Attenuated influenza vectors for the prevention and/or treatment of infectious diseases and for the treatment of oncological diseases

Abstract

The present invention relates to the field of medicine and virology. An attenuated influenza A virus, an influenza virus vector based thereon, and a pharmaceutical composition comprising thereof are provided, which can be used for the prevention and/or treatment of an infectious disease. In addition, the present invention relates to an attenuated influenza A virus, an influenza virus vector based thereon, and a pharmaceutical composition comprising thereof, which can be used for the treatment of oncological diseases.

Claims

1. An attenuated influenza A virus inducing a cross-protective response against influenza A and B viruses, comprising a chimeric NS fragment including a truncated reading frame of an NS1 protein and a Nep gene heterologous sequence, wherein said truncated reading frame of the NS1 protein is derived from H1N1 influenza virus subtype, and the Nep gene heterologous sequence is derived from H2N2 influenza virus subtype and wherein said truncated reading frame encodes the NS1 protein consisting of 124 amino acid residues corresponding to SEQ ID NO: 4.

2. An attenuated influenza virus vector expressing a protein or a fragment thereof selected from the group consisting of proteins or fragments thereof from bacteria, viruses, and protozoa, wherein the vector is an attenuated influenza A virus according to claim 1, in which the truncated reading frame of the NS1 protein gene is elongated by an insertion of a sequence of at least one transgene encoding the protein or the fragment thereof from bacteria, viruses, and protozoa, wherein the bacteria, virus, or protozoa is pathogenic.

3. The attenuated influenza virus vector according to claim 2, wherein the protein or the fragment thereof is selected from the group consisting of proteins of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes virus, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Trypanosoma, Leishmania, Chlamydia, brucellosis causative agent, or a combination thereof.

4. The attenuated influenza virus vector according to claim 2, wherein the protein or the fragment thereof consists of 10 to 400 amino acids.

5. The attenuated influenza virus vector according to claim 2, wherein the insertion encodes an HA protein region from influenza virus.

6. The attenuated influenza virus vector according to claim 5, wherein the HA protein region is an HA2 subunit region selected from the group consisting of 1-185 amino acids from influenza A virus, 1-186 amino acids from influenza B virus, 23-185 amino acids from influenza A virus, or 65-222 amino acids from influenza A virus.

7. The attenuated influenza virus vector according to claim 2, wherein the insertion encodes a sequence of an influenza A or B virus HA2 subunit region of from 1 to 21 amino acids and a sequence of an influenza A virus NP protein region of from 243 to 251 amino acids.

8. The attenuated influenza virus vector according to claim 2, wherein the insertion encodes protein ESAT-6, Ag85A, Ag85B, Mpt64, HspX, Mtb8.4, or 10.4 of mycobacterium tuberculosis, or a fragment thereof.

9. The attenuated influenza virus vector according to claim 8, wherein the viral genome sequence further comprises a sequence encoding a self-cleaving 2A peptide between the truncated reading frame of the NS1 protein gene and the insertion encoding protein ESAT6.

10. An attenuated influenza virus vector expressing an influenza virus protein or a fragment thereof, wherein said vector is an attenuated influenza virus according to claim 1, wherein the truncated reading frame of an NS1 protein gene is elongated by an insertion of a sequence encoding 1-21 aa of an influenza B HA2 protein and 243-251 aa of an influenza A NP protein.

11. An attenuated influenza virus vector having oncolytic activity, wherein said vector is an attenuated influenza A virus according to claim 1, wherein the truncated reading frame of an NS1 protein gene is elongated by an insertion of a sequence encoding a mycobacterium tuberculosis protein ESAT-6 or a fragment thereof.

12. The attenuated influenza virus vector according to claim 11, wherein the protein or a fragment thereof consists of 10 to 400 amino acids.

13. The attenuated influenza virus vector according to claim 11, wherein the truncated reading frame of an NS1 protein gene is further elongated by an insertion of a sequence encoding self-cleaving 2A peptide.

14. An attenuated influenza virus vector inducing a cross-protective response against influenza A and B viruses, comprising: a nucleotide sequence of a PB2 protein gene of SEQ ID NO: 14 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 14; a nucleotide sequence of a PB1 protein gene of SEQ ID NO: 15 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 15; a nucleotide sequence of a PA protein gene of SEQ ID NO: 16 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 16; a nucleotide sequence of an NP protein gene of SEQ ID NO: 17 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 17; a nucleotide sequence of an M protein gene of SEQ ID NO: 18 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18; a nucleotide sequence of an HA protein gene of SEQ ID NO: 19 virus or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19; a nucleotide sequence of an NA protein gene of SEQ ID NO: 20 virus or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 20; a nucleotide sequence of an NS protein chimeric gene of SEQ ID NO: 21 including: an NS1 protein reading frame derived from influenza A/PR/8/34 (H1N1), wherein said reading frame is truncated and encodes an NS1 protein consisting of 124 amino acid residues, and a Nep gene sequence derived from influenza A/Singapore/1/57-like (H2N2) virus, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 21; wherein said NS1 protein truncated reading frame is elongated by an insertion of a nucleotide sequence encoding a fusion peptide of an influenza B subunit HA2 region and a nucleotide sequence encoding a conservative B-cell epitope of influenza A virus nucleoprotein (NP).

15. The attenuated influenza virus vector according to claim 14, wherein the nucleotide sequence of the NS protein chimeric gene is set forth in SEQ ID NO:21.

16. An immunogenic composition for the induction of an immune response against an infectious pathogen in a subject, comprising an effective amount of an attenuated influenza virus vector according to claim 2, and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition for the prophylaxis of influenza disease, comprising in an effective amount of an attenuated influenza virus vector according to claim 14 and a pharmaceutically acceptable carrier.

18. The immunogenic or pharmaceutical composition according to claim 16 or 17, respectively, comprising 6.5 to 10.5 log EID50/ml of the attenuated influenza A virus and a buffer solution comprising 0 to 1.5 wt. % of a monovalent salt, 0 to 5 wt. % of an imidazole-containing compound, 0 to 5 wt. % of a carbohydrate component, 0 to 2 wt. % of a protein component, 0 to 2 wt. % of an amino acid component, and 0 to 10 wt. % of hydroxyethylated starch.

19. The immunogenic composition according to claim 16, wherein the buffer solution comprises 0.5 to 1.5 wt. % of a monovalent salt, 0.01 to 5 wt. % of an imidazole-containing compound, 1 to 5 wt. % of a carbohydrate component, 0.1 to 2 wt. % of a protein component, 0.01 to 2 wt. % of an amino acid component, and 1 to 10 wt. % of hydroxyethylated starch.

20. The immunogenic composition according to claim 19, wherein the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose, or lactose, the protein component is a human albumin, casitone, lactalbumin hydrolysate, or gelatin, the amino acid component is arginine, glycine, or sodium glutamate, and the imidazole-containing compound is L-carnosine or N,N-bis[2-(1H-imidazol-5yl)ethyl]propanediamide.

21. The immunogenic composition according to claim 16, wherein the infectious pathogen is selected from the group consisting of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes simplex virus types I and II, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Chlamydia, Trypanosoma, Leishmania, or a brucellosis causative agent.

22. The immunogenic composition according to claim 16, wherein the subject is a mammal or a bird.

23. The immunogenic composition according to claim 22, wherein the subject is a human subject.

24. A vaccine against an influenza, comprising an effective amount of an attenuated influenza virus vector according to claim 1, and a pharmaceutically acceptable carrier.

25. A vaccine against influenza comprising an effective amount of an attenuated influenza virus vector according to claim 14 and a pharmaceutically acceptable carrier.

26. The vaccine according to claim 24 or 25, comprising 6.5 to 10.5 log EID50/ml of the attenuated influenza virus vector and a buffer solution comprising 0 to 1.5 wt. % of a monovalent salt, 0 to 5 wt. % of an imidazole-containing compound, 0 to 5 wt. % of a carbohydrate component, 0 to 2 wt. % of a protein component, 0 to 2 wt. % of an amino acid component, and 0 to 10 wt. % of hydroxyethylated starch.

27. The vaccine according to claim 24, wherein the buffer solution comprises 0.5 to 1.5 wt. % of a monovalent salt, 0.01 to 5 wt. % of an imidazole-containing compound, 1 to 5 wt. % of a carbohydrate component, 0.1 to 2 wt. % of a protein component, 0.01 to 2 wt. % of an amino acid component, and 1 to 10 wt. % of hydroxyethylated starch.

28. The vaccine according to claim 27, wherein the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose, or lactose, the protein component is a human albumin, casitone, lactalbumin hydrolysate, or gelatin, the amino acid component is arginine, glycine, or sodium glutamate, and the imidazole-containing compound is L-carnosine or N,N-bis[2-(1H-imidazol-5yl)ethyl]propanediamide.

29. A method for treating and/or prophylaxis of influenza disease in a subject in need thereof, comprising administering to said subject an effective amount of an attenuated influenza virus vector according to claim 2.

30. The method according to claim 29, wherein the influenza disease is caused by a pathogen selected from the group consisting of an influenza A virus and an influenza B virus.

31. The method according to claim 30, wherein the subject is a mammal or a bird.

32. The method according to claim 31, wherein the subject is a human subject.

33. A pharmaceutical composition for the treatment of an oncological disease in a subject, comprising an attenuated influenza virus vector according to claim 11 in an effective amount, and a pharmaceutically acceptable carrier.

34. The composition according to claim 33, comprising 8.5 to 10.5 log EID50/ml of the attenuated influenza virus vector, and a buffer solution comprising 0 to 1.5 wt. % of a monovalent salt, 0 to 5 wt. % of an imidazole-containing compound, 0 to 5 wt. % of a carbohydrate component, 0 to 2 wt. % of a protein component, 0 to 2 wt. % of an amino acid component, and 0 to 10 wt. % of hydroxyethylated starch.

35. The composition according to claim 34, wherein buffer solution comprises 0.5 to 1.5 wt. % of a monovalent salt, 0.01 to 5 wt. % of an imidazole-containing compound, 1 to 5 wt. % of a carbohydrate component, 0.1 to 2 wt. % of a protein component, 0.01 to 2 wt. % of an amino acid component, and 1 to 10 wt. % of hydroxyethylated starch.

36. The composition according to claim 35, wherein the monovalent salt is sodium chloride, the carbohydrate component is starch, the protein component is a human albumin, the amino acid component is arginine, and the an imidazole-containing compound is L-carnosine or N,N-bis[2-(1H-imidazol-5yl)ethyl]propanediamide.

37. A method for treating an oncological disease in a subject in need thereof, comprising administering to said subject an effective amount of an attenuated influenza virus vector according to claim 11.

38. The method according to claim 37, wherein the administration is intratumor administration, administration to a cavity formed after surgical removal of a tumor, or intravenous administration.

