NUCLEIC ACID MOLECULES AND USES THEREOF
20210401966 · 2021-12-30
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention is directed to an artificial nucleic acid and to polypeptides suitable for use in treatment or prophylaxis of an infection with Norovirus or a disorder related to such an infection. In particular, the present invention concerns a Norovirus vaccine. The present invention is directed to an artificial nucleic acid, polypeptides, compositions and vaccines comprising the artificial nucleic acid or the polypeptides. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptides, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial nucleic acid, polypeptides, compositions and vaccines.
Claims
1. Artificial nucleic acid comprising at least one coding region encoding at least one polypeptide derived from a Norovirus, and/or a fragment or variant thereof.
2. The artificial nucleic acid according to claim 1, wherein the at least one encoded polypeptide is selected from the group consisting of a non-structural protein derived from a Norovirus and/or a capsid protein derived from a Norovirus, and/or a fragment or variant thereof.
3. The artificial nucleic acid according to claim 1 or 2, wherein the at least one encoded polypeptide is selected from the group consisting of Norovirus non-structural proteins NS1/NS2, NS3, NS4, NS5, NS6, NS7, Norovirus capsid protein VP1 and Norovirus capsid protein VP2, and/or a fragment or variant thereof.
4. The artificial nucleic acid according to any one of claims 1 to 3, wherein the artificial nucleic acid is derived from a Norovirus selected from the group consisting of genogroup I Norovirus, genogroup II Norovirus, genogroup III Norovirus, genogroup IV Norovirus, and genogroup V Norovirus; preferably the artificial nucleic acid is derived from a Norovirus selected from the group consisting of a GI.1 to GI.17 Norovirus, GII.1 to GII.24 Norovirus, GIII.1 to GIII.4 Norovirus, GIV.1 to GIV.4 Norovirus and GV.1 to GV.4 Norovirus; more preferably, the artificial nucleic acid is derived from a Norovirus selected from the group consisting of GI.1 Norovirus and GII.4 Norovirus, even more preferably, the artificial nucleic acid is derived from a GII.4 Norovirus, still more preferably, the artificial nucleic acid is derived from a GII.4 CIN-1 Norovirus or a GII.4 Sydney Norovirus or a GII.4 Sydney 2012 Norovirus.
5. The artificial nucleic acid according to any one of claims 1 to 4, wherein the at least one encoded polypeptide comprises at least one Norovirus capsid protein VP1 or Norovirus capsid protein VP2 and/or a fragment or a variant thereof.
6. The artificial nucleic acid according to any one of claims 1 to 5, wherein the at least one encoded polypeptide comprises at least one Norovirus capsid protein VP1 and/or a fragment or variant thereof.
7. The artificial nucleic acid according to any one of claims 1 to 6, wherein the at least one encoded polypeptide comprises (i) at least one of the amino acid sequences according to any one of SEQ ID NOs: 1-4410; and/or (ii) at least one of the amino acid sequences having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NOs: 1-4410; and/or (iii) an orthologue or a paralogue of any one of SEQ ID NOs: 1-39690, 39713-39746; and/or a fragment or variant of any of these sequences.
8. The artificial nucleic acid according to any one of claims 1 to 7, wherein the at least one coding region comprises (i) at least one of the nucleic acid sequences according to any one of SEQ ID NOs: 4411-39690, 39713-39746; and/or (ii) at least one of the nucleic acid sequences having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence represented by any one of SEQ ID NOs: 4411-39690, 39713-39746; and/or (iii) at least one complement of the nucleic acid sequences which are capable of hybridizing with a nucleic acid sequence comprising a sequence as shown in SEQ ID NOs: 4411-39690, 39713-39746, and/or to a nucleic acid encoding a polypeptide having a sequence as shown in SEQ ID NO: 1-4410; and/or (iv) an orthologue or a paralogue of any one of SEQ ID NOs: 1-39690, 39713-39746; and/or a fragment or variant of any of these sequences.
9. The artificial nucleic acid according to any one of claims 1 to 8, wherein the artificial nucleic acid is monocistronic, bicistronic or multicistronic.
10. The artificial nucleic acid according to any one of claims 1 to 9, wherein the artificial nucleic acid is monocistronic and wherein the coding region encodes a polypeptide comprising at least two different Norovirus proteins as defined in any one of claims 1 to 9, or a fragment or variant thereof.
