NOVEL YELLOW FEVER NUCLEIC ACID MOLECULES FOR VACCINATION

Abstract

The present invention is directed to an artificial nucleic acid, particularly to an artificial RNA suitable for use in treatment and/or prophylaxis of an infection with yellow fewer vims (YFV) or a disorder related to such an infection. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial RNA, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial RNA, compositions and vaccines.

Claims

1. An artificial RNA comprising a) at least one heterologous 5′ untranslated region (5′-UTR) and/or at least one heterologous 3′ untranslated region (3′-UTR); and b) at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from a Yellow fever virus prME polyprotein or a fragment or variant thereof or a Yellow fewer virus NS1 protein or a fragment or variant thereof.

2. Artificial RNA according to claim 1, wherein the at least one heterologous 3′-UTR comprises a nucleic acid sequence derived from a 3′-UTR of a gene selected from an ALB7 gene, an alpha-globin gene, a PSMB3 gene, a CASP1 gene, a COX6B1 gene, a NDUFA1 gene, or from a homolog, a fragment or a variant thereof.

3. Artificial RNA according to claim 1, wherein the at least one heterologous 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of a gene selected from a RPL32 gene, a HSD17B4 gene, a ATP5A1 gene, a NDUFA4 gene, a NOSIP gene, RPL31 gene, a SLC7A3 gene, or from a homolog, a fragment or variant of any one of these genes.

4. Artificial RNA according to any one of the preceding claims, comprising a-1. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-4. at least one 5′ UTR derived from a 5′UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′ UTR derived from a 3′UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-5. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-1. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-4. at least one 5′-UTR element derived from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR element derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-4. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or i-2. at least one 5′-UTR derived from a 5′-UTR of a RPL32 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a ALB7 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof. i-3. at least one 3′-UTR derived from a 3′-UTR of a alpha-globin gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof.

5. Artificial RNA according to claim 4 comprising UTR elements according to a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), b-4 (HSD17B4/CASP1), c-5 (ATP5A1/PSMB3), or g-4 (NOSIP/CASP1).

6. Artificial RNA according to any one of the preceding claims, wherein said 5′-UTR derived from a HSD17B4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1, 2 or a fragment or a variant thereof; said 5′-UTR derived from a ATP5A1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 5, 6 or a fragment or a variant thereof; said 5′-UTR derived from a NDUFA4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3, 4 or a fragment or a variant thereof; said 5′-UTR derived from a NOSIP gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 7, 8 or a fragment or a variant thereof; said 5′-UTR derived from a RPL31 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 9, 10 or a fragment or a variant thereof; said 5′-UTR derived from a RPL32 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13, 14 or a fragment or a variant thereof; said 5′-UTR derived from a SLC7A3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 11, 12 or a fragment or a variant thereof; said 3′-UTR derived from a PSMB3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 15, 16 or a fragment or a variant thereof; said 3′-UTR derived from a CASP1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 17, 18 or a fragment or a variant thereof; said 3′-UTR derived from a COX6B1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 21, 22 or a fragment or a variant thereof; said 3′-UTR derived from a NDUFA1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 19, 20 or a fragment or a variant thereof; said 3′-UTR derived from a ALB7 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 23, 24 or a fragment or a variant thereof; said 3′-UTR derived from a alpha-globin gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 25, 26 or a fragment or a variant thereof.

7. Artificial RNA according to any one of the preceding claims, wherein the at least one antigenic peptide or protein derived from a Yellow fever virus prME polyprotein is pr, M, E, ME, prM or prME, or a fragment or variant of any of these.

8. Artificial RNA according to any one of the preceding claims, wherein the at least one antigenic peptide or protein is prME or prME additionally comprising a C-terminal overhang comprising a fragment of YFV non-structural protein NS1 and/or an N-terminal overhang comprising a fragment of YFV capsid protein C.

9. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 120-126 or a fragment or variant of any of these sequences.

10. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence additionally encodes at least one heterologous signal sequence, preferably signal sequence derived from IgE or Japanese encephalitis virus (JEV), or preferably selected from SEQ ID NOs: 56-61, 1330-1357 or a fragment or variant of any of these sequences.

11. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence additionally encodes at least one heterologous further virus element, preferably a JEV stem sequence, preferably selected from SEQ ID NO: 110 or a fragment or variant of any of these sequences.

12. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 127-157, 1587, 1588 or a fragment or variant of any of these sequences.

13. Artificial RNA according to claims 1-6, wherein the at least one antigenic peptide or protein is NS1 or NS1 additionally comprising a C-terminal overhang comprising a fragment of YFV non-structural protein NS2A and/or an N-terminal overhang comprising a fragment of YFV envelope protein E.

14. Artificial RNA according to claims 1-6 and 13, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 2201-2204 or a fragment or variant of any of these sequences.

15. Artificial RNA according to claims 1-6 and 13-14, wherein the at least one coding sequence additionally encodes at least one heterologous signal sequence, preferably signal sequence derived from IgE or Japanese encephalitis virus (JEV), preferably selected from SEQ ID NOs: 56-61, 1330-1356 or a fragment or variant of any of these sequences.

16. Artificial RNA according to claim 15, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2005 or a fragment or variant of thereof.

17. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence is located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR.

18. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 158-527, 1589-1608, 2206-2245 or a fragment or a fragment or variant of any of these sequences.

19. Artificial RNA according to any one of the preceding claims, wherein the artificial RNA is a modified and/or stabilized artificial RNA.

20. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence is a codon modified coding sequence, wherein the amino acid sequence encoded by the at least one codon modified coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.

21. Artificial RNA according to claim 20, wherein the at least one codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.

22. Artificial RNA according to claim 20 or 21, wherein the at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 195-527, 1591-1608, 2211-2245 or a fragment or variant of any of these sequences.

23. Artificial RNA according to claim 22, wherein the at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 195-305, 1591-1596, 2211-2215 or a fragment or variant of any of these sequences.

24. Artificial RNA according to any one of the preceding claims, wherein the RNA is an mRNA, a self-replicating RNA, a circular RNA, or a replicon RNA.

25. Artificial RNA according to claim 24, wherein the RNA is an mRNA.

26. Artificial RNA according to any one of the preceding claims, which comprises a 5′-cap structure, preferably m7G, cap0, cap1 or cap2.

27. Artificial RNA according to any one of the preceding claims, which comprises at least one histone stem-loop, wherein the histone stem-loop preferably comprises a nucleic acid sequence according to SEQ ID NOs: 27, 28 or a fragment or variant thereof.

28. Artificial RNA according to any one of the preceding claims which comprises a 3′-terminal sequence element according to SEQ ID NOs: 32-51, 1323-1329 or a fragment or variant thereof.