39. The method according to claim 37, wherein the oncological disease is selected from the group consisting of colorectal cancer, cardioesophageal cancer, pancreatic cancer, cholangiocellular cancer, glioma, glioblastoma, and melanoma.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the principle of designing an attenuated influenza vector. FIG. 1A shows a scheme of the NS genomic fragment of influenza A/PR/8/34 (H1N1) virus. FIG. 1B shows a scheme of a genetically modified chimeric NS genomic fragment in which the NS1 reading frame is truncated and can be elongated by an insertion of a foreign sequence. The sequence encoding the Nep protein is replaced with a heterologous sequence derived from another influenza A virus subtype.

(2) FIG. 2 shows the nucleotide sequences of NS genomic fragments of the wild-type virus and examples of two chimeric genetic constructs. FIG. 2A shows the NS fragment of influenza A/PR/8/34 (H1N1) virus. FIG. 2B shows a chimeric NS fragment of influenza A virus in which the reading frame of the NS1 protein is truncated, and the Nep sequence (marked in bold) is derived from A/Singapore/1/57 (H2N2) virus.

(3) FIG. 2C shows a chimeric NS fragment of influenza A virus, wherein the reading frame of the NS1 protein is truncated, and the Nep sequence (marked in bold type) is derived from A/Leningrad/134/47/57 (H2N2) virus.

(4) FIG. 3 shows the amino acid sequences of proteins translated in the reading frame of NS1 chimeric influenza vectors containing heterologous Nep from virus A/Leningrad/134/47/57 (H2N2) virus.

(5) FIG. 4 shows data demonstrating the pathogenicity and ts-phenotype of viruses with a heterologous Nep gene. FIG. 4A shows data of reproduction of viruses at an optimal temperature of 34 C. and at an elevated temperature of 39 C. temperature in Vero cells. FIG. 4B shows data of reproduction of viruses in mouse lungs on Day 2 after infection.

(6) FIG. 5 shows graphs demonstrating a protective effect of a single immunization of mice with vectors expressing HA2 subunit regions from the NS1 reading frame in the control infection with heterologous pathogenic influenza strains. FIG. 5A shows the lethality in the control infection with A/Mississippi/85/1 (H3N2) virus, and FIG. 5B shows the lethality with the control infection with B/Lee/40 virus.

(7) FIG. 6 presents data on the oncolytic effect of recombinant influenza viruses on melanoma induced in mice by the introduction of 110.sup.6 B16 cells into the foot of a hind paw. The therapy was carried out by intra-tumor administration of the virus on day 5 after the tumor implantation. FIG. 6A shows the average foot size on Day 20 after the tumor implantation and four-time treatment with oncolytic vectors; and FIG. 6B shows the survival of mice after four-time treatment with oncolytic vectors.

(8) FIG. 7 shows the structure of an attenuated influenza vector. There are shown eight fragments of the virus genome and their peculiarities.

(9) It is shown that genome fragments PB2, PB1, PA, Np and M are derived from the A/PR/8/34 (H1N1) virus; the surface HA and NA glycoprotein genes are derived from the A/California/7/09-like (H1N1pdm) virus; the NS genomic fragment has a chimeric structure encoding two proteins: 1) NS1 protein truncated to 124 amino acid residues, elongated by an insertion of a sequence of the N-terminal region of influenza B HA2 protein and by an insertion of a conservative B-cell epitope of influenza A NP protein; and 2) Nep protein having a sequence derived from a heterologous influenza A strain, H2N2 A/Singapore/1/57-like serological subtype.

(10) FIG. 8 shows the nucleotide sequences of genomic fragments of a vaccine vector: PB2, PB1, PA, NP, and M from A/PR/8/34 (H1N1) virus; HA and NA from A/California/7/09-like (H1N1pdm) virus; and a chimeric NS (an insertion in the NS1 reading frame is marked in bold).

(11) FIG. 9 shows results reflecting the protective properties of a vaccine vector after intranasal immunization of mice against various variants of influenza A virus and influenza B virus. The diagrams show mortality (in %) in vaccinated mice after the control infection with the indicated serotypes of influenza A virus or influenza B virus in comparison with the control animals. The vaccine was administered once1 or twice2.

(12) FIG. 10 shows data demonstrating the protective properties of a vaccine vector after intranasal immunization of ferrets. Diagram A shows the dynamics of the mean value of temperature fluctuations after the control infection with A/St.Petersburg/224/2015 (H3N2) virus in vaccinated and control animals. Diagram B shows the inoculation results of the control virus from the ferret nasal washings on Days 2, 4 and 6 after infection. The titers are expressed as the virus mean concentration in nasal washings, expressed as 50% cytopathic dose/ml after titration on MDCK cells. The vaccine was administered once1 or twice2.

DETAILED DESCRIPTION OF THE INVENTION

(13) The present invention relates to attenuated influenza A viruses that are produced by genetically engineered methods and that can be used for the treatment and/or prevention of infectious diseases, as well as for the treatment of oncological diseases.

(14) In particular, the present invention relates to an attenuated influenza A virus inducing a cross-protective response against influenza A and B viruses, comprising a chimeric NS fragment including an NS1 truncated reading frame and a heterologous sequence of the Nep gene derived from influenza A subtype that differs from the subtype of said attenuated influenza A virus. Thus, the influenza A virus subtype for the sequence encoding a truncated NS1 protein differs from the virus subtype from which the Nep protein sequence was derived. In particular, one embodiment of the present invention relates to an attenuated influenza A virus, wherein said NS1 truncated reading frame is derived from influenza H1N1 subtype, and the heterologous sequence of Nep gene is derived from a human or animal influenza subtype of from H2 to H18 subtype.

(15) Said truncated reading frame encodes an NS1 protein comprising from 80 to 130 amino acid residues, more preferably said truncated reading frame encodes an NS1 protein comprising 124 amino acid residues.

(16) The present invention is particularly based on the fact that the inventors have found that the problem of insufficient attenuation (the absence of temperature sensitivity and a high reproduction level in mouse lungs) of influenza vectors, in particular the vector NS1-124, may be solved by modification of the second spliced protein product of an NS genomic fragment of influenza virusNep protein (NS2). A replacement of the Nep genomic sequence of influenza A virus, in particular A/PR/8/34 (H1N1) influenza virus, with the Nep sequence derived from heterologous influenza strains, for example from A/Singapore/1/57 (H2N2) or A/Leningrad/134/47/57 (H2N2) virus, leads to the appearance of temperature-sensitivity phenotype and attenuation in influenza A virus, in particular A/PR/8/34 (H1N1) virus. Based on this phenomenon, chimeric NS fragments of influenza virus were constructed that encode a truncated reading frame, NS1-124, of A/PR/8/34 (H1N1) virus in combination with the Nep protein reading frame derived from H2N2 serological subtype. Reassortant influenza viruses based on A/PR/8/34 virus, regardless of the origin of surface antigens H1N1, H5N1 or H1N1pdm, carrying a chimeric NS genomic fragment were unable to provide active reproduction at 39 C. and in the mouse lungs (attenuation phenotype), but still provided reproduction to high titers in 10-day-old chicken embryos.

(17) The present invention also relates to an attenuated influenza virus vector expressing an antigen or a fragment thereof selected from the group consisting of antigens or fragments thereof from bacteria, viruses, and protozoa, wherein the vector is an attenuated influenza A virus according to the present invention, in which a truncated reading frame of an NS1 protein gene is elongated by an insertion of a sequence of at least one transgene encoding the antigen or a fragment thereof from bacteria, viruses, and protozoa. In general, the attenuated virus can be inserted into a transgene encoding a protein or a fragment thereof from any bacteria, virus or protozoa, pathogenic or non-pathogenic for animals and humans, in particular, the protein may be selected from the group consisting of proteins or their fragments from an influenza A virus, influenza B virus, mycobacterium tuberculosis, Brucella abortus, herpes virus, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Trypanosoma, Leishmania, Chlamydia, brucellosis causative agent, or a combination thereof. In particular, the sequence of an insertion can encode an HA protein fragment of influenza virus, mycobacterium tuberculosis protein ESAT-6, Ag85A, Ag85B, Mpt64, HspX, Mtb8.4 or 10.4, or fragments thereof. The genomic sequence of an attenuated vector according to the present invention may further comprise a sequence encoding a self-cleaving 2A peptide between sequences encoding NS1-124 and ESAT6.

(18) The antigen or fragment thereof encoded by the sequence of an insertion may have any size that is limited only by the ability of the genomic fragment to receive the nucleotide sequence encoding the antigen or fragment thereof. Preferably, the size of the antigen is from 10 to 400 amino acids. For example, the insertion may encode an HA protein fragment representing an HA2 subunit region selected from the group consisting of 1 to 185 amino acids of influenza A virus, 1 to 186 amino acids of influenza B virus, 23 to 185 amino acids of influenza A virus, or 65 to 222 amino acids of influenza A virus. The numbering of amino acids is given in accordance with the positions of the amino acids in HA2 subunit region of influenza virus from which the transgene is originated.

(19) Another specific embodiment of an attenuated influenza virus vector according to the present invention is a vector in which an insertion encodes a sequence of an influenza A or B virus HA2 subunit region of from 1 to 21 amino acids and a sequence of an influenza A virus NP protein region of from 243 to 251 amino acids. These vector variants, despite a short insertion therein, have been surprisingly found to exhibit the best protective effectiveness against influenza B virus and heterologous antigenic subtypes of influenza A virus after a single immunization of mice, i.e. they exhibit the properties of a universal influenza vaccine.

(20) The inventors found that insertions of foreign antigenic sequences into the NS1 reading frame, for example, after amino acid position 124, did not significantly affect the attenuation phenotype of a chimeric virus produced according to the present invention. Thus, various influenza vectors were obtained that possessed required production characteristics and manifested phenotypic and genotypic markers of attenuation in accordance with the requirements for live influenza vaccines. Regardless of the nature of insertions, the viruses showed their harmlessness for laboratory animals and the similarity of the manifested phenotypic marker of attenuationthe presence of is phenotype. The similarity in their genetic markers of attenuation was determined by the presence of a truncated reading frame of NS1 protein and by the presence of a heterologous sequence of Nep gene derived from another influenza A subtype. Depending on an insertion, the resulting vectors exhibited the properties of a universal influenza vaccine, a vaccine against tuberculosis, etc.

(21) In particular, the present invention relates to an influenza A virus vaccine vector obtained by the genetic engineering method, which can be used to prevent influenza caused by all known strains, including influenza A and B viruses. In particular, the present invention relates to an attenuated influenza A virus inducing a cross-protective response against influenza A and B viruses, comprising a chimeric NS fragment including a truncated reading frame of an NS1 protein and a Nep gene heterologous sequence derived from H2N2 influenza A virus subtype. Thus, the influenza A virus subtype of the sequence encoding a truncated NS1 protein differs from the virus subtype from which the sequence encoding Nep protein was derived. In particular, in the vaccine vector, the NS1 truncated reading frame is from influenza H1N1 subtype, and the Nep heterologous sequence is from H2N2 influenza subtype.