11. The artificial nucleic acid according to any one of claims 1 to 9, wherein the artificial nucleic acid is bi- or multicistronic and comprises at least two coding regions, wherein the at least two coding regions encode at least two polypeptides, wherein each of the at least two polypeptides comprises at least one Norovirus protein as defined in any one of claims 1 to 9, or a fragment or variant of any one of these proteins, wherein the at least two polypeptides are preferably different polypeptides.
12. The artificial nucleic acid according to any one of claims 1 to 11, wherein the artificial nucleic acid is an RNA, preferably an mRNA.
13. The artificial nucleic acid according to any one of claims 1 to 12, wherein the artificial nucleic acid comprises a 5′-cap structure.
14. The artificial nucleic acid according to any one of claims 1 to 13, wherein the G/C content of the coding region of the mRNA sequence 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 sequence 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 sequence 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 sequence, wherein the encoded amino acid sequence of the mRNA sequence is preferably not being modified compared to the encoded amino acid sequence of the wild type mRNA.
15. The artificial nucleic acid according to any one of claims 1 to 14, wherein (i) the at least one coding region comprises a nucleic acid sequence, which is codon-optimized; and/or (ii) the at least one coding sequence comprises a nucleic acid 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: 8821-13230, 26461-39690, 39715, 39716, 39717, 39720, 39721, 39724, 39725, 39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741, 39742, 39746 and 39746, or a fragment or variant of any of these sequences; and/or (iii) the at least one coding sequence comprises a nucleic acid 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 NO: 13231-17640, or a fragment or variant of any of these sequences; and/or (iv) the artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence comprises a nucleic acid 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 NO: 17641-22050, or a fragment or variant of any of these sequences; and/or (v) the artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence comprises a nucleic acid 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 NO: 22051-26460, or a fragment or variant of any of these sequences.
16. The artificial nucleic acid according to any one of claims 1 to 15, wherein the artificial nucleic acid comprises at least one histone stem-loop.
17. The artificial nucleic acid according to claim 16, 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; stem 1 [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 [N.sub.0-4(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.
18. The artificial nucleic acid according to claim 17, 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##
19. The artificial nucleic acid according to any one of claims 16 to 18, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710, or a fragment or variant thereof.
20. The artificial nucleic acid molecule according to any one of claims 1 to 19, wherein the artificial nucleic acid comprises an untranslated region (UTR).
21. The artificial nucleic acid according to claim 20, wherein the artificial nucleic acid comprises a 3′-UTR.
22. The artificial nucleic acid according to claim 21, wherein the 3′-UTR comprises at least one heterologous 3′-UTR element.
23. The artificial nucleic acid according to claim 21 or 22, wherein the 3′-UTR comprises a poly(A) sequence and/or a poly(C) sequence.
24. The artificial nucleic acid according to claim 23, wherein the poly(A) sequence comprises 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, and/or the poly(C) sequence comprises 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.
25. The artificial nucleic acid according to any one of claims 1 to 24, wherein the at least one heterologous 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene.
28. The artificial nucleic acid according to any one of claims 1 to 25, wherein the at least one heterologous 3′-UTR element comprises 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.
27. The artificial nucleic acid according to any one of claims 1 to 26, wherein the at least one heterologous 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: 39701, or SEQ ID NO: 39702, a homolog, a fragment, or a variant thereof.
28. The artificial nucleic acid according to any one of claims 1 to 77, wherein the at least one heterologous 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 Genbank Accession number NM 000477.5, or from a fragment or variant thereof.
29. The artificial nucleic acid according to any one of claims 1 to 28, wherein the at least one heterologous 3′-UTR element comprises a nucleic acid sequence according to to any one of SEQ ID NO: 39703 to SEQ ID NO: 39708, or a homolog, a fragment or a variant thereof.
30. The artificial nucleic acid according to any one of claims 1 to 29, wherein the artificial nucleic acid comprises a 5′-UTR.
31. The artificial nucleic acid sequence according to any one of claims 1 to 30, wherein the 5′-UTR comprises at least one heterologous 5′-UTR element.
32. The artificial nucleic acid according to any one of claims 1 to 31, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from the 5′-UTR of a TDP gene, preferably from a corresponding RNA sequence, or a homolog, a fragment, or a variant thereof, preferably lacking the 5′TDP motif.