29. Artificial RNA according to any one of the preceding claims comprising, preferably in 5′- to 3′-direction, the following elements a) to h): a) 5′-cap structure, preferably as defined in claim 26; b) optionally, 5′-UTR, preferably as defined by any one of claim 3 or 6; c) at least one coding sequence, preferably as defined by any one of claims 9 to 23; d) 3′-UTR, preferably as defined by any one of claim 2 or 6; e) optionally, a poly(A) sequence; f) optionally, a poly(C) sequence, g) optionally, a histone stem-loop, preferably as defined by any one of claim 27; h) optionally, a 3′-terminal sequence element as defined by claim 28.

30. Artificial RNA according claims 1 to 29 comprising the following elements: a) 5′-cap structure, preferably as defined in claim 26; b) a 5′-UTR and a 3′-UTR according to a-1, a-2, a-4, b-4, c-5, d-1, d-5, g-4, h-4, h-5, i-2, or i-3; c) at least one coding sequence as defined in claims 9 to 23, wherein said coding sequence is located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR. e) optionally, a poly(A) sequence f) optionally, poly(C) sequence g) optionally, histone stem-loop, preferably as defined by any one of claim 27; h) optionally, a 3′-terminal sequence element as defined by claim 28.

31. Artificial RNA according claims 1 to 30 comprising the following elements: a) 5′-cap structure, preferably as defined in claim 26; b) a 5′-UTR and a 3′-UTR according to a-1, a-4, b-4, c-5, or g-4; c) at least one coding sequence as defined in claims 9 to 23, wherein said coding sequence is located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR. d) optionally, a poly(A) sequence, e) optionally, poly(C) sequence, f) optionally, histone stem-loop, preferably as defined by any one of claim 27; g) optionally, a 3′-terminal sequence element as defined by claim 28.

32. Artificial RNA according to any one of claims 25 to 31, wherein the artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 528-1320, 1609-2200, 2246-2593 or a fragment or variant of any of these sequences.

33. Artificial RNA according to claim 32, wherein said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 528-587, 1308, 1609-1648, 2129-2134, 2141-2146, 2153-2158, 2165-2170, 2177-2182, 2189-2194, 2246-2269, 2558-2560, 2576-2578, 2564-2566, 2582-2584, 2570-2572, 2588-2590 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 1248-1307, 1320, 2089-2128, 2534-2557 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 648-707, 1310, 1689-1728, 2294-2317 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 708-767, 1311, 1729-1768, 2318-2341 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 888-947, 1314, 1849-1888, 2390-2413 or a fragment or variant of any of these sequences; or said artificial RNA comprises or consists of an RNA sequence which is identical or at least 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: 1188-1247, 1319, 2049-2088, 2135-2140, 2147-2152, 2159-2164, 2171-2176, 2183-2188, 2195-2200, 2510-2533, 2561-2563, 2579-2581, 2567-2569, 2585-2587, 2573-2575, 2591-2593 or a fragment or variant of any of these sequences.

34. A composition comprising at least one artificial RNA as defined in any one of claims 1 to 33, wherein the composition optionally comprises at least one pharmaceutically acceptable carrier.

35. Composition according to claim 34, wherein the composition comprises at least one YFV prME RNA construct, preferably selected from X-SS-prME-XX, X-SS-prME, SS-prME, SSjev(V3)-prME-XX, X-SS-prMEdelstem_TM-JEV or SSjev(V3)-prMEdelstem_TM-JEV and, in addition, at least one YFV NS1 RNA construct, preferably selected from eSS-NS1-Y, eSS-NSE and SSIgE-NS1.

36. Composition according to claim 35 or 34, wherein the at least one artificial RNA is complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound, preferably cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.

37. Composition according to claim 36, wherein the cationic or polycationic peptide is protamine.

38. Composition according to any one of claim 36 or 37 comprising the at least one artificial RNA, which is complexed with one or more cationic or polycationic compounds, preferably protamine, and at least one free artificial RNA.

39. Composition according to claim 36, wherein the cationic or polycationic peptide is selected from CHHHHHHRRRRHHHHHHC (SEQ ID NO: 55), CRRRRRRRRRRRRC (SEQ ID NO: 52), CRRRRRRRRRRRR (SEQ ID NO: 53), or WRRRRRRRRRRRRC (SEQ ID NO: 54), or a fragment or variant of any of these sequences.

40. Composition according to claim 36, wherein the cationic or polycationic polymer is a polyethylene glycol/peptide polymer selected from HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (SEQ ID NO: 55 of the peptide monomer), HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)4-S-PEG5000-0H (SEQ ID NO: 55 as peptide monomer), HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)7-S-PEG5000-0H (SEQ ID NO: 1321 as peptide monomer), or HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-0H (SEQ ID NO: 1321 as peptide monomer).

41. Composition according to claim 40, wherein the composition comprises a lipid component, preferably a lipidoid component, wherein the lipidoid component is a compound according to formula A ##STR00056##

42. Composition according to claim 41, wherein the lipidoid component is 3-C12-OH according to formula B. ##STR00057##

43. Composition according to claim 36, wherein the artificial RNA is complexed or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes, and/or nanoliposomes.

44. Composition according to claim 43, wherein the artificial RNA is complexed with one or more lipids thereby forming lipid nanoparticles (LNP).

45. Composition according to claim 44, wherein the LNP comprises a cationic lipid with the formula ##STR00058## or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L.sup.1 or L.sup.2 is each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)X—, —S—S—, —C(═O)S—, —SC(═O)—, —NR.sup.aC(═O)—, —C(═O)NR.sup.a—, —NR.sup.aC(═O)NR.sup.a—, —OC(═O)NR.sup.a— or —NR.sup.aC(═O)O—, preferably L.sup.1 or L.sup.2 is —O(C═O)— or —(C═O)O—; G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3 is C.sub.1-C.sub.24 alkylene, alkenylene, C.sub.3-C.sub.8 cycloalkylene, or C.sub.3-C.sub.8 cycloalkenylene; R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; R.sup.3 is H, OR.sup.5, CN, —C(═O)OR.sup.4, —OC(═O)R.sup.4 or —NR.sup.5C(═O)R.sup.4; R.sup.4 is C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and x is 0, 1 or 2;

46. Composition according to claim 45, wherein the cationic lipid is a compound of formula III, and wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)— or (C═O)—O—; G.sup.3 is C.sub.1-C.sub.24 alkylene or C.sub.1-C.sub.24 alkenylene; and R.sup.3 is H or OR.sup.5.