(22) In one embodiment, the present invention relates to an attenuated influenza vector inducing a cross-protective response against influenza A and B viruses, comprising:

(23) a nucleotide sequence of a PB2 protein gene derived from influenza A/PR/8/34 (H1N1) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the PB2 protein gene;

(24) a nucleotide sequence of a PB1 protein gene derived from influenza A/PR/8/34 (H1N1) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the PB1 protein gene;

(25) a nucleotide sequence of a PA protein gene derived from influenza A/PR/8/34 (H1N1) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the PA protein gene;

(26) a nucleotide sequence of an NP protein gene derived from influenza A/PR/8/34 (H1N1) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the NP protein gene;

(27) a nucleotide sequence of an M protein gene derived from influenza A/PR/8/34 (H1N1) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the M protein gene;

(28) a nucleotide sequence of an HA protein gene derived from influenza A/California/7/09-like (H1N1pdm) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the HA protein gene;

(29) a nucleotide sequence of an NA protein gene derived from influenza A/California/7/09-like (H1N1pdm) virus or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the NA protein gene; and

(30) a nucleotide sequence of an NS protein chimeric gene comprising

(31) an NS1 protein reading frame derived from influenza A/PR/8/34 (H1N1) virus, wherein said reading frame is truncated and encodes an NS1 protein consisting of 124 amino acid residues,

(32) and a Nep gene sequence derived from influenza A/Singapore/1/57-like (H2N2) virus, or

(33) a nucleotide sequence having at least 95% or more (for example, 96, 97, 98, or 99%) sequence identity to said nucleotide sequence of the NS chimeric gene;

(34) wherein said NS1 protein truncated reading frame is elongated by an insertion of a nucleotide sequence encoding a fusion peptide of an influenza B virus HA2 subunit region and a nucleotide sequence encoding a conservative B-cell epitope of influenza A virus nucleoprotein (NP).

(35) This truncated reading frame encodes an NS1 protein having 124 amino acid residues that is elongated by two glycines, an insertion of the N-terminal region of the second hemagglutinin subunit HA2 of influenza B virus (23 amino acid residues) and an insertion of a sequence of the conservative B-cell epitope of influenza A virus (7 amino acid residues).

(36) Surface glycoprotein genes of this vector are derived from influenza A/California/7/09 (H1N1pdm) virus. The genes of internal proteins PB2, PB1, RA, NP and M are derived from influenza A/PR/8/34 (H1N1) virus. Thus, the influenza vector according to the invention is a complex genetic construct consisting of genomic sequences of various influenza strains, namely: 1) genes encoding PB2, PB1, PA, NP, and M are from A/PR/8/34 (H1N1) virus (PB2 (Genbank accession number: AB671295), PB1 (Genbank accession number: CY033583), PA (Genbank accession number: AF389117), NP (Genbank accession number: AF389119), M (Genbank accession number: AF389121)), 2) genes encoding HA and NA are from the A/California/7/09-like H1N1pdm virus (HA (GenBank: KM408964.1) and (NA GenBank: KM408965.1)), 3) NS gene is chimeric, wherein the NS protein reading frame of A/PR/8/34 (H1N1) virus is truncated to 124 amino acid residues and is elongated by an insertion of a sequence encoding a fusion peptide of an influenza B virus HA2 subunit region and a sequence encoding a conservative B-cell epitope of influenza A virus nucleoprotein (NP), and the NEP protein reading frame is from H2N2 influenza virus subtype.

(37) The present invention is based, in particular, on the fact that the inventors have unexpectedly found that in intranasal immunization of mice and ferrets with a vector having said structure, without adjuvants, protects the animals against the control infection not only with influenza A (H1N1) viruses but also with influenza A (H3N2) viruses, and influenza B viruses. Therefore, the vaccine vector has the properties of a universal influenza vaccine.

(38) The term universal vaccine in the context of the present invention means a vaccine capable of protecting against all known and unknown variants of influenza virus. The usual seasonal vaccines protect only against viruses similar to those that are included in the vaccine composition.

(39) The term mucosal vaccine means that the vaccine can be administered into the cavities of the respiratory and digestive tracts and applied to the mucous membranes of the mouth and nose, i.e. applied intranasally, orally, or sublingually.

(40) An influenza vector based on A/PR/8/34 virus carrying a chimeric NS genomic fragment were unable to provide active reproduction at 39 C. and in the mouse lungs (attenuation phenotype), but still provided reproduction to high titers in 10-day-old chicken embryos.

(41) The present invention also relates to an attenuated influenza virus vector having oncolytic activity, comprising an attenuated influenza A virus according to the present invention, in which a truncated reading frame of an NS1 protein gene is elongated by an insertion of a sequence of at least one transgene encoding an antigen or a fragment thereof of pathogenic bacteria, viruses, and protozoa. Said antigen can be derived from any bacteria, viruses or protozoa that are pathogenic for animals, in particular the antigen can be selected from the group consisting of antigens of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes virus, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Trypanosoma, Leishmania, Chlamydia, or a combination thereof. In particular, the inserted transgene can encode mycobacterium tuberculosis protein ESAT-6, Ag85A, Ag85B, Mpt64, HspX, Mtb8.4 or 10.4 or fragments thereof; in addition, the truncated reading frame of an NS1 protein gene can be elongated by an insertion of a sequence encoding mycobacterium tuberculosis protein ESAT-6.

(42) The antigen or fragment thereof encoded by the sequence of an insertion may have any size that is limited only by the ability of an NS genomic fragment to receive the nucleotide sequence encoding the antigen or fragment thereof. Preferably, the size of the antigen is from 10 to 400 amino acids.

(43) The inventors unexpectedly found that attenuated influenza vectors carrying a chimeric NS genomic fragment possess an enhanced oncolytic activity due to incorporation of a heterologous Nep gene, provided that the pathogenic antigen, in particular a bacterial antigen from the NS1 protein reading frame, is expressed. For example, a viral vector encoding mycobacterium tuberculosis protein Esat6 had higher activity than the known recombinant virus having a truncated NS1 protein but without an insertion. Without being bound to any theory, it can be assumed that a strong antituberculous immunity in a mammal contributes to the immune attack of a tumor infected with a virus expressing a tubercular protein.

(44) The present invention also relates to pharmaceutical compositions that contain an effective amount of an attenuated influenza A virus according to the present invention or an attenuated influenza vector according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions according to the present invention can be used in the treatment and/or prevention of an infectious disease in a subject, in particular an infectious disease caused by a pathogen selected from the group consisting of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes simplex virus types I and II, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Chlamydia, Trypanosoma, or Leishmania.

(45) In addition, the pharmaceutical compositions according to the present invention can be used in the treatment of oncological diseases of various etiologies; in particular, an oncological disease can be selected from the group consisting of colorectal cancer, cardioesophageal cancer, pancreatic cancer, cholangiocellular cancer, glioma, glioblastoma, and melanoma.

(46) A pharmaceutical composition according to the present invention can be formulated as a vaccine containing an effective amount of an attenuated influenza A virus according to the present invention or an attenuated influenza vector according to the present invention and a pharmaceutically acceptable carrier.

(47) The term subject or animal as used herein means vertebrates that are prone to infection caused by pathogenic bacteria, viruses or protozoa, including birds (waterfowl, chickens, etc.) and representatives of various mammalian species such as dogs, felines, wolves, ferrets, rodents (rats, mice, etc.), horses, cows, sheep, goats, pigs and primates. In one embodiment of the invention, the subject is a human subject.

(48) The term effective amount means the amount of a virus or vector that, when administered to a subject in a single dose or as a part of a treatment cycle, is effective for the treatment and/or prevention with a therapeutic result. This amount can vary depending on the health status and physical condition of a patient, its age, taxonomic group of the subject being treated, a formulation, the estimation of medical situation by a treating physician and other important factors. It is believed that the amount can vary within a relatively wide range, which a skilled person can determine by standard methods. The pharmaceutical composition may contain from 6 to 10.5 log EID50/ml, more particularly from 6.5 to 10.5 log EID50/ml, in particular from 6 to 9.5 log EID50/ml, more particularly from 6.5 to 8.5 log EID50/ml of a chimeric influenza A virus according to the invention or influenza vector according to the invention.

(49) The term pharmaceutically acceptable carrier, as used herein, means any carrier used in the field, in particular water, physiological saline, a buffer solution and the like. In one embodiment, the pharmaceutically acceptable carrier is a buffer solution containing from 0 to 1.5 wt. % of a monovalent salt, from 0 to 5 wt. % of an imidazole-containing compound, from 0 to 5 wt. % of a carbohydrate component, from 0 to 2 wt. % of a protein component, from 0 to 2 wt. % of an amino acid component and from 0 to 10 wt. % of hydroxyethyl starch, preferably said buffer solution contains from 0.5 to 1.5 wt. % of a monovalent salt, from 0.01 to 5 wt. % of an imidazole compound, from 1 to 5 wt. % of a carbohydrate component, from 0.1 to 2 wt. % of a protein component, from 0.01 to 2 wt. % of an amino acid component and from 1 to 10 wt. % of hydroxyethyl starch, most preferably the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component is arginine, glycine or sodium glutamate.

(50) The imidazole-containing compound is L-carnosine or N,N-bis[2-(1H-imidazol-5-yl)ethyl]-propandiamide having formula:

(51) ##STR00001##

(52) Human albumin can be a recombinant albumin or donor albumin.

(53) The present invention also relates to use of an attenuated influenza A virus, attenuated influenza virus vector or pharmaceutical composition according to the present invention for the treatment and/or prevention of an infectious disease in a subject, in particular an infectious disease caused by a pathogen selected from the group consisting of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes simplex virus types I and II, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Chlamydia, Trypanosoma, or Leishmania.

(54) The present invention also relates to the use of an attenuated influenza vector or pharmaceutical composition according to the present invention for the prevention of influenza.

(55) Additionally, the present invention also relates to methods of treatment, comprising administering to a subject an attenuated influenza A virus, attenuated influenza vector or pharmaceutical composition according to the present invention. The methods are intended for the treatment and/or prevention of an infectious disease caused by a pathogen viruses, bacteria, or protozoa, in particular infectious diseases caused by a pathogen selected from the group consisting of an influenza A virus, influenza B virus, mycobacterium tuberculosis, herpes simplex virus types I and II, respiratory syncytial virus, human immunodeficiency virus, hepatitis C virus, malaria parasite, Trichomonas, Chlamydia, Trypanosoma, or Leishmania. In addition, the methods are intended for the treatment of oncological diseases in a subject, in particular, an oncological disease can be selected from the group consisting of colorectal cancer, cardioesophageal cancer, pancreatic cancer, cholangiocellular cancer, glioma, glioblastoma, and melanoma.

(56) The administration to a subject can be made by any standard methods, in particular intramuscularly, intravenously, orally, sublingually, inhalationally or intranasally. The influenza vector or pharmaceutical composition can be administered to a subject one, two or more times; a single administration is preferred.