33. The artificial nucleic acid according to any one of claims 1 to 32, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from a 5′-UTR of a TDP 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′TDP motif.
34. The artificial nucleic acid according to any one of claims 1 to 33, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from a 5′-UTR of a TDP gene encoding a ribosomal Large protein (RPL), preferably RPL32 or RPL35A, or from a gene selected from the group consisting of HSD17B4, ATPSAI, AIGI, ASK COX6C or ABCB7 (MDR), or from a homolog, a fragment or variant of any one of these genes, preferably lacking the 5′TDP motif.
35. The artificial nucleic acid according to any one of claims 1 to 34, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence according to SEQ ID NO: 39691 to SEQ ID NO: 39694, or a homolog, a fragment or a variant thereof.
36. The artificial nucleic acid according to any one of claims 1 to 35 comprising, preferably in 5′ to 3′ direction, the following elements: a) optionally a 5′-cap structure, preferably m7GpppN, b) a coding region encoding at least one polypeptide derived from a Norovirus as described herein, preferably VP1, or a fragment or variant thereof, c) optionally 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 7D, 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: 39709 to SEQ ID NO: 39710.
37. The artificial nucleic acid according to any one of claims 1 to 36 comprising, preferably in 5′ to 3′ direction, the following elements: a) optionally a 5′-cap structure, preferably m7GpppN, b) a coding region encoding at least one polypeptide derived from a Norovirus, preferably VP1 as described herein, or a fragment or variant thereof, c) a 3′-UTR element comprising a nucleic acid sequence, which is derived from an α-globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 39701, or SEQ ID NO: 39702, or a homolog, a fragment or a variant thereof, d) optionally a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, e) 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 f) optionally a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.
38. The artificial nucleic acid according to any one of claims 1 to 37, wherein the artificial nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID NOs: 39713-39746, preferably a nucleic acid sequence according to any one of SEQ ID NDs: 39716, 39721, 39729, 39734, 39738, 39725, or a fragment or variant of any of these sequences.
39. The artificial nucleic acid according to any one of claims 1 to 38, comprising, preferably in 5′ to 3′ direction,the following elements: a) optionally 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 TDP gene, preferably comprising a nucleic acid sequence according to SEQ ID NO: 39891, or SEQ ID NO: 39892, or a homolog, a fragment or a variant thereof, c) a coding region encoding at least one polypeptide derived from a Norovirus, preferably VP1 as described herein, or a fragment or variant thereof, d) a 3′-UTR element comprising a nucleic acid sequence, which is derived from an albumin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 39705, or SEQ ID NO: 39708, or a homolog, a fragment or a variant thereof, e) optionally a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 60 or 50 to 70 adenosine nucleotides, f) optionally a poly(B) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and g) optionally a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.
40. The artificial nucleic acid according to any one of claims 1 to 39, wherein the artificial nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID NOs: 39713-39746, preferably a nucleic acid sequence according to any one of SEQ ID NOs: 39716, 39721, 39729, 39734, 39738, 39725, or a fragment or variant of any of these sequences.
41. The artificial nucleic acid according to any one of claims 1 to 40, wherein the coding region comprises a modified nucleic acid sequence.
42. The artificial nucleic acid according to any one of claims 1 to 41, wherein the at least one coding region comprises a nucleic acid sequence encoding a molecular tag and wherein the molecular tag is selected from the group consisting of a FLAG tag, a glutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, a Strep tag, a green fluorescent protein (GFP) tag and an HA tag.
43. Composition comprising at least one artificial nucleic acid as defined by any one of claims 1 to 42 and a pharmaceutically acceptable carrier.
44. The composition according to claim 43, 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.
45. The composition according to any one of claims 43 to 44, 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.
46. The composition according to any one of claims 43 to 45 comprising the at least one mRNA, which is complexed with one or more cationic or polycationic compounds, and at least one free mRNA.
47. The composition according to any one of claims 43 to 46, wherein the at least one complexed mRNA is identical to the at least one free mRNA.
48. The composition according to any one of claims 43 to 47, wherein the mRNA is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.
