IMMUNOGENIC CONSTRUCTIONS AND VACCINES FOR USE IN THE PREVENTIVE AND THERAPEUTIC TREATMENT OF DISEASES CAUSED BY SARS-COV-2

Claims

1. An immunogenic construct consisting of: (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T-cell epitopes with amino acid sequences that are at least 73% identical to the amino acid sequences of SEQ ID NOs: 1-77; or (ii) a polypeptide encoded by the nucleotide sequence defined in (i).

2. The immunogenic construct according to claim 1, wherein the nucleotide sequence does not encode a targeting unit that targets antigen-presenting cells and does not encode a multimerization unit.

3. An immunogenic construct according to paragraph 1, which is: (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T-cell epitopes with amino acid sequences that are at least 73% identical to the amino acid sequences of SEQ ID NOs: 1-77, and wherein the nucleotide sequence further encodes a targeting unit that targets antigen-presenting cells and a multimerization unit; or (ii) a polypeptide encoded by the nucleotide sequence defined in (i); or (iii) a multimeric protein consisting of multiple polypeptides defined in (ii).

4. The immunogenic construct of any one of the preceding claims, wherein the 77 SARS-CoV-2 T-cell epitopes have the amino acid sequences of SEQ ID NO: 1-77.

5. An immunogenic construct according to any one of the preceding claims, wherein the antigenic unit further comprises one or more T-cell epitopes with amino acid sequences at least 73% identical to the amino acid sequences of SEQ ID NO: 78-96, preferably wherein the antigenic unit further comprises all of said T-cell epitopes.

6. The immunogenic construct of claim 5, wherein one or more T-cell epitopes have the amino acid sequences of SEQ ID NO: 78-96.

7. The immunogenic construct according to claims 1-4, wherein the antigenic unit additionally comprises one or more T-cell epitopes with amino acid sequences that are at least 73% identical to the amino acid sequences of SEQ ID NO: 97-160, wherein preferably the antigenic unit additionally comprises all of said T-cell epitopes.

8. The immunogenic construct of claim 7, wherein one or more T-cell epitopes have the amino acid sequences of SEQ ID NO: 97-160.

9. The immunogenic construct according to claims 7 and 8, wherein the antigen unit further comprises one or more T-cell epitopes with amino acid sequences at least 73% identical to the amino acid sequences of SEQ ID NO: 97-160, preferably all of said T-cell epitopes.

10. The immunogenic construct of claim 9, wherein one or more T-cell epitopes have the amino acid sequences of SEQ ID NO: 97-160.

11. An immunogenic construct according to any of the preceding claims, wherein some or all of the T-cell epitopes are present as individual discrete epitopes and / or wherein some or all of the T-cell epitopes are present as groups of T-cell epitopes, each group comprising at least 2 T-cell epitopes.

12. An immunogenic construct according to any of the preceding claims, wherein some or all of the T-cell epitopes are present as groups of T-cell epitopes, wherein each group contains at least 2 T-cell epitopes.

13. The immunogenic construct of claim 12, wherein at least 2 T-cell epitopes are derived from the same SARS-CoV-2 protein, preferably from the same portion of the same SARS-CoV-2 protein.

14. An immunogenic construct according to any one of claims 11-13, wherein the T-cell epitopes within the group are arranged sequentially, optionally separated by T-cell epitope linkers.

15. An immunogenic construct according to any one of claims 11-13, wherein the T-cell epitopes within the group are aligned to form a continuous amino acid sequence that corresponds to the sequence of the naturally occurring protein from which the epitopes are derived.

16. An immunogenic construct according to any one of the preceding claims, wherein the one or more T-cell epitopes are flanked by amino acid sequences that also flank said epitopes in the naturally occurring protein from which the epitope is derived.

17. The immunogenic construct according to claim 16, wherein one or more T-cell epitopes are flanked by amino acid sequences at the N-terminus and / or C-terminus of the epitope.

18. The immunogenic construct of any one of claims 16 and 17, wherein such flanking sequences comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids, preferably from 1 to 10 amino acids, such as from 2 to 8 amino acids or from 3 to 7 amino acids or from 4 to 5 amino acids.

19. An immunogenic construct according to any of the preceding claims, wherein some or all of the T-cell epitopes and / or some or all of the groups of T-cell epitopes are separated from each other by T-cell epitope linkers, preferably non-immunogenic T-cell epitope linkers, which are preferably flexible linkers.