(57) Additionally, in the case of treating cancer, the administration may be intratumor administration, administration to a cavity formed after surgical removal of a tumor, or intravenous administration.

(58) The invention is illustrated below by its embodiments that are not intended to limit the scope of the invention.

EXAMPLES

Example 1

(59) Production of Influenza Vectors with a Modified NS Genomic Fragment

(60) Recombinant viruses were assembled in several steps. At the first step, complementary DNA (cDNA) copies of all eight genomic fragments of influenza virus A/PR/8/34 (H1N1) were synthetically produced by using data from a genetic bank: pHbank-PR8-HA (Genbank accession number: EF467821.1), pHW-PR8-NA (Genbank accession number: AF389120.1), pHW-PR8-PB2 (Genbank accession number: AB671295), pHW-PR8-PB1 (Genbank accession number: CY033583), pHW-PR8-PA (Genbank accession number: AF389117), pHW-PR8-NP (Genbank accession number: AF389119), pHW-PR8-M (Genbank accession number: AF389121), pHW-PR8-NS (Genbank accession number: J02150.1)). At the second step, the synthesized sequences were cloned into a bidirectional plasmid pHW2000-based vector (Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster R G, A DNA from eight plasmids, Proc Natl Acad Sci USA. 2000; 97 (11): 6108-13). This plasmid vector, due to the presence of Pol I and Pol II promoters, provided simultaneous intracellular transcription of viral and corresponding messenger RNAs upon transfection of mammalian cells.

(61) There were produced 7 plasmid clones encoding PB1, PB2, PA, HA, NA, NP, and M without modifications, and a set of variants of an NS genomic fragment with modifications, the principle of which is presented in FIG. 1.

(62) FIG. 1A shows schemes of the NS genomic fragment of influenza A/PR/8/34 (H1N1) virus. The full-length genomic fragment of negative-polarity viral RNA (vRNA) has a length of 230 nucleotides (nt). The transcription of the NS fragment by the influenza virus polymerase complex leads to the formation of 2 types of messenger RNA: 1. A direct transcript, which is mRNA of an NS1 protein encoding an NS1 protein having 230 amino acid residues (aa), and spliced mRNA of a Nep protein encoding a protein having 121 aa. FIG. 1B shows a scheme of a genetically modified chimeric NS genomic fragment, where the reading frame of an NS1 protein comprises up to 398 nt and can be elongated by an insertion of a foreign sequence terminated with a triple stop-codon. The sequence encoding a Nep protein is replaced with a heterologous sequence derived from another influenza A virus subtype. As a result of modification, the chimeric NS genomic fragment has a length depending on the length of an insertion of a foreign sequence into the NS1 reading frame.

(63) The nucleotide sequence of influenza A/PR/8/34 (H1N1) virus, including the encoding region and the 5- and 3-terminal non-coding regions (sequence number J02150 in the GenBank database), was used as the basis for the development of a chimeric construct of an NS genomic segment. Depending on the purpose, various variants of chimeric constructs of an NS genomic fragment were constructed, with the following common features: 1) replacement of the sequence encoding the Nep protein of A/PR/8/34 (H1N1) virus with a sequence derived from H2N2 influenza virus subtype (strains: A/Singapore/1/57 and A/Leningrad/134/47/57) (FIGS. 2B and 2C); 2) deletion of a sequence consisting of 30 nucleotides (positions 499-428 nt) from the NS1-encoding region, up to the Nep splicing site; 3) limitation of the reading frame of NS1 protein to 124 amino acid residues by inserting a cassette of three consecutive stop codons (TGATAATAA) after nucleotide position 399 (FIG. 2A and FIG. 2B); 4) the presence or absence of a foreign genetic sequence inserted into the NS1 reading frame after nucleotide position 398, just prior to the stop codon cassette.

(64) FIG. 2A shows the sequence of SEQ ID NO:1 of an NS fragment of influenza A/PR/8/34 (H1N1) virus, in which the sequence consisting of 30 nt to be deleted to produce the constructs of B and C is highlighted and underlined. The sequence of Nep gene to be replaced by a heterologous analogue from another influenza A virus subtype is marked in bold. FIG. 2B shows the sequence of SEQ ID NO: 2 of a recombinant NS fragment of influenza A virus in which the reading frame of an NS1 protein is truncated to 398 nt by means of an insertion consisting of three consecutive stop codons (underlined), and the Nep sequence (marked in bold) is borrowed from A/Singapore/1/57 (H2N2) virus. FIG. 2C shows the sequence of SEQ ID NO: 3 of a recombinant NS fragment of influenza A virus in which the reading frame of an NS1 protein is truncated to 398 nt by means of an insertion consisting of three consecutive stop codons (underlined), and the Nep sequence (marked in bold) is borrowed from A/Leningrad/134/47/57 (H2N2) virus.

(65) TABLE-US-00001 (SEQIDNO:1) AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAG CTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGAC CAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGA AATCCCTAAGAGGAAGGGGCAGCACTCTTGGTCTGGACATCGAGACAGC CACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCC GATGAGGCACTTAAAATGACCATGGCCTCTGTACCTGCGTCGCGTTACC TAACCGACATGACTCTTGAGGAAATGTCAAGGGAATGGTCCATGCTCAT ACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCG ATCATGGATAAAAACATCATACTGAAAGCGAACTTCAGTGTGATTTTTG ACCGGCTGGAGACTCTAATATTGCTAAGGGCTTTCACCGAAGAGGGAGC AATTGTTGGCGAAATTTCACCATTGCCTTCTCTTCCAGGACATACTGCT GAGGATGTCAAAAATGCAGTTGGAGTCCTCATCGGAGGACTTGAATGGA ATGATAACACAGTTCGAGTCTCTGAAACTCTACAGAGATTCGCTTGGAG AAGCAGTAATGAGAATGGGAGACCTCCACTCACTCCAAAACAGAAACGA GAAATGGCGGGAACAATTAGGTCAGAAGTTTGAAGAAATAAGATGGTTG ATTGAAGAAGTGAGACACAAACTGAAGGTAACAGAGAATAGTTTTGAGC AAATAACATTTATGCAAGCCTTACATCTATTGCTTGAAGTGGAGCAAGA GATAAGAACTTTCTCATTTCAGCTTATTTAATAATAAAAAACACCCTTG TTTCTACT (SEQIDNO:2) AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAG CTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGAC CAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGA AATCCCTAAGAGGAAGGGGCAGCACTCTTGGTCTGGACATCGAGACAGC CACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCC GATGAGGCACTTAAAATGACCATGGCCTCTGTACCTGCGTCGCGTTACC TAACCGACATGACTCTTGAGGAAATGTCAAGGGAATGGTCCATGCTCAT ACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCG ATCATGTGATAATAAAGTGTGATTTTTGACCGGCTGGAGACTCTAATAT TGCTAAGGGCTTTCACCGAAGAGGGAGCAATTGTTGGCGAAATTTCACC ATTGCCTTCTCTTCCAGGACATACTAATGAGGATGTCAAAAATGCAATT GGGGTCCTCATCGGAGGACTTGAATGGAATGATAACACAGTTCGAGTCT CTAAAACTCTACAGAGATTCGCTTGGTGAAACAGTAATGAGAATGGGAG ACCTCCACTCACTCCAAAACAGAAACGGAAAATGGCGAGAACAATTAGG TCAAAAGTTCGAAGAAATAAGATGGCTGATTGAAGAAGTGAGACACAAA TTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATGCAAGCCT TACAGCTACTATTTGAAGTGGAACAAGAGATAAGAACTTTCTCGTTTCA GCTTATTTAATAATAAAAAACACCCTTGTTTCTACT (SEQIDNO:3) AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAG CTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGAC CAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGA AATCCCTAAGAGGAAGGGGCAGCACTCTTGGTCTGGACATCGAGACAGC CACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCC GATGAGGCACTTAAAATGACCATGGCCTCTGTACCTGCGTCGCGTTACC TAACCGACATGACTCTTGAGGAAATGTCAAGGGAATGGTCCATGCTCAT ACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCG ATCATGTGATAATAAAGTGTGATTTTTGACCGGCTGGAGACTCTAATAT TGCTAAGGGCTTTCACCGAAGAGGGAGCAATTGTTGGCGAAATTTCACC ATTGCCTTCTCTTCCAGGACATACTAATGAGGATGTCAAAAATGCAATT GGGGTCCTCATCGGAGGACTTGAATGGAATGATAACACAGTTCGAGTCT CTAAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAG ACCTCCACTCACTCCAAAACAGAAACGGAAAATGGCGAGAACAATTAGG TCAAAAGTTCGAAGAAATAAGATGGCTGATTGAAGAAGTGAGACACAAA TTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATACAAGCCT TACAGCTACTATTTGAAGTGGAACAAGAGATAAGAACTTTCTCGTTTCA GCTTATTTAATAATAAAAAACACCCTTGTTTCTACT

(66) Thus, the constructed chimeric NS genomic fragments, when transcribed by the polymerase influenza virus complex, formed two types of messenger RNA: 1) NS1 mRNA translated in the form of an NS1 protein truncated to 124 amino acid residues and limited by stop codons or elongated by an insertion of sequences transgenes of different origin, the translation of which is limited by the stop codon cassette; 2) heterologous Nep mRNA derived from influenza A virus of another antigenic subtype. The translation variants of the recombinant NS1 protein with insertions are shown in FIG. 3 and in Table 1 below.

