MERS CORONAVIRUS VACCINE

Abstract

The present invention relates to mRNAs suitable for use as mRNA-based vaccines against infections with MERS coronaviruses. Additionally, the present invention relates to a composition comprising the mRNAs and the use of the mRNAs or the composition for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the prophylaxis or treatment of MERS coronavirus infections. The present invention further describes a method of treatment or prophylaxis of infections with MERS coronavirus using the mRNA sequences.

Claims

1. mRNA comprising at least one coding region, encoding at least one antigenic peptide or protein derived from a Middle East respiratory syndrome coronavirus (MERS coronavirus/MERS-CoV).

2. The mRNA according to claim 1 suitable for use as a vaccine.

3. The mRNA according to claim 1 or 2, wherein the at least one antigenic peptide or protein comprises a spike protein (S), a spike S1 fragment (S1), an envelope protein (E), a membrane protein (M) or a nucleocapsid protein (N) of a MERS coronavirus, or a fragment or variant of any one of these proteins.

4. The mRNA according to any one of claims 1 to 3, wherein the at least one antigenic peptide or protein comprises a spike protein (S) of a MERS coronavirus, or a fragment or variant thereof.

5. The mRNA according to claim 4, wherein the antigenic peptide or protein comprises the spike S1 fragment (S1) (S1 subunit of a spike protein (S)) of a MERS coronavirus, or a fragment or variant thereof.

6. The mRNA according to claim 5, wherein the antigenic peptide or protein comprises the receptor binding domain (RBD) of the S1 subunit of a spike protein (S) of a MERS coronavirus, or a fragment or variant thereof.

7. The mRNA according to any one of claims 1 to 6, wherein the at least one coding region encodes at least one antigenic peptide or protein comprising a stabilized spike protein (S_stabilized) of a MERS coronavirus, wherein the respective amino acid sequences according to SEQ ID NOs: 1-101 are modified by changing the amino acid residue at position 1060 to Proline and the amino acid residue at position 1061 to Proline, or a fragment or variant thereof.

8. The mRNA according to any one of claims 1 to 7, wherein the at least one coding region encodes an antigenic peptide or protein comprising a spike protein (S or S_stabilized) of a MERS coronavirus, or a fragment or variant thereof, which comprises an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NO: 1 to 101, or a fragment or variant of any one of these amino acid sequences.

9. The mRNA according to claim 8, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 153 to 253.

10. The mRNA according to any one of claims 1 to 9, wherein the at least one coding region encodes an antigenic peptide or protein comprising a spike S1 fragment (S1) of a MERS coronavirus, or a fragment or variant thereof, which comprises an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NO: 1448-1548, or a fragment or variant of any one of these amino acid sequences.

11. The mRNA according to claim 10, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 1549 to 1649.

12. The mRNA according to any one of claims 1 to 11, wherein the at least one coding region encodes an antigenic peptide or protein comprising an envelope protein (E) of a MERS coronavirus, or a fragment or variant thereof, which comprises an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NO: 102 to 108, or a fragment or variant of any one of these amino acid sequences.

13. The mRNA according to claim 12, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 254 to 260.

14. The mRNA according to any one of claims 1 to 13, wherein the at least one coding region encodes an antigenic peptide or protein comprising a membrane protein (M) of a MERS coronavirus, or a fragment or variant thereof, which comprises an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NO: 109 to 124, or a fragment or variant of any one of these amino acid sequences.

15. The mRNA according to claim 14, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 261 to 276.

16. The mRNA according to any one of claims 1 to 15, wherein the at least one coding region encodes an antigenic peptide or protein comprising a nucleocapsid protein (N) of a MERS coronavirus, or a fragment or variant thereof, which comprises an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NO: 125 to 152, or a fragment or variant of any one of these amino acid sequences.

17. The mRNA according to claim 16, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 277 to 304.

18. The mRNA according to any one of claims 1 to 17, wherein the mRNA is an artificial mRNA.

19. The mRNA according to any one of claims 1 to 18, wherein the mRNA is a modified mRNA, preferably a stabilized mRNA.

20. The mRNA according to any one of claims 1 to 19, wherein the G/C content of the coding region of the mRNA is increased compared to the G/C content of the corresponding coding sequence of the wild type mRNA, or wherein the C content of the coding region of the mRNA is increased compared to the C content of the corresponding coding sequence of the wild type mRNA, or wherein the codon usage in the coding region of the mRNA is adapted to the human codon usage, or wherein the codon adaptation index (CAI) is increased or maximised in the coding region of the mRNA, wherein the amino acid sequence encoded by the mRNA is preferably not being modified compared to the amino acid sequence encoded by the wild type mRNA.