47. Composition according to any one of claims 45 to 46, wherein the cationic lipid is a compound of formula III, and wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)— or (C═O)—O—; and R.sup.1 and R.sup.2 each independently have one of the following structures: ##STR00059##

48. Composition according to any one of claims 45 to 47, wherein the cationic lipid is a compound of formula III, and wherein R.sup.3 is OH.

49. Composition according to any one of claims 45 to 48, wherein the cationic lipid is selected from structures III-1 to III-36: TABLE-US-00015 No. Structure III-1 III-2 III-3 III-4 III-5 III-6 III-7 III-8 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 III-17 III-18 III-19 III-20 III-21 III-22 III-23 III-24 III-25 III-26 III-27 III-28 III-29 III-30 III-31 III-32 III-33 III-34 III-35 III-36

50. Composition according to any one of claims 45 to 49, wherein the cationic lipid is ##STR00096##

51. Composition according to any one of claims 44 to 50, wherein the LNP additionally comprises a PEG lipid with the formula (IV): ##STR00097## wherein R.sup.8 and R.sup.9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

52. Composition according to claim 51, wherein in the PEG lipid R.sup.8 and R.sup.9 are saturated alkyl chains.

53. Composition according to claim 51 or 52, wherein the PEG lipid is ##STR00098## wherein n has a mean value ranging from 30 to 60, preferably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most preferably wherein n has a mean value of 49.

54. Composition according to any one of claims 44 to 53, wherein the LNP additionally comprises one or more neutral lipids and/or a steroid or steroid analogues.

55. Composition according to claim 54, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).

56. Composition according to claim 54 or 55 wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and wherein the molar ratio of the cationic lipid to DSPC is optionally in the range from about 2:1 to 8:1.

57. Composition according to claim 54, wherein the steroid is cholesterol, and wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range from about 2:1 to 1:1

58. Composition according to any one of claims 44 to 57, wherein the LNP essentially consists of (i) at least one cationic lipid, preferably as defined in any one of claims 47 to 50; (ii) a neutral lipid, preferably as defined in any one of claims 54 to 56; (iii) a steroid or steroid analogue, preferably as in claim 57; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, preferably as defined in any one of claims 51 to 53, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.

59. A vaccine comprising the artificial RNA as defined in any one of claims 1 to 33, or the composition as defined in any one of claims 34 to 58.

60. Vaccine according to claim 56, wherein the artificial RNA as defined in any one of claims 1 to 33 or the composition as defined in any one of claims 34 to 58 elicits an adaptive immune response.

61. Vaccine according to claim 59 or 60, wherein the vaccine further comprises a pharmaceutically acceptable carrier and optionally at least one adjuvant.

62. A Kit or kit of parts comprising the artificial RNA as defined in any one of claims 1 to 33, the composition as defined in any one of claims 34 to 58, or the vaccine as defined in any one of claims 59 to 61, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.

63. Kit or kit of parts according to claim 62 further comprising Ringer lactate solution.

64. Artificial RNA as defined in any one of claims 1 to 33, the composition as defined in any one of claims 34 to 58, the vaccine as defined in any one of claims 59 to 61, or the kit or kit of parts as defined in claims 62 to 63 for use as a medicament.

65. Artificial RNA as defined in any one of claims 1 to 33, the composition as defined in any one of claims 34 to 58, the vaccine as defined in any one of claims 59 to 61, or the kit or kit of parts as defined in claims 62 to 63 for use in the treatment or prophylaxis of an infection with Yellow fever virus, or a disorder related to such an infection.

66. A method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof the artificial RNA as defined in any one of claims 1 to 33, the composition as defined in any one of claims 34 to 58, the vaccine as defined in any one of claims 59 to 61, or the kit or kit of parts as defined in claims 62 to 63.

67. Method according to claim 66, wherein the disorder is an infection with a Yellow fever virus, or a disorder related to such an infection.

68. Method according to claim 66 or 67, wherein the subject in need is a mammalian subject or an avian subject.

69. Method according to claim 68, wherein the mammalian subject is a human subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0565] FIG. 1 shows that LNP-formulated mRNA encoding X-SS-prME-XX induces PRNT titers in non-human primates after i.m. injection. Further details are provided in Example 2.

[0566] FIG. 2 shows that LNP-formulated mRNA encoding X-SS-prME-XX induces long lasting PRNT titers in non-human primates after i.m. injection. Further details are provided in Example 2.

[0567] FIG. 3 shows that UTR combinations according to the invention increase the expression of YFV prME in vitro. Data shows the result of an ICW assay on HeLa cells. Values show % of the detected YFV signal. Values normalized to 100% according to the expression of the reference (UTR combination RPL32/ALB7), also indicated by dashed horizontal line. Water for injection (WFI) serves as a control. N=4. Further details are provided in Example 3.

[0568] FIG. 4 shows that UTR combinations according to the invention increase the expression of YFV prME in vitro. Data shows the result of an ICW assay on HDF cells. Values show % of the detected YFV signal. Values normalized to 100% according to the expression of the reference (UTR combination RPL32/ALB7), also indicated by dashed horizontal line. WFI serves as a control. N=6. Further details are provided in Example 3.

[0569] FIG. 5 shows that UTR combinations according to the invention increase the expression of YFV prME in vitro. Data shows the result of a dot-blot assay using supernatants of transfected HeLa cells. Values show % of the detected YFV signal. Values normalized to 100% according to the expression of the reference (UTR combination RPL32/ALB7), also indicated by dashed horizontal line. Further details are provided in Example 3.

[0570] FIG. 6 shows that UTR combinations according to the invention increase the expression of YFV prME in vivo. Data shows the result of a FACS-based immunoassay using supernatants of vaccinated mice.

[0571] FIG. 6A shows the result of serum samples of day 14 (1:100), FIG. 6B shows the result of serum samples of day 28 (1:150). Values show % of the detected YFV signal. Values normalized to 100% according to the reference UTR combination (RPL32/ALB7) expression (also indicated by dashed horizontal line). Further details are provided in Example 4.

[0572] FIG. 7 shows that UTR combinations according to the invention increase the expression of YFV prME in vivo. Data shows the result of a FACS-based immunoassay using supernatants of vaccinated mice.

[0573] FIG. 7A shows the result of serum samples of day 56 (1:200), FIG. 7B shows the result of serum samples of day 70 (1:500). Further details are provided in Example 4.

[0574] FIG. 8 shows that a dose of 1 ug mRNA encoding X-SS-prME-XX (LNP formulated) is sufficient to induce binding antibody titers. Further details are provided in Example 5.

[0575] FIG. 9 shows that LNP-formulated mRNA encoding X-SS-prME-XX induces PRNT titers in in mice after i.m. injection Further details are provided in Example 5.