(67) TABLE-US-00002 TABLE1 AminoacidsequencesofproteinstranslatedintheNS1reading frame,recombinantviruseshavingaheterologousNepfrom A/Leningrad/134/47/57(H2N2)virus Designation Aminoacidsequence Description NS124/Nep-Len MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM truncatedto124aa, (SEQIDNO:4) withoutaninsertion ofaforeignsequence NS124-HA2(A)- MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe 185 RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, GLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAA elongatedbythe DQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNK translatedsequence LEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTL ofinfluenzaAvirus DFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKC HA2subunit(shownin DNECMESVRNGTYDYPKYSEESKLNREKVDGVKLES bold),from1to185 MGIYQ aa (SEQIDNO:5) NS124-HA2(A)- MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe 65-222 RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, AVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLV elongatedbythe LLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNG translatedsequence CFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREK ofinfluenzaAvirus VDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISF HA2subunit(shownin WMCSNGSLQCRICI bold),from65to222 (SEQIDNO:6) aa NS124-HA2(A)- MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe 23-185 RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, GYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEK elongatedbythe MNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTY translatedsequence NAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNA ofinfluenzaAvirus KEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEES HA2subunit(shownin KLNREKVDGVKLESMGIYQ bold),from23to185 (SEQIDNO:7) aa NS124-HA2(B)- MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe 186 RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, GFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAA elongatedbythe DLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMNG translatedsequence LHDEILELDEKVDDLRADTISSQIELAVLLSNEGII ofinfluenzaBvirus NSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKC HA2subunit(shownin NQTCLDRIAAGTFNAGDFSLPTFD bold),from1to186 (SEQIDNO:8) aa NS124-Fus(A)-NP MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, GLFGAIAGFIEGGWTGMIDGW-GG-RESRNPGNA elongatedbythe (SEQIDNO:9) translatedsequence ofinfluenzaAvirus HA2subunit(shownin bold),from1to186 aa,andwiththe sequenceofa conservativeB-cell epitopeofinfluenza AvirusNPprotein. GGmeansglycine insertionsseparating theconstruct components NS124-Fus(B)-NP MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, GFFGAIAGFLEGGWEGMIAGW-GG-RESRNPGNA elongatedbythe (SEQIDNO:10) translatedsequence ofinfluenzaBvirus HA2subunit(shownin bold),from1to21 aa,andwiththe sequenceofa conservativeB-cell epitopeofinfluenza AvirusNPprotein. GGmeansglycine insertionsseparating theconstruct components NS124-Esat6 MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSL elongatedbythe TKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNL translatedsequence ARTISEAGQAMASTEGNVTGMFA ofmycobacterium (SEQIDNO:11) tuberculosisprotein Esat6(showninbold) NS24-2A-Esat6 MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-GG- truncatedto124aa, NFDLLKLAGDVESNPGP- elongatedbythe MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSL translatedsequence TKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNL ofaself-cleaving2A ARTISEAGQAMASTEGNVTGMFA peptide(from (SEQIDNO:12) picornavirus)and withthesequenceof mycobacterium tuberculosisprotein Esat6 NS124-HSV-2ASY MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRL Viruswiththe RRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEE readingframeofan SDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPK NS1protein, QKVAGPLCIRMDQAIM-AAA-NLLTTPKFT-AAA- truncatedto124aa, RMLGDVMAV-AAA-NLLTTPKFT-AAA- elongatedbythe RMLGDVMAV translatedsequences (SEQIDNO:13) (inbold)ofaT-cell epitopesofherpes simplexvirus(HSV)1 and2types.Epitope insertionsaredone withrepetition

(68) Recombinant viruses were assembled by transfection of VERO cells with seven plasmids encoding genomic unmodified fragments of influenza virus, and with one of variants of a chimeric NS genomic fragment by the plasmid DNA electroporation method (Cell Line Nucleofector Kit V (Lonza)) according to the instruction for use. After transfection, the cells were incubated in Optipro medium (Invitrogen) for 96 hours at 34 C. with the addition of 1 g/ml trypsin to ensure post-translational cleavage of the hemagglutinin precursor into HA1 and HA2 subunits. The viral harvest from Vero cells was used to infect 10-day-old chicken embryos (SPF). Embryos were incubated for 48 hours at 34 C., after which allantoic fluids having a positive titer in the haemagglutination reaction were used for the second passage on chicken embryos. Allantoic liquids of the second passage were aliquoted and stored at 80 C. The second passage material was used to control the genetic structure of the chimeric NS fragment and the presence of the transgene by producing the RT-PCR product and its sequencing. In addition, the second passage material was used to determine the phenotypic markers of recombinant viral strains and vectors and to determine the genetic stability of the transgene for 5 passages in chicken embryos.

Example 2

(69) Determination of Temperature-Sensitivity Phenotype and Attenuation of Heterologous Nep-Carrying Recombinant Viruses

(70) The temperature sensitivity of viruses was determined by comparative titration of the infectious activity of viruses on Vero cells at an optimal temperature of 34 C. and an elevated temperature of 39 C., in 96-well plates. The virus titers were counted by the Reed-Muench method after incubation for 96 hours, taking into account the development of the cytopathic effect in the plate wells (Reed, L J, Muench, H. (1938). The A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 27: 493-497.). FIG. 4A shows virus titers at these temperatures, expressed in 50% tissue cytopathic doses (Log TCD50/ml). Both viruses carrying heterologous Nep from A/Singapore/1/57 (H2N2) or A/Leningrad/134/47/57 (H2N2) strains surprisingly showed a significant decrease of more than 4 log in infectious titers at 39 C., compared with the optimal temperature of 34 C. Control strainswild-type A/PR/8/34 (H1N1) virus, and recombinant NS124/Nep PR8 virus with a truncated NS1 protein and a homologous Nep protein did not show temperature sensitivity, replicating effectively at a high temperature. Thus, the replacement of Nep resulted in the appearance of is phenotype in viruses.

(71) Moreover, in intranasal infection of mice under mild anesthesia with said viruses in a dose of 6 log/mouse, virusescarriers of a heterologous Nep gene had a decreased reproduction ability in the lung tissues (p<0.002), compared with the wild-type virus or NS124/Nep PR8 virus having a homologous Nep (FIG. 4B). Virus titers in the lungs were assessed 2 days after infection of the animals by titration of clarified lung homogenates, in Vero cells. Pulmonary titers were expressed in log TCD50/g lung tissue. Thus, the introduction of the chimeric NS genomic fragment into influenza A/PR/8/34 (H1N1) strain led to the attenuation of the virus, manifested in a decrease in its reproduction ability in the lower respiratory tract of animals.

Example 3

(72) Determination of the ts Phenotype and Attenuation of Vectors Carrying a Chimeric NS Genomic Fragment and Various Insertions in the Reading Frame of an NS1 Protein

(73) A wide set of vectors encoding insertions of different nature was produced to determine the effect of insertions of foreign sequences into the reading frame of an NS1 protein on the ts phenotype of viruses comprising a chimeric Nep gene. The viruses with insertions shown in FIG. 3 were studied. The ts-phenotype was studied by titration of the virus infectious activity at temperatures of 34 and 39 C. in 10-day-old chicken embryos (ChE), by determining the haemagglutinating activity of allantoic fluids collected 48 hours after incubation. The titer was calculated by the Reed-Muench method and expressed in log of 50% embryonic infectious doses (log EID50/ml). As can be seen from the data presented in Table 2, all vectors, contrary to the wild-type A/PR/8/34 (H1N1) virus, had a significantly reduced reproduction ability at a high temperature and corresponded in the ts phenotypic marker to the prototype chimeric strains that did not have insertions but carried a heterologous Nep.

(74) TABLE-US-00003 TABLE 2 NS fragment composition* NS1 Yield in ChE (Log length Nep origin from EID50/ml) at T: ts Virus/vector (aa) strain** 34 C. 39 C. phenotype*** A/PR/8/34 230 A/PR/8/34 (H1N1) 9.8 9.5 no NS124/Nep-Len 124 Len 8.8 2.8 yes NS124/Nep- 124 Sing 8.8 3.3 yes Sing NS124-HA2(A)- 124 Len 8.3 2.8 yes 185 NS124-HA2(A)- 124 Len 8.5 3.0 yes 65-222 NS124-HA2(A)- 124 Len 8.3 3.5 yes 23-185 NS124-HA2(B)- 124 Len 8.8 3.5 yes 186 NS124-Fus(A)- 124 Len 8.0 2.8 yes NP NS124-Fus(B)- 124 Len 8.5 2.5 yes NP NS124-Esat6 124 Len 9.5 3.8 yes NS124-2A- 124 Len 9.8 4.0 yes Esat6 NS124-HSV- 124 Len 8.0 2.5 yes 2ASY Designation: *Length (in amino acid residues) of the natural NS1 protein sequence before an insertion; **Origin of the Nep gene from a strain: A/PR/8/34 (H1N1) or A/Singapore/1/57(H2N2), or A/Leningrad/134/47/57 (H2N2); ***ts-phenotype is considered positive if a difference in the virus growth at 34 and 39 C. exceeds 2 log

(75) To determine the effect of insertions on the attenuation (att) of vector strains for animals, the mice were challenged intranasally, under mild anesthesia, with virus-containing allantoic fluids of chicken embryos infected with the viruses or vectors represented in FIG. 3. Allantoic fluids were preliminarily characterized by the level infectious virus titers contained therein. The titers were expressed in log EID50/ml. Mice were injected with 0.05 ml of each virus sample. Each group of mice contained 8 animals. The lethal activity of viruses was assessed for 12 days. It was found that, unlike the wild-type A/PR/8/34 (H1N1) virus that caused a 50% lethal effect when using a material with a titer of 3.2 log EID50/ml, none of the vectors showed 50% lethal activity in mice at an infective dose of more than 7.5 log. Thus, all vectors carrying a chimeric NS genomic fragment, regardless of an insertion, were highly attenuated for mice (Table 3).

(76) TABLE-US-00004 TABLE 3 Mouse-lethal dose of the virus 50% lethal virus Virus/vector dose (LD50/ml) Att-phenotype* A/PR/8/34 3.2 No NS124/Nep-Len >7.5 Yes NS124/Nep-Sing >7.5 Yes NS124-HA2(A)-185 >7.5 Yes NS124-HA2(A)-65-222 >7.5 Yes NS124-HA2(A)-23-185 >7.5 Yes NS124-HA2(B)-186 >8.0 Yes NS124-Fus(A)-NP >8.0 Yes NS124-Fus(B)-NP >8.0 Yes NS124-Esat6 >7.5 Yes NS124-2A-Esat6 >7.5 Yes NS124-HSV-2ASY >7.5 Yes *attenuation phenotype is determined by the absence of lethal activity in protective dose exceeding 7.0 log EID50/mouse

Example 4

(77) Protective Response to Heterologous Strains of Influenza A and B Viruses in Control Infection of Mice

(78) The protective activity to heterologous antigen variants of influenza virus was determined by using viruses with surface antigens from A/PR/8/34 (H1N1) virus carrying a chimeric NS genomic fragment with a Nep sequence from virus A/Leningrad/134/47/57 (H2N2). The following recombinant viruses were used that encoded hemagglutinin HA2 subunit regions in the NS1 reading frame: 1) vector NS124-HA2(A)-185 expressing the full-length influenza A virus HA2 ectodomain of 185 amino acid residues (FIG. 3, SEQ ID NO: 5), 2) vector NS124-HA2(A)-185 expressing the full-length influenza B virus HA2 ectodomain of 186 amino acid residues (FIG. 3, SEQ ID NO: 8) 3) vector NS124-Fus(A)-NP expressing a sequence consisting of the N-terminal 21 amino acid residues of HA2 (fusion domain) in combination with the sequence of a conserved B-cell epitope from influenza A virus NP protein (FIG. 3, SEQ ID NO: 9), and 4) NS124/Nep-Len virus having a stop codon cassette at position 399 of the nucleotide sequence of an NS genomic fragment, limiting translation of the NS1 protein to 124 amino acid residues (FIG. 3, SEQ ID NO: 4). The control groups included mice infected with the wild-type A/PR/8/34 (H1N1) virus without genetic modifications, or mice received a phosphate buffer solution containing no active ingredient. The mice were immunized intranasally under mild anesthesia, with a single viral dose of 6.5 log/mouse. After 28 days, the animals were subjected to a control infection with mouse-pathogenic heterologous influenza strains: A/Mississippi/85/1(H3N2) or B/Lee/40 in a dose corresponding to 3-5 LD50, respectively.