21. The mRNA according to any one of claims 1 to 20, wherein the at least one coding region encodes an antigenic peptide or protein comprising a spike protein (S or S_stabilized) of a MERS coronavirus, or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence according to any one of SEQ ID NO: 305 to 405 or 2365, 457 to 557, 609 to 709, 761 to 861, 913 to 1013, 1065 to 1165 or 1217 to 1317, or a fragment or variant of any one of these RNA sequences.

22. The mRNA according to claim 21, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 305 to 405 or 2365, 457 to 557, 609 to 709, 761 to 861, 913 to 1013, 1065 to 1165 or 1217 to 1317.

23. The mRNA according to any one of claims 1 to 22, wherein the at least one coding region encodes an antigenic peptide or protein comprising a spike 51 fragment (51) of a MERS coronavirus, or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence according to any one of SEQ ID NO: 1650 to 1750 or 2366, 1751 to 1851, 1852 to 1952, 1953 to 2053, 2054 to 2154, 2155 to 2255, 2256 to 2356, or a fragment or variant of any one of these RNA sequences.

24. The mRNA according to claim 23, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 1650 to 1750 or 2366, 1751 to 1851, 1852 to 1952, 1953 to 2053, 2054 to 2154, 2155 to 2255, 2256 to 2356.

25. The mRNA according to any one of claims 1 to 24, wherein the at least one coding region encodes an antigenic peptide or protein comprising envelope protein (E) of a MERS coronavirus, or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence according to any one of SEQ ID NO: 406 to 412, 558 to 564, 710 to 716, 862 to 868, 1014 to 1020, 1166 to 1172 or 1318 to 1324, or a fragment or variant of any one of these RNA sequences.

26. The mRNA according to claim 25, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 406 to 412, 558 to 564, 710 to 716, 862 to 868, 1014 to 1020, 1166 to 1172 or 1318 to 1324.

27. The mRNA according to any one of claims 1 to 26, wherein the at least one coding region encodes an antigenic peptide or protein comprising a membrane protein (M) of a MERS coronavirus, or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence according to any one of SEQ ID NO: 413 to 428, 565 to 580, 717 to 732, 869 to 884, 1021 to 1036, 1173 to 1188 or 1325 to 1340, or a fragment or variant of any one of these RNA sequences.

28. The mRNA according to claim 27, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 413 to 428, 565 to 580, 717 to 732, 869 to 884, 1021 to 1036, 1173 to 1188 or 1325 to 1340.

29. The mRNA according to any one of claims 1 to 28, wherein the at least one coding region encodes an antigenic peptide or protein comprising a nucleocapsid protein (N) of a MERS coronavirus, or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence according to any one of SEQ ID NO: 429 to 456, 581 to 608, 733 to 760, 885 to 912, 1037 to 1064, 1189 to 1216 or 1341 to 1368, or a fragment or variant of any one of these RNA sequences.

30. The mRNA according to claim 29, wherein the at least one coding region comprises an RNA sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with any one of the nucleic acid sequences according to SEQ ID NO: 429 to 456, 581 to 608, 733 to 760, 885 to 912, 1037 to 1064, 1189 to 1216 or 1341 to 1368.

31. The mRNA according to any of claims 1 to 30 comprising additionally a) a 5′-cap structure, b) a poly(A) sequence, and c) optionally a poly (C) sequence.

32. The mRNA according to claim 31, wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.

33. The mRNA according to any of claims 1 to 32 comprising additionally at least one histone stem-loop.

34. The mRNA according to claim 33, wherein the at least one histone stem-loop comprises a nucleic acid sequence according to the following formulae (I) or (II): formula (I) (stem-loop sequence without stem bordering elements): ##STR00011## formula (II) (stem-loop sequence with stem bordering elements): ##STR00012## wherein: stem1 or stem2 bordering elements N.sub.1-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof; stem1 [N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine; loop sequence [No-4(U/T)N.sub.0-4] is located between elements stem1 and stem2, and is a consecutive sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides; wherein each N.sub.0-4 is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein U/T represents uridine, or optionally thymidine; stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially reverse complementary with element stem1, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleotide guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2.