[0576] FIG. 10 shows that Capt mRNA encoding YFV prME lead to an increased expression of YFV prME in vitro in comparison to m7G (Cap0) mRNA. Further details are provided in Example 6. (FIG. 10a: Western blot, FIG. 10b: relative signal intensity).

EXAMPLES

[0577] 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 and Compositions for In Vitro and In Vivo Experiments

[0578] The present Example provides methods of obtaining the artificial RNA of the invention as well as methods of generating a composition or a vaccine of the invention.

1.1. Preparation of DNA and mRNA Constructs:

[0579] For the present examples, DNA sequences encoding YFV prME were prepared and used for subsequent RNA in vitro transcription reactions. Said DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a G/C optimized or C-maximized sequence for stabilization. Sequences were introduced into a pUC19 derived vector to comprise stabilizing 3′-UTR sequences derived from an ALB7 gene, an alpha-globin gene, a PSMB3 gene, a CASP1 gene, a COX6B1 gene, or a NDUFA1 gene and 5′-UTR sequences derived from a RPL32 gene, a HSD17B4 gene, an ATP5A1 gene, a NDUFA4 gene, a NOSIP gene, a RPL31 gene, or a SLC7A3 gene, additionally comprising, a stretch of adenosines (64A or 75A), and, optionally, a histone-stem-loop (hSL) structure and/or, a stretch of 30 cytosines (C30) as listed in Table 3.

[0580] The obtained plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA constructs were extracted, purified, and used for subsequent RNA in vitro transcription (see section 1.2).

[0581] Alternatively, DNA plasmids prepared according to paragraph 1 are used as DNA template for PCR-based amplification. Eventually, the generated PCR products are purified and used for subsequent RNA in vitro transcription (see section 1.3).

1.2. RNA In Vitro Transcription from Plasmid DNA Templates:

[0582] DNA plasmids prepared according to paragraph 1.1 were enzymatically linearized using EcoRI or SapI and used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG, or m7G(5′)ppp(5)(2′OMeG)pG) under suitable buffer conditions. The obtained mRNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. RNA for clinical development (see Example 6) is produced under current good manufacturing practice e.g. according to WO2016/180430, implementing various quality control steps on DNA and RNA level. The generated RNA sequences/constructs are provided in Table 3 with the encoded YFV protein, the UTR elements, and the 3′-terminal end indicated therein. In addition to the information provided in Table 3, further information relating to specific mRNA construct SEQ-ID NOs may be derived from the information provided under <223> identifier provided in the ST.25 sequence listing.

[0583] Alternatively, EcoRI or SapI linearized DNA is used for DNA dependent RNA in vitro transcription using an RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m14P) or pseudouridine (Ψ) and cap analog (m7GpppG, m7G(5)ppp(5′)(2′OMeA)pG, or m7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. The obtained m14Ψ- or Ψ-modified mRNAs are purified using RP-HPLC (PureMessenger®, CureVac, Tubingen, Germany; WO2008/077592) and used for further experiments.

[0584] Some mRNA constructs are in vitro transcribed in the absence of a cap analog. The cap-structure (cap1) is added enzymatically using Capping enzymes as commonly known in the art. In short, in vitro transcribed mRNA is capped using an m7G capping kit with 2′-O-methyltransferase to obtain cap1-capped mRNA. Cap1-capped mRNA is purified using RP-HPLC (PureMessenger®, CureVac, Tubingen, Germany; WO2008/077592) and used for further experiments.

1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates:

[0585] Purified PCR amplified DNA templates prepared according to paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppGunder suitable buffer conditions. Alternatively, PCR amplified DNA is transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m14′) or pseudourinde (Ψ)) and cap analog (m7GpppG, m7G(5′)ppp(5)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. Some mRNA constructs are in vitro transcribed in the absence of a cap analog and the cap-structure (cap1) is added enzymatically using capping enzymes as commonly known in the art e.g. using an m7G capping kit with 2′-O-methyltransferase. The obtained mRNAs are purified e.g. using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments.

TABLE-US-00008 TABLE 3 mRNA constructs used in the present examples (Example 1) 5′-UTR/3′-URT; SEQ RNA ID construct UTR Design 3′-end ID NO: R2388/ X-SS-prME-XX (opt1) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1194 R2571/ globin gene (muag); i-3 R6711 R2581/ X-SS-prME-XX (opt1) RPL32/ALB7; i-2 A64-N5-C30-hSL-N5 1074 R2582/ R3911 R5351 X-SS-prME-XX (opt1) HSD17B4/PSMB3; a-1 A64-N5 1308 R5353 X-SS-prME-XX (opt1) HSD17B4/CASP1; b-4 A64-N5 1310 R5356 X-SS-prME-XX (opt1) SLC7A3/NDUFA1; d-5 A64-N5 1313 R5357 X-SS-prME-XX (opt1) ATP5A1/PSMB3; c-5 A64-N5 1311 R5360 X-SS-prME-XX (opt1) N0SIP/CASP1; g-4 A64-N5 1314 R5362 X-SS-prME-XX (opt1) RPL31/PSMB3; d-1 A64-N5 1312 R5364 X-SS-prME-XX (opt1) N0SIP/PSMB3; a-4 A64-N5 1320 R5367 X-SS-prME-XX (opt1) SLC7A3/CASP1; h-4 A64-N5 1315 R5368 X-SS-prME-XX (opt1) SLC7A3/COX6B1; h-5 A64-N5 1316 R5369 X-SS-prME-XX (opt1) NDUFA4/PSMB3; a-2 A64-N5 1309 R5372 X-SS-prME-XX (opt1) RPL32/ALB7; i-2 A64-N5 1318 R7229 X-SS-prME-XX (opt1) NDUFA4/PSMB3; a-2 A64-N5-C30-hSL-N5 594 R7230 X-SS-prME-XX (opt1) N0SIP/PSMB3; a-4 A64-N5-C30-hSL-N5 1254 R7231 X-SS-prME-XX (opt1) ATP5A1/PSMB3; c-5 A64-N5-C30-hSL-N5 714 R7232 X-SS-prME-XX (opt1) HSD17B4/CASP1; b-4 A64-N5-C30-hSL-N5 654 R7233 X-SS-prME-XX (opt1) HSD17B4/PSMB3; a-1 A64-N5-C30-hSL-N5 534 R8481/ X-SS-prME-XX (opt1) 3′-UTR of an alpha- hSL-A64-N5 2171 R8482 globin gene (muag); i-3 R8483 X-SS-prME-XX (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2190 R8484 X-SS-prME-XX (opt1) 3′-UTR of an alpha- A64-N5-hSL-N5 2183 globin gene (muag); i-3 R7253 X-SS-prME-XX (opt11) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1242 globin gene (muag); i-3 R7250 X-SS-prME-XX (opt2) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1212 globin gene (muag); i-3 R7251 X-SS-prME-XX (opt4) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1224 globin gene (muag); i-3 R7252 X-SS-prME-XX (opt6) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1236 globin gene (muag); i-3 R2387 X-SS-prME-XX (wt) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1188 globin gene (muag); i-3 R8491/ X-SS-prME (opt1) 3′-UTR of an alpha- hSL-A64-N5 2173 R8492 globin gene (muag); i-3 R8493 X-SS-prME (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2191 R2605/ SS-prME (opt1) 3′-UTR of an alpha- A64-N5-C30-hSL-N5 1195 R2606 globin gene (muag); i-3 R2607/ SS-prME (opt1) RPL32/ALB7; i-2 A64-N5-C30-hSL-N5 1075 R2608 R8488/ SSjev(V3)-prME-XX (opt1) 3′-UTR of an alpha- hSL-A64-N5 2174 R8489 globin gene (muag); i-3 R8490 SSjev(V3)-prME-XX (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2192 R8494/ X-SS-prMEdelstem_TM-JEV (opt1) 3′-UTR of an alpha- hSL-A64-N5 2175 R8495 globin gene (muag); i-3 R8496 X-SS-prMEdelstem_TM-JEV (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2193 R8497/ SSjev(V3)-prMEdelstem_TM-JEV 3′-UTR of an alpha- hSL-A64-N5 2176 R8498 (opt1) globin gene (muag); i-3 R8499 SSjev(V3)-prMEdelstem_TM-JEV HSD17B4/PSMB3; a-1 hSL-A100 2194 (opt1) R8501/ eSS-NS1-Y (opt1) 3′-UTR of an alpha- hSL-A64-N5 2579 R8502 globin gene (muag); i-3 R8503 eSS-NS1-Y (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2588 R8504/ eSS-NS1 (opt1) 3′-UTR of an alpha- hSL-A64-N5 2580 R8505 globin gene (muag); i-3 R8506 eSS-NS1 (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2589 R8507/ SSIgE-NS1 (opt1) 3′-UTR of an alpha- hSL-A64-N5 2581 R8508 globin gene (muag); i-3 R8509 SSIgE-NS1 (opt1) HSD17B4/PSMB3; a-1 hSL-A100 2590
1.4. Preparation of an LNP Formulated mRNA Composition:

[0586] Lipid nanoparticles (LNP), cationic lipids, and polymer conjugated lipids (PEG-lipid) were prepared and tested essentially according to the general procedures described in WO2015/199952, WO2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein by reference. LNP formulated mRNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Briefly, cationic lipid compound of Formula III-3, DSPC, cholesterol, and PEG-lipid of Formula IVa were solubilized in ethanol at a molar ratio (%) of approximately 50:10:38.5:1.5 or 47.5:10:40.9:1.7. LNPs comprising cationic lipid compound III-3 and PEG-lipid compound IVa were prepared at a ratio of mRNA to total Lipid of 0.03-0.04 w/w. The mRNA was diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15m1/min. The ethanol was then removed and the external buffer replaced with a PBS buffer comprising Sucrose by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 um pore sterile filter and the LNP-formulated mRNA composition was adjusted to about 1 mg/ml total mRNA. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is essentially similar. The obtained LNP-formulated mRNA composition (1 mg/ml total mRNA) was diluted to the desired target concentration using Saline before in vivo application.

1.5. Preparation of a Protamine Complexed mRNA Composition:

[0587] mRNA constructs are complexed with protamine prior to use in in vivo immunization experiments. The mRNA formulation 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 is added, and the final concentration is adjusted with Ringer's lactate solution.

1.6. Preparation of Polymer-Lipidoid Complexed mRNA Composition:

[0588] mRNA constructs are complexed with a polymer-lipidoid prior to use in in vivo immunization experiments. 20 mg peptide (CHHHHHHRRRRHHHHHHC-NH2; SEQ ID NO: 55) TFA salt are dissolved in 2 mL borate buffer pH 8.5 and stirred at room temperature for approximately 18h. 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 18h 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) are used for further formulation. Lipidoid component 3-C12 is 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 is prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine essentially according to e.g. Love et al., pp. 1864-1869, PNAS, vol. 107 (2010). Ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of said polymer are mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the polymer-lipidoid carrier compositions are used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier by incubation of 10 min at RT. 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 2: Vaccination of NHP with LNP Formulated YFV mRNA Vaccine

[0589] The present Example shows that LNP-formulated mRNA encoding YFV prME according to the invention induces long lasting PRNT titers in vaccinated NHPs.

[0590] Cynomolgus monkeys (Macaca fascicularis, two male and two female) were vaccinated intramuscularly on days 0, 28 and 56 with 10 ug LNP-formulated YFV X-SS-prME-XX mRNA (R3911). Animals received a single intramuscular injection in biceps femoris muscle at the day of vaccination. Serum samples were collected from all animals at day 0, 28, 56, and 77 to determine immunogenicity. Immune response to yellow fever virus was assessed in a plaque reduction neutralizing titer (PRNT) using an attenuated YFV 17D strain. The results of the PRNT assay are shown in FIG. 1. For assessing the durability of the immune responses, serum samples collected from all animals at day 294 were analyzed (see FIG. 2).

2.1. Plaque Reduction Neutralization Test (PRNT50):

[0591] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated NHPs were incubated with YFV. That mixture is used to infect cultured cells, and the reduction in the number of plaques was determined. The results of the PRNT assay are shown in FIGS. 1 and 2.

2.2. Results:

[0592] As shown in FIG. 1, immunization of Cynomolgus macaques with LNP-formulated mRNA based vaccine led to the production of YF-specific neutralizing antibodies. The results of FIG. 2 further demonstrate that induced immune responses have a remarkable longevity as all vaccinated animals maintained high PRNT titers at day 294 post prime vaccination.

[0593] To further improve the efficiency of the mRNA-based vaccine, several alternative YFV prME mRNA constructs were designed harboring different UTR combinations to potentially increase translation efficiency of the mRNA. Those mRNA constructs were tested in vitro (see Example 3) and in vivo (see e.g. Example 4).

Example 3: In Vitro Expression Screen of YFV mRNA Constructs in Cell Western (ICW) and Dot-Blot Analysis

[0594] The present Example shows that the UTR combinations according to the invention strongly improve the expression performance of said mRNA constructs compared to a reference mRNA construct (harboring RPL32/ALB7 UTRs) used e.g. in Example 2.