(79) As can be seen in FIG. 5A, the control infection of non-immune mice with the virus (H3N2) resulted in their death in 80% of cases. At the same time, mice immunized with viral preparations were fully protected from death caused by infection with a heterologous influenza A (H3N2) virus strain. Immunization with the wild-type virus also resulted in a statistically significant level of protection against infection by a heterologous strain. When control infection was performed by using influenza B/Lee/40 virus, the immunization of mice with the wild-type A/PR/8/34 (H1N1) virus did not protect animals from death, as well as the mice received in immunization a phosphate buffer solution (FIG. 5B). It was surprisingly found that recombinant viruses carrying insertions in the reading frame of an NS1 protein was protective against influenza B virus. The vector NS124-Fus(A)-NP showed the best protective level. Thus, in single intranasal immunization of mice, the vector strains carrying a chimeric NS genomic fragment, showed the properties of a universal influenza vaccine effective against heterologous antigenic subtypes of both influenza A virus and influenza B virus.

Example 5

(80) Production of an Influenza Vector with a Modified NS Genomic Fragment Encoding a Sequence of Influenza B Virus HA2 Region and H1N1pdm Virus Surface Glycoproteins

(81) A recombinant virus was assembled in several steps. At the first step, complementary DNA (cDNA) copies of 5 genomic fragments (PB2, PB1, PA, NP, M) of influenza A/PR/8/34 (H1N1) virus (PB2 (Genbank accession number: AB671295), PB1 (Genbank accession number: CY033583), PA (Genbank accession number: AF389117), NP (Genbank accession number: AF389119), M (Genbank accession number: AF389121)) and 2 genomic fragments (HA, NA) of A/California/7/09-like virus (HA (GenBank: KM408964.1) and (NA GenBank: KM408965.1)) were produced, and a chimeric NS genomic fragment composed of the sequences related to H1N1 virus (NS1 gene), H2N2 virus (Nep gene) and the sequences of two peptides from an influenza B virus HA2 region and an influenza A virus NP region was synthesized.

(82) At the second step, the synthesized sequences were cloned into a bidirectional plasmid pHW2000-based vector (Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster R G, A DNA from eight plasmids, Proc Natl Acad Sci USA. 2000; 97 (11):6108-13.). This plasmid vector, due to the presence of Pol I and Pol II promoters, provides simultaneous intracellular transcription of viral and corresponding messenger RNAs upon transfection of mammalian cells. FIG. 7 shows a genetic diagram of the influenza virus. FIG. 8 shows the nucleotide sequences of all eight genomic fragments of the vaccine vector.