35. The mRNA according to claim 34, wherein the at least one histone stem-loop comprises a nucleic acid sequence according to the following formulae (Ia) or (IIa): formula (Ia) (stem-loop sequence without stem bordering elements): ##STR00013## formula (IIa) (stem-loop sequence with stem bordering elements): ##STR00014##

36. The mRNA according to any one of claims 33 to 35, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NO: 1387 and most preferably a RNA sequence according to SEQ ID NO: 1388.

37. The mRNA according to any one of claims 1 to 36 comprising a poly(A) sequence, preferably comprising 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, and/or a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.

38. The mRNA according to any one of claims 1 to 37 comprising, preferably in 5′ to 3′ direction, the following elements: a) a 5′-cap structure, preferably m7GpppN, b) at least one coding region encoding at least one antigenic peptide or protein comprising a protein of MERS coronavirus, or fragment or variant thereof, c) a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, d) optionally a poly(C) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and e) optionally a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 1388.

39. The mRNA according to any one of claims 1 to 38 comprising additionally a 3′-UTR element.

40. The mRNA according to claim 39, wherein the at least one 3′-UTR element comprises or consists of a sequence element according to SEQ ID NO: 1444, 1445, 1446 or 1447 or a homolog, a fragment or a variant thereof.

41. The mRNA according to claim 39 or 40, wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′-UTR of a gene providing a stable mRNA or from a homolog, a fragment or a variant thereof.

42. The mRNA according to claim 41, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence derived from a 3′-UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof.

43. The mRNA according to any one of claims 39 to 42, wherein the 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of an α-globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 1379, a homolog, a fragment, or a variant thereof.

44. The mRNA according to any of claims 1 to 43 comprising, preferably in 5′- to 3′-direction: a) a 5′-cap structure, preferably m7GpppN; b) at least one coding region encoding at least one antigenic peptide or protein comprising a protein of a MERS coronavirus, or a fragment or variant thereof, c) a 3′-UTR element comprising or consisting of a nucleic acid sequence which is derived from an alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 1379, a homolog, a fragment or a variant thereof; d) optionally, a poly(A) sequence, preferably comprising 64 adenosines; e) optionally, a poly(C) sequence, preferably comprising 30 cytosines; and f) optionally, a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 1388

45. The mRNA according to claim 1 to 44, wherein the RNA comprises or consists of an RNA sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2367, 2368, 2369, 2370, 2371, 2372 or a fragment or variant of any of these sequences.

46. The mRNA according to claim 39, wherein the at least one 3′-UTR element comprises a nucleic acid sequence, which is derived from the 3′-UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3′-UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3′-UTR of a human albumin gene or from a variant thereof, even more preferably from the 3′-UTR of the human albumin gene according to Genebank Accession number NM_000477.5, or from a fragment or variant thereof.

47. The mRNA according to claim 46, wherein the 3-′UTR element is derived from a nucleic acid sequence according to SEQ ID NO: 1383 or 1385, preferably from a corresponding RNA sequence, or a homolog, a fragment or a variant thereof

48. The mRNA according to claim 1 to 47, wherein the mRNA comprises a 5′-UTR element.

49. The mRNA according to 48, wherein the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a TOP gene preferably from a corresponding RNA sequence, a homolog, a fragment, or a variant thereof, preferably lacking the 5′TOP motif.

50. The mRNA according to claim 49, wherein the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a TOP gene encoding a ribosomal protein, preferably from a corresponding RNA sequence or from a homolog, a fragment or a variant thereof, preferably lacking the 5′TOP motif.

51. The mRNA according to claim 50, wherein the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a homolog, a fragment or variant thereof, preferably lacking the 5′TOP motif and more preferably comprising or consisting of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 1369, or a homolog, a fragment or variant thereof.

52. The mRNA according to claim 51, wherein the 5′-UTR element which is derived from a 5′-UTR of a TOP gene comprises or consists of the nucleic acid sequence according to SEQ ID NO: 1369 or the corresponding RNA sequence according to SEQ ID NO: 1370, or a homolog, a fragment or variant thereof.