20. The immunogen construct according to any one of the preceding claims, wherein the antigen unit comprises an amino acid sequence that is at least 73% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166 and 167, such as at least 75-80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166 and 167, preferably at least 81-85%, more preferably at least 86-90% or at least 91-95% and most preferably at least 96-99%.

21. An immunogenic construct according to any one of the preceding claims, wherein the antigenic unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 161, 162, 163, 164, 165, 166 and 167, wherein preferably the antigenic unit has an amino acid sequence selected from the group consisting of SEQ ID NO: 161, 162, 163, 164, 165, 166 and 167, wherein more preferably the antigenic unit comprises the amino acid sequence of SEQ ID NO: 167 or wherein even more preferably the antigenic unit has the amino acid sequence of SEQ ID NO:

167.

22. An immunogenic construct according to any one of the preceding claims, wherein the targeting unit is or comprises a fragment that interacts with cell surface molecules on antigen-presenting cells, preferably wherein the targeting unit is or comprises a fragment that interacts with cell surface molecules on human antigen-presenting cells.

23. The immunogenic construct of claim 22, wherein the cell surface molecule is selected from the group consisting of MHC, CD14, CD40, CLEC9A, chemokine receptors such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1, and Toll-like receptors such as TLR-2, TLR-4 or TLR-5, preferably wherein the cell surface molecule is selected from the group consisting of HLA; hCD14, hCD40, hCLEC9A, human chemokine receptors such as hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR1, and Toll-like receptors such as hTLR-2, hTLR-4 or hTLR-5.

24. The immunogen construct according to any one of the preceding claims, wherein the targeting unit comprises or consists of a soluble CD40 ligand, preferably a human soluble CD40 ligand, CCL4 and its isoforms, preferably human CCL4 and its isoforms, CCL5, preferably human CCL5, CCL19, preferably human CCL19, CCL20, preferably human CCL20, CCL21, preferably human CCL21, macrophage inflammatory protein alpha, including its isoforms such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, preferably human XCL1, XCL2, preferably human XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CO40, preferably antibodies to human CD40, anti-TLR-2, preferably antibodies to human TLR-2, anti-TLR-4, preferably antibodies to human TLR-4, anti-TLR-5, preferably,antibodies to human TLR-5 or anti-CLEC9A, preferably antibodies to human CLEC9A., 25. An immunogenic construct according to any of the preceding claims, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

26. An immunogenic construct according to any one of the preceding claims, wherein the multimerization unit is selected from the group consisting of a dimerization unit, a trimerization unit and a tetramerization unit, and wherein said multimerization unit optionally comprises a hinge region that has the ability to form one or more covalent bonds.

27. The immunogenic construct of claim 26, wherein the multimerization unit is a trimerization unit, such as a human collagen-based trimerization unit selected from the group consisting of a trimerization domain derived from human collagen XVIII and a trimerization domain derived from human collagen XV, or the C-terminal domain of bacteriophage T4 fibritin.

28. The immunogenic construct of claim 26, wherein the multimerization unit is a tetramerization unit that is a domain derived from p53.

29. An immunogenic construct according to any one of the preceding claims, wherein the multimerization unit comprises a hinge region that has the ability to form one or more covalent bonds, preferably a hinge region derived from Ig, for example derived from human Ig, for example derived from hIgG1 or hIgG2, or hIgG3, or from hIgM.

30. An immunogenic construct according to any one of claims 26 and 29, wherein the multimerization unit is a dimerization unit, and said dimerization unit further comprises another domain that facilitates dimerization.

31. The immunogenic construct of claim 30, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain, more preferably a carboxyl-terminal C-domain derived from IgG, preferably from IgG3, more preferably from hIgG3.

32. The immunogenic construct according to any one of claims 30 and 31, wherein the dimerization unit further comprises a dimerization unit linker, for example a glycine and serine rich linker, for example GGGSSGGGSG, preferably, wherein the dimerization unit linker connects the hinge region and another domain that facilitates dimerization.

33. The immunogenic construct according to any one of claims 30-32, wherein the dimerization unit comprises a hinge exon h1 and a hinge exon h4, a linker of the dimerization unit and the CH3 domain of human IgG3.