(83) TABLE-US-00005 NucleotidesequeneofgenomicPB2 (SEQIDNO:14) 1 agcgaaagcaggtcaattatattcaatatggaaagaataaaagaactacgaaatctaatg 61 tcgcagtctcgcacccgcgagatactcacaaaaaccaccgtggaccatatggccataatc 121 aagaagtacacatcaggaagacaggagaagaacccagcacttaggatgaaatggatgatg 181 gcaatgaaatatccaattacagcagacaagaggataacggaaatgattcctgagagaaat 241 gagcaaggacaaactttatggagtaaaatgaatgatgccggatcagaccgagtgatggta 301 tcacctctggctgtgacatggtggaataggaatggaccaataacaaatacagttcattat 361 ccaaaaatctacaaaacttattttgaaagagtcgaaaggctaaagcatggaacctttggc 421 cctgtccattttagaaaccaagtcaaaatacgtcggagagttgacataaatcctggtcat 481 gcagatctcagtgccaaggaggcacaggatgtaatcatggaagttgttttccctaacgaa 541 gtgggagccaggatactaacatcggaatcgcaactaacgataaccaaagagaagaaagaa 601 gaactccaggattgcaaaatttctcctttgatggttgcatacatgttggagagagaactg 661 gtccgcaaaacgagattcctcccagtggctggtggaacaagcagtgtgtacattgaagtg 721 ttgcatttgactcaaggaacatgctgggaacagatgtatactccaggaggggaagtgagg 781 aatgatgatgttgatcaaagcttgattattgctgctaggaacatagtgagaagagctgca 841 gtatcagcagatccactagcatctttattggagatgtgccacagcacacagattggtgga 901 attaggatggtagacatccttaggcagaacccaacagaagagcaagccgtggatatatgc 961 aaggctgcaatgggactgagaattagctcatccttcagttttggtggattcacatttaag 1021 agaacaagcggatcatcagtcaagagagaggaagaggtgcttacgggcaatcttcaaaca 1081 ttgaagataagagtgcatgagggatatgaagagttcacaatggttgggagaagagcaaca 1141 gccatactcagaaaagcaaccaggagattgattcagctgatagtgagtgggagagacgaa 1201 cagtcgattgccgaagcaataattgtggccatggtattttcacaagaggattgtatgata 1261 aaagcagtcagaggtgatctgaatttcgtcaatagggcgaatcaacgattgaatcctatg 1321 catcaacttttaagacattttcagaaggatgcgaaagtgctttttcaaaattggggagtt 1381 gaacctatcgacaatgtgatgggaatgattgggatattgcccgacatgactccaagcatc 1441 gagatgtcaatgagaggagtgagaatcagcaaaatgggtgtagatgagtactccagcacg 1501 gagagggtagtggtgagcattgaccgttttttgagaatccgggaccaacgaggaaatgta 1561 ctactgtctcccgaggaggtcagtgaaacacagggaacagagaaactgacaataacttac 1621 tcatcgtcaatgatgtgggagattaatggtcctgaatcagtgttggtcaatacctatcaa 1681 tggatcatcagaaactgggaaactgttaaaattcagtggtcccagaaccctacaatgcta 1741 tacaataaaatggaatttgaaccatttcagtctttagtacctaaggccattagaggccaa 1801 tacagtgggtttgtaagaactctgttccaacaaatgagggatgtgcttgggacatttgat 1861 accgcacagataataaaacttcttcccttcgcagccgctccaccaaagcaaagtagaatg 1921 cagttctcctcatttactgtgaatgtgaggggatcaggaatgagaatacttgtaaggggc 1981 aattctcctgtattcaactataacaaggccacgaagagactcacagttctcggaaaggat 2041 gctggcactttaactgaagacccagatgaaggcacagctggagtggagtccgctgttctg 2101 aggggattcctcattctgggcaaagaagacaagagatatgggccagcactaagcatcaat 2161 gaactgagcaaccttgcgaaaggagagaaggctaatgtgctaattgggcaaggagacgtg 2221 gtgttggtaatgaaacggaaacgggactctagcatacttactgacagccagacagcgacc 2281 aaaagaattcggatggccatcaattagtgtcgaatagtttaaaaacgaccttgtttctac 2341 t NucleotidesequenceofgenomicPB1 (SEQIDNO:15) 1 atggatgtcaatccgaccttacttttcttaaaagtgccagcacaaaatgctataagcaca 61 actttcccttatactggagaccctccttacagccatgggacaggaacaggatacaccatg 121 gatactgtcaacaggacacatcagtactcagaaaagggaagatggacaacaaacaccgaa 181 actggagcaccgcaactcaacccgattgatgggccactgccagaagacaatgaaccaagt 241 ggttatgcccaaacagattgtgtattggaggcgatggctttccttgaggaatcccatcct 301 ggtatttttgaaaactcgtgtattgaaacgatggaggttgttcagcaaacacgagtagac 361 aagctgacacaaggccgacagacctatgactggactctaaatagaaaccaacctgctgca 421 acagcattggccaacacaatagaagtgttcagatcaaatggcctcacggccaatgagtct 481 ggaaggctcatagacttccttaaggatgtaatggagtcaatgaacaaagaagaaatgggg 541 atcacaactcattttcagagaaagagacgggtgagagacaatatgactaagaaaatgata 601 acacagagaacaatgggtaaaaagaagcagagattgaacaaaaggagttatctaattaga 661 gcattgaccctgaacacaatgaccaaagatgctgagagagggaagctaaaacggagagca 721 attgcaaccccagggatgcaaataagggggtttgtatactttgttgagacactggcaagg 781 agtatatgtgagaaacttgaacaatcagggttgccagttggaggcaatgagaagaaagca 841 aagttggcaaatgttgtaaggaagatgatgaccaattctcaggacaccgaactttctttc 901 accatcactggagataacaccaaatggaacgaaaatcagaatcctcggatgtttttggcc 961 atgatcacatatatgaccagaaatcagcccgaatggttcagaaatgttctaagtattgct 1021 ccaataatgttctcaaacaaaatggcgagactgggaaaagggtatatgtttgagagcaag 1081 agtatgaaacttagaactcaaatacctgcagaaatgctagcaagcatcgatttgaaatat 1141 ttcaatgattcaacaagaaagaagattgaaaaaatccgaccgctcttaatagaggggact 1201 gcatcattgagccctggaatgatgatgggcatgttcaatatgttaagcactgtattaggc 1261 gtctccatcctgaatcttggacaaaagagatacaccaagactacttactggtgggatggt 1321 cttcaatcctctgacgattttgctctgattgtgaatgcacccaatcatgaagggattcaa 1381 gccggagtcgacaggttttatcgaacctgtaagctacttggaatcaatatgagcaagaaa 1441 aagtcttacataaacagaacaggtacatttgaattcacaagttttttctatcgttatggg 1501 tttgttgccaatttcagcatggagcttcccagttttggggtgtctgggatcaacgagtca 1561 gcggacatgagtattggagttactgtcatcaaaaacaatatgataaacaatgatcttggt 1621 ccagcaacagctcaaatggcccttcagttgttcatcaaagattacaggtacacgtaccga 1681 tgccatagaggtgacacacaaatacaaacccgaagatcatttgaaataaagaaactgtgg 1741 gagcaaacccgttccaaagctggactgctggtctccgacggaggcccaaatttatacaac 1801 attagaaatctccacattcctgaagtctgcctaaaatgggaattgatggatgaggattac 1861 caggggcgtttatgcaacccactgaacccatttgtcagccataaagaaattgaatcaatg 1921 aacaatgcagtgatgatgccagcacatggtccagccaaaaacatggagtatgatgctgtt 1981 gcaacaacacactcctggatccccaaaagaaatcgatccatcttgaatacaagtcaaaga 2041 ggagtacttgaggatgaacaaatgtaccaaaggtgctgcaatttatttgaaaaattcttc 2101 cccagcagttcatacagaagaccagtcgggatatccagtatggtggaggctatggtttcc 2161 agagcccgaattgatgcacggattgatttcgaatctggaaggataaagaaagaagagttc 2221 actgagatcatgaagatctgttccaccattgaagagctcagacggcaaaaatagtgaatt 2281 tagcttgt NucleotidesequenceofgenomicPA (SEQIDNO:16) 1 agcgaaagcaggtactgatccaaaatggaagattttgtgcgacaatgcttcaatccgatg 61 attgtcgagcttgcggaaaaaacaatgaaagagtatggggaggacctgaaaatcgaaaca 121 aacaaatttgcagcaatatgcactcacttggaagtatgcttcatgtattcagattttcac 181 ttcatcaatgagcaaggcgagtcaataatcgtagaacttggtgatccaaatgcacttttg 241 aagcacagatttgaaataatcgagggaagagatcgcacaatggcctggacagtagtaaac 301 agtatttgcaacactacaggggctgagaaaccaaagtttctaccagatttgtatgattac 361 aaggagaatagattcatcgaaattggagtaacaaggagagaagttcacatatactatctg 421 gaaaaggccaataaaattaaatctgagaaaacacacatccacattttctcgttcactggg 481 gaagaaatggccacaaaggcagactacactctcgatgaagaaagcagggctaggatcaaa 541 accagactattcaccataagacaagaaatggccagcagaggcctctgggattcctttcgt 601 cagtccgagagaggagaagagacaattgaagaaaggtttgaaatcacaggaacaatgcgc 661 aagcttgccgaccaaagtctcccgccgaacttctccagccttgaaaattttagagcctat 721 gtggatggattcgaaccgaacggctacattgagggcaagctgtctcaaatgtccaaagaa 781 gtaaatgctagaattgaaccttttttgaaaacaacaccacgaccacttagacttccgaat 841 gggcctccctgttctcagcggtccaaattcctgctgatggatgccttaaaattaagcatt 901 gaggacccaagtcatgaaggagagggaataccgctatatgatgcaatcaaatgcatgaga 961 acattctttggatggaaggaacccaatgttgttaaaccacacgaaaagggaataaatcca 1021 aattatcttctgtcatggaagcaagtactggcagaactgcaggacattgagaatgaggag 1081 aaaattccaaagactaaaaatatgaagaaaacaagtcagctaaagtgggcacttggtgag 1141 aacatggcaccagaaaaggtagactttgacgactgtaaagatgtaggtgatttgaagcaa 1201 tatgatagtgatgaaccagaattgaggtcgctagcaagttggattcagaatgagtttaac 1261 aaggcatgcgaactgacagattcaagctggatagagctcgatgagattggagaagatgtg 1321 gctccaattgaacacattgcaagcatgagaaggaattatttcacatcagaggtgtctcac 1381 tgcagagccacagaatacataatgaagggggtgtacatcaatactgccttgcttaatgca 1441 tcttgtgcagcaatggatgatttccaattaattccaatgataagcaagtgtagaactaag 1501 gagggaaggcgaaagaccaacttgtatggtttcatcataaaaggaagatcccacttaagg 1561 aatgacaccgacgtggtaaactttgtgagcatggagttttctctcactgacccaagactt 1621 gaaccacataaatgggagaagtactgtgttcttgagataggagatatgcttataagaagt 1681 gccataggccaggtttcaaggcccatgttcttgtatgtgagaacaaatggaacctcaaaa 1741 attaaaatgaaatggggaatggagatgaggcgttgcctcctccagtcacttcaacaaatt 1801 gagagtatgattgaagctgagtcctctgtcaaagagaaagacatgaccaaagagttcttt 1861 gagaacaaatcagaaacatggcccattggagagtcccccaaaggagtggaggaaagttcc 1921 attgggaaggtctgcaggactttattagcaaagtcggtattcaacagcttgtatgcatct 1981 ccacaactagaaggattttcagctgaatcaagaaaactgcttcttatcgttcaggctctt 2041 agggacaaccttgaacctgggacctttgatcttggggggctatatgaagcaattgaggag 2101 tgcctgattaatgatccctgggttttgcttaatgcttcttggttcaactccttccttaca 2161 catgcattgagttagttgtggcagtgctactatttgctatccatactgtccaaaaaagta 2221 ccttgtttctact NucleotidesequenceofgenomicNP (SEQIDNO:17) 1 agcgaaagcaggtagatattgaaagatgagtcttctaaccgaggtcgaaacgtacgtact 61 ctctatcatcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctt 121 tgcagggaagaacactgatcttgaggttctcatggaatggctaaagacaagaccaatcct 181 gtcacctctgactaaggggattttaggatttgtgttcacgctcaccgtgcccagtgagcg 241 aggactgcagcgtagacgctttgtccaaaatgcccttaatgggaacggggatccaaataa 301 catggacaaagcagttaaactgtataggaagctcaagagggagataacattccatggggc 361 caaagaaatctcactcagttattctgctggtgcacttgccagttgtatgggcctcatata 421 caacaggatgggggctgtgaccactgaagtggcatttggcctggtatgtgcaacctgtga 481 acagattgctgactcccagcatcggtctcataggcaaatggtgacaacaaccaatccact 541 aatcagacatgagaacagaatggttttagccagcactacagctaaggctatggagcaaat 601 ggctggatcgagtgagcaagcagcagaggccatggaggttgctagtcaggctagacaaat 661 ggtgcaagcgatgagaaccattgggactcatcctagctccagtgctggtctgaaaaatga 721 tcttcttgaaaatttgcaggcctatcagaaacgaatgggggtgcagatgcaacggttcaa 781 gtgatcctctcgctattgccgcaaatatcattgggatcttgcacttgacattgtggattc 841 ttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggc 901 cttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtg 961 ctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaaaaactaccttgt 1021 ttctact NucleotidesequenceofgenomicM (SEQIDNO:18) 1 agcgaaagcaggtagatattgaaagatgagtcttctaaccgaggtcgaaacgtacgtact 61 ctctatcatcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctt 121 tgcagggaagaacactgatcttgaggttctcatggaatggctaaagacaagaccaatcct 181 gtcacctctgactaaggggattttaggatttgtgttcacgctcaccgtgcccagtgagcg 241 aggactgcagcgtagacgctttgtccaaaatgcccttaatgggaacggggatccaaataa 301 catggacaaagcagttaaactgtataggaagctcaagagggagataacattccatggggc 361 caaagaaatctcactcagttattctgctggtgcacttgccagttgtatgggcctcatata 421 caacaggatgggggctgtgaccactgaagtggcatttggcctggtatgtgcaacctgtga 481 acagattgctgactcccagcatcggtctcataggcaaatggtgacaacaaccaatccact 541 aatcagacatgagaacagaatggttttagccagcactacagctaaggctatggagcaaat 601 ggctggatcgagtgagcaagcagcagaggccatggaggttgctagtcaggctagacaaat 661 ggtgcaagcgatgagaaccattgggactcatcctagctccagtgctggtctgaaaaatga 721 tcttcttgaaaatttgcaggcctatcagaaacgaatgggggtgcagatgcaacggttcaa 781 gtgatcctctcgctattgccgcaaatatcattgggatcttgcacttgacattgtggattc 841 ttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggc 901 cttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtg 961 ctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaaaaactaccttgt 1021 ttctact NucleotidesequenceofgenomicHA (SEQIDNO:19) 1 atgaaggcaatactagtagttctgctatatacatttgcaaccgcaaatgcagacacatta 61 tgtataggttatcatgcaaacaattcaacagacactgtagacacagtactagaaaagaat 121 gtaacagtaacacactctgttaaccttctagaagacaagcataacgggaaactatgcaaa 181 ctaagaggggtagccccattgcatttgggtaaatgtaacattgctggctggatcctggga 241 aatccagagtgtgaatcactctccacagcaagttcatggtcctacattgtggaaacatct 301 agttcagacaatggaacgtgttacccaggagatttcatcaattatgaggagctaagagag 361 caattgagctcagtgtcatcatttgaaaggtttgagatattccccaaaacaagttcatgg 421 cccaatcatgactcgaacaaaggtgtaacggcagcatgtcctcacgctggagcaaaaagc 481 ttctacaaaaatttaatatggctagttaaaaaaggaaattcatacccaaagctcagccaa 541 tcctacattaatgataaagggaaagaagtcctcgtgctgtggggcattcaccatccatct 601 actactgctgaccaacaaagtctctatcagaatgcagatgcatatgtttttgtggggaca 661 tcaagatacagcaagaagttcaagccggaaatagcaataagacccaaagtgagggatcaa 721 gaagggagaatgaactattactggacactagtagagccgggagacaaaataacattcgaa 781 gcaactggaaatctagtggtaccgagatatgcattcacaatggaaagaaatgctggatct 841 ggtattatcatttcagatacaccagtccacgattgcaatacaacttgtcagacacccgag 901 ggtgctataaacaccagcctcccatttcagaatatacatccgatcacaattggaaaatgt 961 ccaaagtatgtaaaaagcacaaaattgagactggccacaggattgaggaatgtcccgtct 1021 attcaatctagaggcctattcggggccattgccggcttcattgaaggggggtggacaggg 1081 atggtagatggatggtacggttatcaccatcaaaatgagcaggggtcaggatatgcagcc 1141 gacctgaagagcacacaaaatgccattgacaagattactaacaaagtaaactctgttatt 1201 gaaaagatgaatacacagttcacagcagtgggtaaagagttcaaccacctggaaaaaaga 1261 atagagaatttaaataaaaaagttgatgatggtttcctggacatttggacttacaatgcc 1321 gaactgttggttctattggaaaatgaaagaactttggactaccatgattcaaatgtgaag 1381 aacttgtatgaaaaggtaagaaaccagttaaaaaacaatgccaaggaaattggaaacggc 1441 tgctttgaattttaccacaaatgcgataacacgtgcatggaaagtgtcaaaaatgggact 1501 tatgactacccaaaatactcagaggaagcaaaattaaacagagaaaaaatagatggggta 1561 aagctggaatcaacaaggatttaccagattttggcgatctattcaactgtcgccagttca 1621 ttggtgctggtagtctccctgggggcaatcagcttctggatgtgctctaatgggtctcta 1681 cagtgtagaatatgtatttaa NucleotidesequenceofgenomicNA (SEQIDNO:20) 1 atgaatccaaaccaaaagataataaccattggttcggtctgtatgacaattggaatggct 61 aacttaatattacaaattggaaacataatctcaatatggattagccactcaattcaagtt 121 gggaatcaaagtcagatcgaaacatgcaatcaaagcgtcattacttatgaaaacaacact 181 tgggtaaatcagacatatgttaacatcagcaacaccaactttgctgctgggcagccagtg 241 gtttccgtgaaattagcgggcaattcctctctctgccctgttagtggatgggctatatac 301 agtaaagacaacagtgtaagagtcggttccaagggggatgtgtttgtcataagggaacca 361 ttcatatcatgctcccccttggaatgcagaaccttcttcttgactcaaggggccttgcta 421 aatgacaaacattccaatggaaccattaaagacaggagcccatatcgaaccttaatgagc 481 tgtcctattggtgaagttccctctccatacaactcaagatttgagtcagtcgcttggtca 541 gcaagtgcttgtcatgatggcatcaattggctaacaattggaatttctggcccagacagt 601 ggggcagtggctgtgttaaagtacaacggcataataacagacactatcaagagttggaga 661 aacgatatattgagaacacaagagtctgaatgtgcatgtgtaaatggttcttgctttacc 721 ataatgaccgatggaccaagtgatggacaggcctcatacaagatcttcagaatagaaaag 781 ggaaagatagtcaaatcagtcgaaatgaatgcccctaattatcactatgaggaatgctcc 841 tgttatcctgattctagtgaaatcacatgtgtgtgcagggataactggcatggctcgaat 901 cgaccgtgggtgtctttcaaccagaatctggaatatcagataggatacatatgcagtggg 961 attttcggagacaatccacgccctaatgataagacaggcagttgtggtccagtatcgtct 1021 aatggagcaaatggagtaaaaggattttcattcaaatacggcaatggtgtttggataggg 1081 agaactaaaagcattagttcaagaaaaggttttgagatgatttgggatccaaatggatgg 1141 actgggacagacaataacttctcaataaagcaagatatcgtaggaataaatgagtggtca 1201 ggatatagcgggagttttgttcagcatccagaactaacagggctggattgtataagacct 1261 tgcttctgggttgaactaatcagagggcgacccaaagagaacacaatctggactagtggg 1321 agcagcatatccttttgtggtgtaaacagtgacactgtgggttggtcttggccagacggt 1381 gctgagttgccatttaccattgacaagtaa NucleotidesequenceofachimericNSfragmentgene (insertionisshowninboldtype) (SEQIDNO:21) AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTC TTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCC GAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACTCTTGGTCTGGACATCGAGACAGCCACACGTGCTG GAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGCCTCTG TACCTGCGTCGCGTTACCTAACCGACATGACTCTTGAGGAAATGTCAAGGGAATGGTCCATGCTCATAC CCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCGATCATGGGAGGAGGTTTCTTCG GAGCTATTGCTGGTTTCTTGGAAGGAGGATGGGAAGGAATGATTGCAGGTTGGGGAGGAAGAGAGAGCC GGAACCCAGGGAATGCTTGATAATAAGCGGCCGCAGTGTGATTTTTGACCGGCTGGAGACTCTAATATT GCTAAGGGCTTTCACCGAAGAGGGAGCAATTGTTGGCGAAATTTCACCATTGCCTTCTCTTCCAGGACA TACTAATGAGGATGTCAAAAATGCAATTGGGGTCCTCATCGGAGGACTTGAATGGAATGATAACACAGT TCGAGTCTCTAAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTCAC TCCAAAACAGAAACGGAAAATGGCGAGAACAATTAGGTCAAAAGTTCGAAGAAATAAGATGGCTGATTG AAGAAGTGAGACACAAATTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATACAAGCCTTAC AGCTACTATTTGAAGTGGAACAAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAATAATAAAAAACAC CCTTGTTTCTACT