53. The mRNA according to any one of claims 1 to 52 comprising, preferably in 5′- to 3′-direction: a) a 5′-cap structure, preferably m7GpppN; b) a 5′-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a TOP gene, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 1369 or 1370, a homolog, a fragment or a variant thereof; c) at least one coding region encoding at least one antigenic peptide or protein comprising a protein of a MERS coronavirus, or a fragment or variant thereof, d) a 3′-UTR element comprising or consisting of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 1383 or 1385, a homolog, a fragment or a variant thereof; e) a poly(A) sequence preferably comprising 64 adenosines; f) optionally a poly(C) sequence, preferably comprising 30 cytosines; and g) optionally a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 1387.

54. The mRNA according to any one of claims 1 to 38 and 46 to 53, wherein the RNA comprises or consists of an RNA sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2373, 2374, 2375, 2376, 2377, 2378 or a fragment or variant of any of these sequences.

55. Composition comprising at least one mRNA according to any one of claims 1 to 54 and a pharmaceutically acceptable carrier.

56. The composition according to claim 55, wherein the at least one mRNA is complexed with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g. protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids.

57. The composition according to claim 56, wherein the N/P ratio of the at least one mRNA to the one or more cationic or polycationic compounds is in the range of about 0.1 to 20, including a range of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1.5

58. The composition according to claim 56 or 57, wherein the at least one mRNA is complexed with one or more cationic or polycationic compounds in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNA to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9.

59. The composition according to any one of claims 55 to 58 comprising the at least one mRNA, which is complexed with one or more cationic or polycationic compounds, and at least one free mRNA.

60. The composition according to claim 59, wherein the at least one complexed mRNA is identical to the at least one free mRNA.

61. The composition according to claim 59 or 60, wherein the molar ratio of the complexed mRNA to the free mRNA is selected from a molar ratio of about 0.001:1 to about 1:0.001, including a ratio of about 1:1.

62. The composition according to any one of claims 59 to 61, wherein the ratio of the complexed mRNA to the (free) mRNA is selected from a range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of the complexed mRNA to the free (m)RNA is selected from a ratio of 1:1 (w/w).

63. The composition according to any one of claims 55 to 62, wherein the mRNA is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

64. The composition according to any one of claims 55 to 63, wherein the composition comprises at least one adjuvant.

65. The composition according to claim 64, wherein the adjuvant is selected from the group consisting of: cationic or polycationic compounds, comprising cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones, cationic polysaccharides, including chitosan, polybrene, cationic polymers, including polyethyleneimine (PEI), cationic lipids, including DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, including modified polyaminoacids, including β-aminoacid-polymers or reversed polyamides, modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, including pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified Amidoamines including pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE), including diamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers, including polypropylamine dendrimers or pAMAM based dendrimers, polyimine(s), including PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar backbone based polymers, including cyclodextrin based polymers, dextran based polymers, Chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks selected from a cationic polymer as mentioned before, and of one or more hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); or cationic or polycationic proteins or peptides, selected from the following proteins or peptides having the following total formula (III): (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x, wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except from Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide; or nucleic acids having the formula (V): G.sub.lX.sub.mG.sub.n, wherein: G is guanosine (guanine), uridine (uracil) or an analogue of guanosine (guanine) or uridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides); l is an integer from 1 to 40, wherein, when l=1 G is guanosine (guanine) or an analogue thereof, when l>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3× is uridine (uracil) or an analogue thereof, when m>3 at least 3 successive uridines (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1 G is guanosine (guanine) or an analogue thereof, when n>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; or nucleic acids having the formula (VI): C.sub.lX.sub.mC.sub.n, wherein: C is cytidine (cytosine), uridine (uracil) or an analogue of cytidine (cytosine) or uridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides); l is an integer from 1 to 40, wherein when l=1 C is cytidine (cytosine) or an analogue thereof, when l>1 at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3× is uridine (uracil) or an analogue thereof, when m>3 at least 3 successive uridine (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1 C is cytidine (cytosine) or an analogue thereof, when n>1 at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or an analogue thereof; or adjuvants selected from the group consisting of: TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)—N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)—N-acetylmuramyl-L-alanyl-D-isoglutamine); imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-1beta; interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59™; (squalene-water emulsion); MONTANIDE ISA51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5 c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide.