34. An immunogenic construct according to any of the preceding paragraphs, which is a polynucleotide (i).

35. The immunogenic construct according to claim 34, wherein the polynucleotide comprises a nucleotide sequence that additionally encodes a signal peptide.

36. The immunogenic construct according to claim 35, wherein the signal peptide is a natural leader sequence of a targeting unit.

37. An immunogenic construct according to any one of claims 34-36, wherein the polynucleotide is DNA or RNA.

38. An immunogenic construct according to any of the preceding claims, which is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 171, 172, 173, 174, 175, 176 and 177, or a dimeric protein consisting of two such polypeptides, preferably a polypeptide having the amino acid sequence of SEQ ID NO: 177, or a dimeric protein consisting of two such polypeptides.

39. An immunogenic construct according to any one of claims 1-37, which is a polynucleotide containing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 178, 179, 180, 181, 182, 183 and 184, preferably containing a nucleotide sequence encoding a polypeptide that has the amino acid sequence of SEQ ID NO:

184.

40. An immunogenic construct according to any one of claims 1-37, which is a polynucleotide containing the nucleotide sequence SEQ ID NO:

185. 41: An immunogenic construct according to any one of claims 1-37, which is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 161, 162, 163, 164, 165, 166 and 167.

42. An immunogenic construct according to any one of claims 1-37, which is a polynucleotide containing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 161, 162, 163, 164, 165, 166 and 167.

43. A polynucleotide according to any of the preceding paragraphs.

44. A vector containing a polynucleotide according to claim 43.

45. The vector of claim 44, wherein the vector is a polycistronic vector that comprises: a) the polynucleotide of claim 43 and b) one or more nucleotide sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the co-expression of the polypeptide encoded by the polynucleotide and one or more immunostimulatory compounds as separate molecules.

46. ​​The vector of any one of claims 44 and 45, wherein the vector is selected from the group consisting of a DNA vector and an RNA vector, preferably selected from the group consisting of a DNA plasmid and a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus and Sendai virus, or, preferably, selected from the group consisting of an RNA plasmid and an RNA viral vector, such as a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, Moloney murine leukemia virus and a rhabdovirus.

47. A method for obtaining a vector according to any of paragraphs 44-46, including: a) in vitro transfection or transduction of cells with a vector; b) culturing the said cells; c) optionally, lysing the cells to release the vector from the cells; and d) extraction and, optionally, purification of the vector.

48. A host cell comprising a polynucleotide according to claim 43 or a vector according to any one of claims 44-46.

49. A polypeptide encoded by a nucleic acid according to any one of claims 1-42.

50. A multimeric protein consisting of several polypeptides according to claim 49.

51. The multimeric protein of claim 50, which is a dimeric protein consisting of two polypeptides of claim 49.

52. A method for producing a polypeptide according to claim 49, comprising: a) transfection or transduction of cells with a polynucleotide according to claim 43 or a vector containing such a polynucleotide; b) cell cultivation; c) isolating the polypeptide from the cells; and d) optionally, purifying the isolated polypeptide.

53. A method for producing a multimeric protein according to any one of claims 50, 51, comprising: a) transfection or transduction of cells with a polynucleotide according to claim 43 or a vector containing such a polynucleotide; b) cell cultivation; c) isolation of the multimeric protein from the cells; and d) optionally, purifying the isolated multimeric protein.

54. An immunogenic construct according to any one of claims 1-42, or a polynucleotide according to claim 43, or a vector according to any one of claims 44-46, or a polypeptide according to claim 49, or a multimeric protein according to any one of claims 50, 51 for use as a medicinal product.

55. A pharmaceutical composition or vaccine comprising an immunogen construct according to any one of claims 1-42, or a polynucleotide according to claim 43, or a vector according to any one of claims 43-46, or a polypeptide according to claim 49, or a multimeric protein according to any one of claims 50, 51 and a pharmaceutically acceptable carrier.

56. The pharmaceutical composition or vaccine of claim 55 for use in the treatment of a disease caused by SARS-CoV-2, wherein said treatment is a prophylactic treatment or a therapeutic treatment.

57. A method for treating a disease caused by SARS-CoV-2, comprising administering to a subject who has previously been vaccinated with a vaccine against SARS-CoV-2, a pharmaceutical composition or vaccine according to any one of paragraphs 55, 56, wherein said method is a prophylactic treatment or a therapeutic treatment.