[0595] To determine in vitro protein expression performance of YFV prME mRNA constructs comprising different UTR combinations (mRNA constructs used: R5351, R5369, R5353, R5357, R5362, R5356, R5360, R5367, R5368, R5372, R5364 see Example 1, Table 3), different cell types were transiently transfected with said mRNA constructs and YFV prME antigen expression was analyzed using in cell western analysis (for HeLa, and HDF cells) and dot-blot analysis (for HeLa cells).

3.1. In Cell Western (ICW) Analysis on HeLa and HDF Cells:

[0596] HeLa cells and HDF cells were analyzed via ICW according to the following protocol: Cells were seeded on 96 well plates with black rim and clear optical bottom (Nunc Microplate; Thermo Fisher). HeLa cells or HDF (10,000 cells in 200 ul/well) were seeded 24h before transfection in a compatible complete cell medium. Cells were maintained at 37° C., 5% CO2. The day of transfection, the complete medium on HeLa or HDF was replaced with serum-free Opti-MEM medium (Thermo Fisher). Lipocomplexed mRNAs (Lipofectamine) were added to cells for transfection with 200 ng of RNA (HeLa & HDF) per well in a total volume of 150 ul. 90 min post start of transfection, 100 ul/well of transfection solution on HeLa or HDF was exchanged for 100 ul/well of complete medium. Cells were further maintained at 37° C., 5% CO2 before performing ICW.

[0597] 36h post start of transfection, YFV prME expression was quantified by ICW according to the following procedure (all steps performed at room temperature): First, cells were washed once with PBS and fixed with 3.7% formaldehyde in PBS for 10 min. After washing once in PBS, cells were permeabilized with Perm/Wash buffer (BD) for 30 min. Cells were blocked for 30 min with a mix of Odyssey blocking buffer (PBS) (LI-COR) and Perm/Wash buffer (BD) (1:1). Next, cells were incubated for 150 min with primary antibody directed against YFV prME (mouse monoclonal anti-YF (3576); Santa Cruz SC-58083/F1714; diluted 1:200 in BD). Cells were then washed 3 times (Perm/Wash buffer (BD)). Subsequently, cells were incubated with a mixture of secondary antibody (IRDye-coupled secondary antibody (IRDye 800CW goat anti-rabbit IgG; LI-COR; diluted 1:200 in BD) and Cell-Tag 700 Stain (LI-COR) (1:1000 in BD) for 1 h in the dark. After washing 4 times in BD, PBS was added to cells and plates scanned using an Odyssey® CLx Imaging system (LI-COR). Fluorescence (800 nm) was quantified using Image Studio Lite Software, normalized to the Cell-Tag 700 Stain and the results compared to expression from a reference construct containing the RPL32/ALB7 UTR-combination, set to a level of 100% expression. The results of the analysis are shown in FIG. 3 (HeLa cells) and FIG. 4 (HDF cells).

3.2. Dot Blot Analysis on HeLa Cells:

[0598] HeLa cells were seeded in a 24 well plate at a density of 300,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24h prior to transfection in a compatible complete cell medium. Cells were maintained at 37° C., 5% CO2. The day of transfection, the complete medium on HeLa was replaced with serum-free Opti-MEM medium (Thermo Fisher). Lipocomplexed mRNA (Lipofectamine) was added to cells for transfection with 500 ng of RNA per well in a total volume of 1000 ul. 90 min post start of transfection, transfection solution was exchanged of complete medium. Cells were further maintained at 37° C., 5% CO2. Supernatants were harvested 24h post transfection and 200 ul of each supernatant was used to performing Dot blot analysis. Non-specific sites of the membrane were blocked in blocking buffer (5% (w/v) skim milk powder in TBS with 0.1% Tween-20) for 1h at 4° C. on a shaker. Next, the membrane was incubated in primary antibody dilution (5 ul mouse anti-Flavivirus group antigen antibody; clone D1-4G2-4-15 (Millipore, 1:2000) in 10m1 dilution buffer (0.5% (w/v) skim milk powder in TBS with 0.1% Tween-20)) for 2h at RT in a 100m1 falcon tube on a rotating shaker. After 3×10 min washing steps in washing buffer (1×TBS with 0.1% Tween 20), the membrane was incubated in secondary antibody (goat anti-mouse IgG (H+L) IRDye 800CW; LI-COR Biosciences; 1:10000) for 1h at RT in the dark. After 3×10 min washing steps in washing buffer, the membrane was placed in TBS and subsequently imaged using an Odyssey CLx image system. The results were compared to the expression from a reference construct containing the RPL32/ALB7 UTR-combination which was set to a level of 100%. The results of the analysis are shown in FIG. 5.

3.3. Results:

[0599] As shown in FIGS. 3-5, the expression performances of the mRNA constructs comprising UTR combinations according to the invention were strongly increased compared to the construct comprising the reference UTR combination (RPL32/ALB7). Notably, the increase in expression was observed in different cell types (HeLa, HDF) using different in vitro assays (ICW, dot-blot). The herein identified advantageous UTR combinations were further analyzed in vivo (see Example 4).

Example 4: Vaccination of Mice with mRNA Encoding YFV prME

[0600] The present Example shows that specific UTR combinations according to the invention also improved the expression performance of mRNA constructs in vivo. The data furthermore shows that mice vaccinated with said improved mRNA constructs show much higher humoral immune responses compared to mice vaccinated with a reference mRNA (harboring RPL32/ALB7 UTRs) used e.g. in Example 2.

4.1. Immunization Procedure:

[0601] Female BALB/c mice (8 animals per group) were injected intramuscularly (i.m.) with 50 ug non-formulated mRNA per dose. As a negative control, one group of mice (5 animals) was injected with buffer (ringer lactate). All animals were injected on day 0, 28 and 56. Blood samples were collected on day 14, 28, 56, and 70 for the determination of antibody titers. Further details are provided in Table 4 below.

TABLE-US-00009 TABLE 4 Vaccination regimen (Example 4): RNA ID/SEQ UTR description Volume per Group ID NO: Construct (5′-UTR/3′-UTR) injection 1 R5353/1310 X-SS-prME-XX (opt1) HSD17B4/CASP1 2 × 25 ul 2 R5356/1313 X-SS-prME-XX (opt1) SLC7A3/NDUFA1 2 × 25 ul 3 R5357/1311 X-SS-prME-XX (opt1) ATP5A1/PSMB3 2 × 25 ul 4 R5360/1314 X-SS-prME-XX (opt1) NOSIP/CASP1 2 × 25 ul 5 R5362/1312 X-SS-prME-XX (opt1) RPL31/PSMB3 2 × 25 ul 6 R5367/1315 X-SS-prME-XX (opt1) SLC7A3/CASP1 2 × 25 ul 7 R5368/1316 X-SS-prME-XX (opt1) SLC7A3/COX6B1 2 × 25 ul 8 R5369/1309 X-SS-prME-XX (opt1) NDUFA4/PSMB3 2 × 25 ul 9 R5372/1318 X-SS-prME-XX (opt1) RPL32/ALB7 2 × 25 ul 10 RiLa 2 × 25 ul

4.2. Detection of Antigen Specific Humoral Immune Responses:

[0602] Hela cells were transfected with 2 ug YFV prME mRNA constructs (R3758) using lipofectamine. The cells were harvested 20h post transfection, and seeded at 1×10.sup.5 per well into a 96 well plate. The cells were incubated with corresponding sera of vaccinated mice (serum of day 14, diluted 1:100; serum of day 28, diluted 1:150; serum of day 56, diluted 1:200; serum of day 70, diluted 1:500) followed by a FITC-conjugated anti-mouse IgG antibody staining. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo. The results are shown in FIG. 6 (day 14 and day 28) and FIG. 7 (day 56 and day 70). As read out MFI of living cells (MFI=geometric mean fluorescence intensity) was used.