66. The composition according to any one of claims 55 to 65, wherein the composition comprises a plurality or more than one of the mRNAs each defined in any one of claims 1 to 48.

67. The composition according to claim 66, wherein each of the mRNAs encodes at least one different antigenic peptide or protein comprising at least one protein of the same MERS coronavirus, or a fragment or variant thereof.

68. The composition according to claim 67, wherein each of the mRNAs encodes at least one different antigenic peptide or protein comprising different proteins of the same MERS coronavirus, or a fragment or variant thereof.

69. The composition according to claim 68, wherein each of the mRNAs encodes at least one different antigenic peptide or protein comprising different proteins of different MERS coronaviruses, or a fragment or variant thereof.

70. The composition according to any one of claims 66 to 69, wherein each of the mRNAs encodes at least one antigenic peptide or protein comprising a spike protein (S or S_stabilized), a spike 51 fragment (51), an envelope protein (E), a membrane protein (M) or a nucleocapsid protein (N) of a MERS-coronavirus, or a fragment or variant of any one of these proteins.

71. The composition according to claim 70, wherein at least one antigenic peptide or protein comprising a spike protein (S or S_stabilized), a spike 51 fragment (51), an envelope protein (E), a membrane protein (M) or a nucleocapsid protein (N), or a fragment or variant thereof, of 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 100 different MERS coronaviruses are encoded by the plurality of mRNAs.

72. The composition according to claim 70 or 71, wherein the composition comprises at least one mRNA encoding at least one antigenic peptide or protein comprising a spike protein (S or S_stabilized) or a spike S1 fragment (S1) of MERS coronavirus, or a fragment or variant thereof, and at least one mRNA encoding at least one antigenic peptide or protein comprising a nucleocapsid protein (N), a membrane protein (M) or an envelope protein (E) of the same MERS coronavirus, or a fragment or variant of any one of these proteins.

73. Vaccine comprising the mRNA according to any one of claims 1 to 54 and a pharmaceutically acceptable carrier, or comprising the composition according to any one of claims 55 to 72.

74. The vaccine according to claim 73, wherein the mRNA according to any one of claims 1 to 54 or the composition according to any one of claims 55 to 72 elicits an adaptive immune response.

75. The vaccine according to claim 73 or 74 comprising an adjuvant.

76. Kit or kit of parts comprising the components of the mRNA as defined according to any one of claims 1 to 54, the composition as defined according to any one of claims 55 to 72, the vaccine as defined according to any one of claims 73 to 75 and optionally technical instructions with information on the administration and dosage of the components.

77. mRNA as defined according to any one of claims 1 to 54, the composition as defined according to any one of claims 55 to 72, the vaccine as defined according to any one of claims 73 to 75, the kit or kit of parts as defined according to claim 76 for use as a medicament.

78. mRNA as defined according to any one of claims 1 to 54, the composition as defined according to any one of claims 55 to 72, the vaccine as defined according to any one of claims 73 to 75, the kit or kit of parts as defined according to claim 76 for use in the treatment or prophylaxis of MERS coronavirus infections or disorders related thereto.

79. mRNA, composition, pharmaceutical composition and kit or kit of parts for use according to claim 77 or 78, wherein the mRNA, the composition, the vaccine or the kit or kit of parts is administered by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

80. mRNA, composition, vaccine and kit or kit of parts for use according to claim 79, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

81. A method of treatment or prophylaxis of MERS coronavirus infections comprising the steps: a) providing the mRNA as defined according to any one of claims 1 to 54, the composition as defined according to any one of claims 55 to 72, the vaccine as defined according to any one of claims 73 to 75, the kit or kit of parts as defined according to claim 76; b) applying or administering the mRNA, the composition, the vaccine or the kit or kit of parts to a tissue or an organism.

82. The method according to claim 81, wherein the mRNA, the composition, the vaccine or the kit or kit of parts is administered to the tissue or to the organism by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

83. The method according to claim 82, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0569] FIG. 1: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in cells after transfection of the mRNA. Shown are the results of a FACS analysis. Panels A and C show cell surface staining; Panels B and D show intracellular staining. Further details are provided in Example 2.

[0570] FIG. 2: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in cells after transfection of the mRNA. Shown are the results of a western blot analysis. Further details are provided in Example 3.