(84) Recombinant viruses were assembled by transfection of VERO cells with eight plasmids encoding genomic unmodified fragments of influenza virus, and with a chimeric NS genomic fragment by the plasmid DNA electroporation method (Cell Line Nucleofector Kit V (Lonza)) according to the instruction for use. After transfection, the cells were incubated in Optipro medium (Invitrogen) for 96 hours at 34 C. with the addition of 1 g/ml trypsin to ensure post-translational cleavage of the hemagglutinin precursor into HA1 and HA2 subunits. The viral harvest from Vero cells was used to infect 10-day-old chicken embryos (SPF). Embryos were incubated for 48 hours at 34 C., after which allantoic fluids having a positive titer in the haemagglutination reaction were used for next passages on chicken embryos. Allantoic fluids of 7 passages were purified with tangential flow filtration and lyophilized for storage. The animals were immunized after dissolution of the lyophilisate with an equivalent volume of distilled water.

Example 6

(85) Protective Response to Heterologous Strains of Influenza A and B Viruses in Control Infection of Mice

(86) The protective activity to heterologous antigen variants of influenza virus was determined by intranasal immunization of mice with an influenza vector at a dose of 6.8 log EID50/mouse in a volume of 50 l under mild anesthesia, once or twice with a 3 week period. At twenty-one days after the last immunization, the animals were subjected to the control infection with mouse-pathogenic heterologous influenza strains: homologous A/California/7/09 (H1N1pdm) or heterologous A/Aichi/2/68 (H3N2), A/Mississippi/85/1(H3N2) or influenza B/Lee/40 virus in a dose corresponding to 3-5 LD50, respectively.

(87) As can be seen in FIG. 9A, the control infection of non-immune mice with the H1N1pdm virus resulted in their death in 90% of cases. However, the mice immunized once or twice with the virus preparation were reliably protected from death.

(88) As can be seen in FIG. 3B9B, the control infection of non-immune mice with A/Aichi/2/68 (H3N2) virus resulted in their death in 100% cases. However, the mice immunized once or twice with the virus preparation were reliably protected from death.

(89) As can be seen in FIG. 9C, the control infection of non-immune mice with A/Mississippi/85/1(H3N2) virus resulted in their death in 100% cases. However, the mice immunized twice with the virus preparation had 100% protection.

(90) As can be seen in FIG. 9D, the control infection of non-immune mice with B/Lee/40 influenza virus resulted in their death in 100% cases. However, the mice immunized twice with the virus preparation had 60% protection significantly different from the control.

(91) Thus, the influenza vector carrying a chimeric NS genomic fragment showed the properties of a universal influenza vaccine effective against heterologous antigenic subtypes of both influenza A virus and influenza B virus.

Example 7

(92) Protective Response to a Heterologous Influenza A (H3N2) Strain in the Control Infection of Ferrets

(93) Ferrets are an optimal, model recommended by the WHO for studying the effectiveness of influenza vaccines and drugs. The protective activity to a heterologous antigen variant of influenza virus was determined by immunization of ferrets (9 animals per group) with the influenza vector produced in Example 5 at a dose of 7.5 log EID50/ferret, administered intranasal in a volume of 500 l under mild anesthesia, once or twice with a 3 week period. At twenty-one days after the last immunization, the animals were subjected to the control infection with the ferret-pathogenic A/St.Petersburg/224/2015 (H3N2) virus. As shown in FIG. 10A, the control infection of non-immune animals resulted in a rise of the body temperature on day 2 after infection, while the vaccinated animals did not have a temperature response.

(94) The effect of the vaccination on the reproduction of the control virus in the respiratory tract of ferrets was studied by using nasal washings taken in animals on Days 2, 4 and 6 to determine the concentration of the infectious virus by titration of 50% cytopathic dose in the MDCK cell culture. As can be seen in FIG. 10B, the control infection of non-immune ferrets resulted in the active reproduction of the virus without a significant reduction in titers up to day 6. In a single immunization of ferrets, a significant reduction was observed in the viral titer on Days 4 and 6 after the challenge. After double immunization, a significant, more than 100-fold decrease in the viral titer was recorded already on day 2 after infection of the animals.

(95) Thus, even a single vaccination of ferrets with the influenza vector resulted in the protection of animals from clinical manifestations in the form of a temperature reaction and facilitated the accelerated elimination of the control heterologous strain from the respiratory tract. Repeated immunization accelerated the process of viral elimination.

Example 8

(96) Oncolytic Effect of Influenza Vector Encoding Mycobacterial Protein Esat6

(97) The oncolytic potential of attenuated influenza vectors carrying a chimeric NS genomic fragment with a heterologous Nep gene was determined by treating with the viruses a mouse melanoma induced by the administration of 10.sup.6 B16 cells in a volume of 30 l to the subcutaneous space of the right hind foot. Each group contained 10 animals. The therapy was performed on day 5 after the administration of tumor cells, by injection of 30 l of the viral preparation or a phosphate buffer solution directly into the tumor growth zone. Injections were performed 4 times every third day, after which the rate of an increase in the volume of the affected foot and the survival rate of the animals were assessed for 85 days. The animals with tumors that reached 2000 mm.sup.3 were euthanized for ethical reasons and were considered dead.

(98) The melanoma was treated with a vector expressing mycobacterial antigen Esat6 in a design providing for 2A-mediated posttranslational cleavage of protein Esat-6 from the C-terminal region of a truncated NS1 protein of influenza NS124-2A-Esat6 virus (FIG. 3, SEQ ID NO:12). The control therapeutic agent was NS124/Nep-Len virus that did not contain insertions of a mycobacterial protein.

(99) FIG. 6A shows the results of measuring the foot volume on day 19 after administration of the tumor cells. It was surprisingly found that the smallest average feet volume was in mice receiving therapy with a vector expressing protein Esat6. This result was found to correlate with the survival of mice over a long observation period consisting of 85 days (FIG. 6B). Three from ten animals of the NS124-2A-Esat6 group were found to be in remission of the tumor growth, while the animals in the other groups died to day 60. Thus, the obtained data demonstrate the advantage of the oncolytic vector encoding the bacterial antigen.

Example 10

(100) Formulation of an Influenza Virus-Based Vaccine for Intranasal Immunization

(101) A vaccine containing the influenza vector produced in Example 1 or Example 5 in an amount of 6.5 to 8.5 log 50% embryo infectious doses (EID50)/ml, and a buffer stabilizing solution containing 0.9 wt. % chloride solution, 0.5 wt. % L-carnosine, 2.5 wt. % sucrose, 1 wt. % recombinant albumin, 1 wt. % L-arginine and 3 wt. % hydroxyethyl starch 130/0.4 (molecular weight is 130 kDa, the degree of molar substitution is 0.4).

Example 11

(102) Formulation of an Influenza Virus-Based Vaccine for Intranasal Immunization

(103) A vaccine containing the influenza vector produced in Example 1 or Example 5 in an amount of 6.5 to 8.5 log 50% embryo infectious doses (EID50)/ml, and a buffer stabilizing solution containing 0.9 wt. % chloride solution, 0.1 wt. % L-carnosine, 2.5 wt. % sucrose, 1 wt. % recombinant albumin, 1 wt. % L-arginine and 3 wt. % hydroxyethyl starch 130/0.4 (molecular weight is 130 kDa, the degree of molar substitution is 0.4).

Example 12

(104) The Formulation of an Influenza Virus-Based Vaccine for Oncolytic Purposes

(105) A vaccine containing the influenza vector produced in Example 1 or Example 5 in an amount of 6.5 to 10.5 log 50% embryo infectious doses (EID50)/ml, and a buffer stabilizing solution containing 1.35 wt. % chloride solution, 0.5 wt. % L-carnosine, 1 wt. % recombinant albumin, 1 wt. % L-arginine and 3 wt. % hydroxyethyl starch 130/0.4 (molecular weight is 130 kDa, the degree of molar substitution is 0.4).