influenza virus and diseases caused by coronavirus. 23. The vaccine according to item 22, wherein the coronavirus is a beta-coronavirus. 24. The vaccine according to item 22, wherein the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2. 25. The vaccine according to item 22, wherein the specific immunity to coronavirus is specific T-cell immunity. - 53 048516 26. A method for inducing specific immunity to the influenza virus and/or preventing diseases caused by the influenza virus, or inducing specific immunity to the influenza virus and coronavirus or combined prevention of diseases caused by the influenza virus and diseases caused by coronavirus, comprising administering a recombinant virus based on the influenza virus according to any of paragraphs 10-14, a pharmaceutical composition according to paragraph 15 or a vaccine according to paragraph 21, in an effective amount. 27. The method according to claim 26, wherein the coronavirus is a beta-coronavirus. 28. The method according to claim 26, wherein the coronavirus is selected from the group that includes human coronavirus OC43, human coronavirus HKU1, MERS-CoV, SARS-CoV or SARS-CoV-2. 29. The method according to claim 26, wherein the specific immunity to coronavirus is specific T-cell immunity.