[0571] FIG. 3: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in vivo and that humoral immune responses are induced in mice. Shown are the results of an ELISA analysis. Panel A shows IgG1 endpoint titers; Panel B shows IgG2 endpoint titers. Further details are provided in Example 4.

[0572] FIG. 4: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins induces specific humoral immune responses after immunization in mice. Further details are provided in Example 5.

EXAMPLES

[0573] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of mRNA Constructs for In Vitro and In Vivo Experiments

[0574] For the present examples, DNA sequences encoding MERS coronavirus/MERS-CoV antigenic proteins were prepared and used for subsequent RNA in vitro transcription reactions. DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithms that increase the GC content of the respective coding sequence. Moreover, sequences were introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 1” in Table X1). Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L45′-UTR ribosomal 5′TOP UTR and 3′-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 2” in Table X1). The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

[0575] RNA in vitro transcription on linearized pDNA:

[0576] The DNA plasmids prepared according to paragraph 1 were enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. RNA production is performed under current good manufacturing practice according to WO2016180430. The obtained mRNAs were purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008077592) and used for in vitro and in vivo experiments.

[0577] RNA constructs used in the present Examples are provided in Table X1. Therein, the respective antigen name is indicated, the corresponding protein SEQ ID NO and the mRNA SEQ ID NO. Three different Spike constructs were tested (S=full length; S(V1060P,K1061P)=mutant comprising two consecutive proline residues at the beginning of the central helix for retaining S protein in the prototypical prefusion conformation; S1) comprising either the endogenous signal peptide (R1-R3) or a heterologous signal peptide (HsIgE; R4-R6).

TABLE-US-00011 TABLE X1 mRNA constructs of the Example section (Example 1): SEQ ID NO of SEQ ID NO: encoded mRNA Name antigen design 2 RNA ID S  16 2377 R1 S(V1060P, K1061P) 2357 2373 R2 S1 1463 2378 R3 HsIgE(1-18)_S(18-1353) 2358 2374 R4 HsIgE(1-18)_S(18-1353, 2359 2375 R5 V1060P, K1061P) HsIgE(1-18)_S(18-747) 2360 2376 R6

Example 2: Expression of MERS Coronavirus/MERS-CoV Proteins in HeLa Cells and Analysis by FACS

[0578] The results of the present Example show that mRNA encoding MERS coronavirus/MERS-CoV proteins are translated in cells after transfection of the mRNA.

[0579] To determine in vitro protein expression of the mRNA constructs, HeLa cells were transiently transfected with mRNA encoding MERS coronavirus/MERS-CoV antigens and stained using a suitable anti-S protein antibodies (raised in mouse), counterstained with a FITC-coupled secondary antibody (F5262 from Sigma). HeLa cells were seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. HeLa cells were transfected with 1 μg and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs prepared according to Example 1 and listed in Table X1 were used in the experiment (SEQ ID NOs: 2373-2378; RNA ID: R1-R6), including a negative control (water for injection). 24 hours post transfection, HeLa cells are stained with suitable anti anti-NiV or anti-HeV antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.). The results are shown in FIG. 1.

[0580] Results:

[0581] The results show that the used constructs led to a detectable protein expression at the cell surface for full length S construct (R1 and R4) and the stabilized S construct (R2 and R5). Moreover, a detectable intracellular protein expression for full length S construct (R1 and R4), the stabilized S construct (R2 and R5), and the S1 construct (R3 and R6) was shown. The results exemplify that the inventive mRNA encoding S proteins is translated in cells and that alternative RNA constructs as described in the present invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 3: Expression Analysis of MERS Coronavirus/MERS-CoV Proteins Using Western Blot

[0582] The results of the present Example shows that mRNA encoding Nipah virus G protein and Hendra virus G protein are expressed in HeLa cells after transfection.

[0583] For the analysis of MERS coronavirus/MERS-CoV, HeLa cells were transfected with unformulated mRNA (wfi as negative control) using Lipofectamine as the transfection agent. The mRNA constructs prepared according to Example 1 and listed in Table X1 were used in the experiment (SEQ ID NOs: 2373-2378; RNA ID: R1-R6), including a negative control (water for injection) and a positive control (S protein). Post transfection, HeLa cells were detached by trypsin-free/EDTA buffer, harvested, and cell lysates were prepared. Cell lysates were subjected to SDS-PAGE followed by western blot detection. Western Blot analysis was performed using an anti-S protein antibody used in combination with a suitable secondary antibody. The presence of αβ-tubulin was analyzed (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted). MERS S protein was used as a positive control.