4.3. Results:

[0603] As shown in FIG. 6 and FIG. 7, the mRNA constructs encoding YFV prME harboring different UTR combinations of the invention are expressed in mice after i.m. administration. Moreover, as specific antigen IgGs were detected in sera of immunized mice, the results also show that the applied mRNA constructs are suitable to induce specific humoral immune responses. Furthermore, the results reveal that the mRNA constructs harboring different UTR combinations (HSD17B4/CASP1, Slc7a3/Ndufa1, ATP5A1/PSMB3, Nosip/CASP1, Rpl31/PSMB3, Slc7a3/CASP1, Slc7a3/COX6B1, Ndufa4/PSMB3) induce stronger immune responses compared to the reference mRNA construct (RPL32/ALB7) showing that these improved mRNA constructs may be particularly suitable for use as a vaccine, e.g. as an LNP-formulated mRNA based YFV vaccine (tested in Example 5).

Example 5: Vaccination of Mice with LNP-Formulated mRNA Encoding YFV prME

[0604] The present example shows that LNP formulated mRNA vaccine efficiently induces binding Antibody titers in vaccinated mice at a low dose (paragraph 5.1). In addition, different mRNA constructs with optimized UTR combinations are tested as LNP-formulated vaccine (paragraph 5.4).

5.1. Immunization Procedure of the Dose Finding Experiment:

[0605] Female BALB/c mice (8 animals per group) were injected intramuscularly (i.m.) with LNP-formulated vaccine. As a negative control, one group of mice (5 animals) was injected with 0.9% NaCl buffer. All animals were injected on day 0 and 21. Blood samples were collected on day 21 and 35 for the determination of antibody titers. Further details are provided in Table 5 below.

TABLE-US-00010 TABLE 5 Vaccination regimen (Example 5.1): RNA ID/SEQ Formu- Dose per Group ID NO: Construct lation injection A R6711/1194 X-SS-prME-XX (opt1) LNP 10 ug  B R6711/1194 X-SS-prME-XX (opt1) LNP 5 ug C R6711/1194 X-SS-prME-XX (opt1) LNP 1 ug D buffer

5.2 Detection of Antigen Specific Humoral Immune Responses:

[0606] HeLa cells were transfected with 2 ug R6711 using lipofectamine. Transfected cells were harvested 20h post transfection and seeded at 1×10.sup.5/well into 96-well V-bottom plate. Cells were stained with live/dead marker, fixed, permeabilized, and subsequently incubated with sera of mRNA vaccinated mice (diluted 1:50) followed by FITC-conjugated anti-mouse IgG antibody. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo. The result is shown in FIG. 8. The results show that the LNP-formulated mRNA vaccine induces binding antibody titers at a dose of 1 ug.

5.3. Plaque Reduction Neutralization Test (PRNT50):

[0607] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice were incubated with YFV. That mixture is used to infect cultured cells, and the reduction in the number of plaques was determined. The result of the PRNT assay is shown in FIG. 9. The result shows that the LNP-formulated mRNA vaccine led to the induction of YF-specific neutralizing antibodies.

5.4. Immunization Procedure of the mRNA Construct Evaluation Experiment:

[0608] Optimized YF mRNA constructs with inventive UTR combinations are used in the present experiment. Female BALB/c mice (6 animals per group) are injected intramuscularly (i.m.) with LNP-formulated mRNA with constructs as indicated in Table 6A and B. As a negative control, one group of mice is injected with buffer (ringer lactate). All animals are injected on day 0 and 21. Blood samples were collected on day 21 and 35 for the determination of antibody titers (as e.g. explained in paragraph 5.2). Splenocytes were isolated on day 35 for analysis of CD4/CD8 T cells. Further details are provided in Table 6A and B below.

TABLE-US-00011 TABLE 6A Vaccination regimen (Example 5.4): mRNA vaccine Gr. Balb/C UTR description RNA ID/SEQ Dose per mice N = 8 Construct (5′-UTR/3′-UTR) ID NO: Formulation injection 1 X-SS-prME-SS (opt1) HSD17B4/CASP1 R7232/654 LNP 5 ug 2 X-SS-prME-SS (opt1) HSD17B4/CASP1 R7232/654 LNP 1 ug 3 X-SS-prME-SS (opt1) ATP5A1/PSMB3 R7231/714 LNP 5 ug 4 X-SS-prME-SS (opt1) ATP5A1/PSMB3 R7231/714 LNP 1 ug 5 X-SS-prME-SS (opt1) NOSIP/PSMB3 R7230/1254 LNP 5 ug 6 X-SS-prME-SS (opt1) NOSIP/PSMB3 R7230/1254 LNP 1 ug 7 X-SS-prME-SS (opt1) HSD17B4/PSMB3 R7233/534 LNP 5 ug 8 X-SS-prME-SS (opt1) HSD17B4/PSMB3 R7233/534 LNP 1 ug 9 buffer

TABLE-US-00012 TABLE 6B Vaccination regimen (Example 5.4): mRNA vaccine Gr. Balb/C UTR description RNA ID/SEQ Dose per mice N = 8 Construct (5′-UTR/3′-UTR) ID NO: Formulation injection 1 X-SS-prME-SS (opt2) —/muag R7250/1212 LNP 5 ug 2 X-SS-prME-SS (opt2) —/muag R7250/1212 LNP 1 ug 3 X-SS-prME-SS (opt4) —/muag R7251/1224 LNP 5 ug 4 X-SS-prME-SS (opt4) —/muag R7251/1224 LNP 1 ug 5 X-SS-prME-SS (opt6) —/muag R7252/1236 LNP 5 ug 6 X-SS-prME-SS (opt6) —/muag R7252/1236 LNP 1 ug 7 X-SS-prME-SS (opt11) —/muag R7253/1242 LNP 5 ug 8 X-SS-prME-SS (opt11) —/muag R7253/1242 LNP 1 ug 9 buffer

Example 6: In Vitro Expression Analysis of YFV mRNA Constructs Comprising Cap1 or Cap0 with Western Blot

[0609] The present Example shows that the use of Cap1 according to the invention strongly improve the expression performance of said mRNA construct compared to a reference mRNA construct comprising Cap0.