[0584] Results:

[0585] As shown in FIG. 2, the mRNA encoding MERS coronavirus/MERS-CoV S proteins is expressed in HeLa cell lysates.

[0586] The results exemplify that the inventive mRNA encoding MERS coronavirus/MERS-CoV S proteins is translated in cells and that alternative mRNA as described in the present invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 4: Vaccination of Mice with mRNA Encoding MERS Coronavirus/MERS-CoV Antigens and ELISA Analysis

[0587] The results of the present Example shows that mRNA encoding MERS coronavirus/MERS-CoV antigens are expressed in mice after intradermal injection. In addition, the expressed antigens provided by the inventive mRNA induces humoral immune responses after immunization in mice.

[0588] Preparation of protamine complexed mRNA:

[0589] Nipah virus mRNA construct (SEQ ID NO: 1353) was prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA was complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

[0590] Immunization:

[0591] Female BALB/c mice (6-8 weeks old) were injected intradermally (i.d.) with mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table X2. As a negative control, one group of mice was vaccinated with buffer (ringer lactate). All animals were vaccinated on day 0, 21 and 42. Blood samples were collected on day 21 (post prime) and 35 (post boost) for the determination of antibody titers (ELISA).

TABLE-US-00012 TABLE X2 Vaccination regimen (Example 4): Group Composition Dose Route Volume 1 Full length S 80 μg id.; 2 × 50 μl SEQ ID NO: 2377; RNA ID: R1 back of the Protamine formulated animal 2 HsIgE(1-18)_S(18-747) 80 μg i.d.; 2 × 50 μl SEQ ID NO: 2360; RNA ID: R6 back of the Protamine formulated animal 3 100% RiLa — id.; 2 × 50 μl Control back of the animal

[0592] Determination of IgG1 and IgG2 antibody titers using ELISA:

[0593] Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the MERS coronavirus/MERS-CoV antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. IgG1 and IgG2 titers directed against MERS coronavirus/MERS-CoV antigens were measured by ELISA on day 21 (post prime vaccination) and 35 (post boost vaccination).

[0594] Results:

[0595] As shown in FIG. 3, the mRNA encoding MERS coronavirus/MERS-CoV antigens are expressed in mice after intradermal injection. In addition, the expressed antigens provided by the inventive mRNA induces humoral immune responses after immunization in mice. In detail, boost vaccination induces clear ELISA responses in 4/6 animals in R1 (full length S) and 6/6 animals in R6 ((HsIgE(1-18)_S(18-747)). The results exemplify that the inventive mRNA encoding MERS coronavirus/MERS-CoV S proteins is translated in vivo and induces humoral immune responses in mice. Notably, alternative mRNA constructs as described in the present invention may also be translated in vivo and induce humoral immune responses in mice, which is a prerequisite for an effective mRNA-based MERS coronavirus/MERS-CoV vaccine.

Example 5: Analysis of Antigen-Specific Humoral Immune Responses in Mice Using a Cell-Based Assay

[0596] The results of the present Example shows that mRNA encoding MERS coronavirus/MERS-CoV antigens induces antigen-specific humoral immune responses after immunization in mice.

[0597] Hela cells were transfected with 2 μg of RNA ID: R1 (SEQ ID NO: 2377) using lipofectamine. The cells were harvested 20 h post transfection, and seeded at 1×10.sup.5 per well into an 96 well plate. The cells were incubated with sera of mice vaccinated with RNA ID: R1 (SEQ ID NO: 2377) (diluted 1:50; day 35) obtained from Example 4, followed by FITC-conjugated anti-mouse IgG antibody. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

[0598] Results:

[0599] As shown in FIG. 4, the mRNA encoding MERS coronavirus/MERS-CoV S1 protein (mRNA construct R1) is expressed in mice after i.d. administration. Moreover, as antigen-specific IgGs were detected in sera of immunized mice, the results also show that the applied mRNA vaccine is suitable to induce antigen-specific humoral immune responses. The results exemplify that the inventive mRNA-based vaccine works and that similar mRNA vaccines comprising alternative mRNA constructs according to the invention may also be suitably used.