6.1. Western Blot Analysis

[0610] To determine in vitro protein expression performance of YFV prME mRNA constructs comprising different Cap analogues (mRNA constructs used: R7233 (cap0) and R7927 (cap1)), see Example 1, Table 3), HeLa cells were transiently transfected with said mRNA constructs and YFV prME antigen expression was analyzed in cell lysates using western blot analysis.

[0611] For the analysis HeLa cells were transfected with 2 μg unformulated mRNA (R7233 (cap0), R7927 (cap1) or WFI (negative control)) using 3 μl of Lipofectamine as the transfection reagent, and cell lysates were prepared 20h post transfection. Western Blot analysis was performed using anti-flavivirus group antigen (4G2; 1:2000 diluted) as primary antibody in combination with secondary anti-mouse IRDye 800CW labelled antibody. The result of the analysis is shown in FIG. 10.

6.2. Results:

[0612] For both of the tested mRNA constructs (R7233 (cap0) and R7927 (cap1) YFV protein was detectable. As shown in FIG. 10, the expression performances of the mRNA constructs comprising Capt according to the invention was strongly increased (2.4×) compared to the construct comprising Cap0.

Example 7: Vaccination of Mice with LNP-Formulated mRNA Encoding YFV NS1

[0613] mRNA vaccines encoding YFV NS1 proteins (eSS-NS1, SSIgE-NS1, and eSS-NS1-Y) are prepared according to Example 1.

[0614] Female BALB/c mice or A129 mice (type-I interferon receptor deficient) (9-10 animals per group) are injected intramuscularly (i.m.) with LNP-formulated mRNA with constructs as indicated in Table 7. As a negative control, one group of mice is injected with LNP-formulated irrelevant mRNA. All animals are injected on day 0 and 21. Blood samples are collected on day 21 and 35 for the determination of antibody titers (as e.g. explained in paragraph 5.2) and NS1-specific antibodies will be detected via ELISA. Splenocytes are isolated on day 35 for analysis of CD4/CD8 T cells. A129 mice are challenged 2 weeks post last vaccination with YFV BeH 622205 strain (human case from Brazil, 2000), 10.sup.4 PFU via s.c. into foodpad. Upon challenge the mice are observed for 2 weeks regarding survival, body weight, morbidity index, temperature, and viremia.

TABLE-US-00013 TABLE 7 Vaccination regimen (Example 7): mRNA vaccine Gr. Balb/C UTR description Dose per mice N = 8 Construct (5′-UTR/3′-UTR) RNA ID Formulation injection 1 eSS-NS1 —/muag R8504/R8505 LNP 1 μg, 2.5 μg or 5 μg 2 SSIgE-NS1 —/muag R8507/R8508 LNP 1 μg, 2.5 μg or 5 μg 3 eSS-NS1-Y —/muag R8501/R8502 LNP 1 μg, 2.5 μg or 5 μg 4 eSS-NS1 HSD17B4/PSMB3 R8506 LNP 1 μg, 2,5 μg or 5 μg 5 SSIgE-NS1 HSD17B4/PSMB3 R8509 LNP 1 μg, 2.5 μg or 5 μg 6 eSS-NS1-Y HSD17B4/PSMB3 R8503 LNP 1 μg, 2.5 μg or 5 μg 7 Irrelevant mRNA LNP 1 μg, 2.5 μg or 5 μg

Example 8: Vaccination of Mice with LNP-Formulated mRNA Encoding YFV prME and YFV NS1

[0615] To broaden and optimize the YFV specific immune response and to potentially reduce the pathogenicity of the YFV, mRNA vaccines encoding different YFV proteins (prME construct: SS-prME, X-SS-prME-XX, SSjev-prME, SSjev-prME-XX, SSIgE-prME, SSIgE-prME-XX, X-SS-prMEdelstem_TM-JEV, SSjev(V3)-prMEdelstem_TM-JEV, X-SS-prME and NS1 construct: eSS-NS1, SSIgE-NS1, and eSS-NS1-Y) are prepared according to Example 1.

[0616] In order to assess the effect of single or combined vaccines, these vaccines are administered i.m. with 2.5 ug mRNA for each antigen either alone or in combination as shown in Table 8.

TABLE-US-00014 TABLE 8 Vaccination regimen (Example 7): Gr. Balb/C UTR description Formu- mice N = 8 Construct (5′-UTR/3′-UTR) lation 1 NS1 construct —/muag or HSD17B4/PSMB3 LNP 2 prME construct —/muag or HSD17B4/PSMB3 LNP 3 prME —/muag or HSD17B4/PSMB3 LNP construct + NS1 construct 8 Irrelevant —/muag or HSD17B4/PSMB3 LNP mRNA

[0617] Female BALB/c mice or A129 mice (type-I interferon receptor deficient) (9-10 animals per group) are injected intramuscularly (i.m.) with LNP-formulated mRNA with constructs as indicated in Table 8. As a negative control, one group of mice is injected with buffer (ringer lactate). All animals are injected on day 0 and 21. Blood samples are collected on day 21 and 35 for the determination of antibody titers (as e.g. explained in paragraph 5.2) and YF-specific neutralizing antibodies are determined using the plaque reduction neutralization test (PRNT50) (as e.g. explained in paragraph 5.3) and NS1-specific antibodies are determined using ELISA, Splenocytes are isolated on day 35 for CD4/CD8 T cells analysis by ICS. A129 mice are challenged 2 weeks post last vaccination with YFV BeH 622205 strain (human case from Brazil, 2000), 10.sup.4 PFU via s.c. into foodpad. Upon challenge the mice are observed for 2 weeks regarding survival, body weight, morbidity index, temperature, and viremia.

Example 9: Clinical Development of a YFV mRNA Vaccine Composition

[0618] To demonstrate safety and efficiency of the YFV mRNA vaccine composition, a clinical trial (phase I) is initiated. For clinical development, RNA is used that has been produced under GMP conditions (e.g. using a procedure as described in WO2016/180430).

[0619] In the clinical trial, a cohort of healthy human volunteers is intramuscularly injected for at least two times with respective LNP formulated vaccine compositions comprising favorable UTR combinations.

[0620] In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signals, vaccination site tolerability assessments, hematologic analysis).

[0621] 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.