Example 6: Preparation of MERS Coronavirus/MERS-CoV Vaccine Compositions

[0600] The results of the previous Examples showed that the inventive MERS coronavirus/MERS-CoV mRNA constructs are expressed in vitro and in vivo, and that the mRNA vaccine (protamine formulated) induces specific humoral immune responses in mice after i.d. administration. For further in vivovaccination experiments, other mRNA vaccine compositions are prepared, preferably using constructs listed in Table X1. One composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs), and one composition comprises polymer-lipidoid complexed mRNA.

[0601] Preparation of LNP Encapsulated mRNA:

[0602] A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

[0603] Preparation of Polymer-Lipidoid Complexed mRNA:

[0604] 20 mg peptide (CHHHHHHRRRRHHHHHHC—NH2; SEQ ID NO: 1443) TFA salt is dissolved in 2 mL borate buffer pH 8.5 and stirred at room temperature for approximately 18 h. Then, 12.6 mg PEG-SH 5000 (Sunbright) dissolved in N-methylpyrrolidone is added to the peptide solution and filled up to 3 mL with borate buffer pH 8.5. After 18 h incubation at room temperature, the reaction mixture is purified and concentrated by centricon procedure (MWCO 10 kDa), washed against water, and lyophilized. The obtained lyophilisate is dissolved in ELGA water and the concentration of the polymer is adjusted to 10 mg/mL. The obtained polyethylene glycol/peptide polymers (HO-PEG 5000-S—(S—CHHHHHHRRRRHHHHHHC—S-)7-S-PEG 5000-OH— amino acid component: SEQ ID NO: 1443) are used for further formulation and are hereinafter referred to as PB83.

[0605] Preparation of 3-C12-OH lipidoid: First, lipidoid 3-C12 was obtained by acylation of tris(2-aminoethyl)amine with an activated lauric (C12) acid derivative, followed by reduction of the amide. Alternatively, it may be prepared by reductive amination with the corresponding aldehyde. Lipidoid 3-C12-OH was prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5. Preparation of compositions with nanoparticles of polymer-lipidoid complexed mRNA: First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of a polymer (PB83) are mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the carrier compositions are used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier and allowing an incubation period of 10 minutes at room temperature such as to enable the formation of a complex between the lipidoid, polymer and mRNA. In order to characterize the integrity of the obtained polymer-lipidoid complexed mRNA particles, RNA agarose gel shift assays are performed. In addition, size measurements are performed (gel shift assay, Zetasizer) to evaluate whether the obtained nanoparticles have a uniform size profile.

Example 7: Vaccination of Mice and Evaluation of Specific Immune Response

[0606] Female BALB/c mice are injected intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 6) with doses, application routes and vaccination schedules as indicated in Table X3. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below).

TABLE-US-00013 TABLE X3 Vaccination regimen - Nipah virus experiment (Example 7) Number Route/ Vaccination of mice Vaccine composition Volume Schedule (day) 8 5 μg RNA construct R1; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R1; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35 8 5 μg RNA construct R2; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R2; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35 8 5 μg RNA construct R3; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R3; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35 8 5 μg RNA construct R4; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R4; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35 8 5 μg RNA construct R5; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R5; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35 8 5 μg RNA construct R6; LNP formulated i. m.; 2 × 25μl 0/21/35 8 20 μg RNA construct R6; polymer-lipidoid complexed i. m.; 2 × 25μl 0/21/35

[0607] Determination of IgG1 and IgG2 antibodies by ELISA:

[0608] ELISA performed essentially as described in Example 4.

[0609] Determination of antigen-specific humoral immune responses using a cell based assay:

[0610] Cell based assay performed essentially as described in Example 5.

[0611] Intracellular cytokine staining:

[0612] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (2×10.sup.6 cells per well). The cells are stimulated with a mixture of four Nipah virus protein specific peptide epitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of a protein transport inhibitor. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

[0613] Plaque reduction neutralization test (PRNT50):

[0614] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art.

[0615] Briefly, obtained serum samples of vaccinated mice are incubated with MERS coronavirus/MERS-CoV. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 8: Clinical Development of a Nipah Virus and Hendra Virus mRNA Vaccine Composition

[0616] To demonstrate safety and efficiency of the mRNA vaccine composition(s), a clinical trial (phase I) is initiated. In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times. In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis). The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.