49. The composition according to any one of claims 43 to 48, wherein the composition comprises at least one adjuvant.
50. The composition according to any one of claims 43 to 49, wherein a) the composition comprises a plurality or more than one of the mRNA sequences each defined in any one of claims 1 to 42; or b) the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acids as defined by any one of claims 1 to 42, wherein each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acids comprises at least one coding region encoding at least one polypeptide comprising a Norovirus protein as defined in any one of claims 1 to 42, and/or a fragment or a variant of any one of these proteins, wherein each coding region preferably encodes a different Norovirus protein, more preferably each coding region encodes a capsid protein, preferably VP1 of a different Norovirus.
51. The composition according to any one of claims 43 to 50, wherein a) wherein each of the mRNA sequences encodes at least one different antigenic peptide or protein derived from proteins of the same Norovirus; and/or b) the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acids as defined by any one of claims 1 to 42, wherein each of the at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acids comprises at least one coding region encoding at least one polypeptide comprising at least two different Norovirus proteins, preferably VP1 and VP2, as defined in any one of claims 1 to 42, and/or a fragment or a variant of any one of these proteins.
52. The composition according to any one of claims 43 to 51, wherein the at least one artificial nucleic acid is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably a cationic protein or peptide.
53. The composition according to any one of claims 43 to 52, wherein (i) the ratio of complexed nucleic acid to free nucleic acid 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), wherein the ratio is most preferably about 1:1 (w/w); or (ii) the 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; and/or wherein the at least one artificial nucleic acid or 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 and/or wherein the at least one artificial nucleic acid or mRNA is complexed with one or more lipids and thereby forming liposomes, lipid nanoparticles and/or lipoplexes.
54. The composition according to any one of claims 43 to 53 wherein the composition comprises (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial nucleic acids as defined in claims 1 to 42; or (ii) at least 10, 15, 20 or 50 artificial nucleic acids as defined in claims 1 to 42; or (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial nucleic acids as defined in claims 1 to 42; and a pharmaceutically acceptable carrier, wherein preferably the artificial nucleic acid encodes a capsid protein VP1 derived from a Norovirus.
55. The composition according to any one of claims 43 to 54, wherein (i) the artificial nucleic acids are derived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI Noroviruses; or (ii) the artificial nucleic acids are derived from a single GII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII Noroviruses; or (iv) the artificial nucleic acids are derived from a single GIII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GIII Noroviruses; or (iv) the artificial nucleic acids are derived from a single GIV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GIV Noroviruses; or (v) the artificial nucleic acids are derived from a single GV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GV Noroviruses; or (vi) the artificial nucleic acids are derived from a single GI Norovirus and additionally from a single GII Norovirus, GIII Norovirus, GIV Norovirus and/or GV Norovirus; or (vii) the artificial nucleic acids are derived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI Noroviruses and additionally from a single GII, GIII, GIV or GV Norovirus and/or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and/or more GII, GIII, GIV or GV Noroviruses; wherein preferably the artificial nucleic acids encode a capsid protein VP1 derived from a Norovirus.
56. The composition according to any one of claims 43 to 55, wherein (i) the artificial nucleic acids are derived from a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1 Noroviruses; or (ii) the artificial nucleic acids are derived from a single GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII.4 Noroviruses; or (iii) the artificial nucleic acids are derived from a single GI.1 Norovirus and additionally from a single GII.4 Norovirus; or (iv) the artificial nucleic acids are derived from a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1 Noroviruses and additionally from a single GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII.4 Noroviruses; and/or wherein (i) at least one of the nucleic acid sequences according to any one of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or (ii) at least one of the nucleic acid sequences having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence represented by any one of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or (iii) at least one complement of the nucleic acid sequences which are capable of hybridizing with a nucleic acid sequence comprising a sequence as shown in SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or (iv) an orthologue or a paralogue of any one of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or a fragment or variant of any of these sequences.
57. Polypeptide encoded by the artificial nucleic acid according to any one of claims 1 to 42.
58. Polypeptide according to any one of claims 1 to 42 comprising at least one protein selected from the group consisting of NS1/NS2, NS3, NS4, NS5, NS6, NS7, VP1, and VP2 derived from Norovirus, or a fragment or variant of any of these proteins, and at least one amino acid sequence selected from the group consisting of: a) an amino acid sequence derived from a C-terminal fragment from mature Norovirus capsid protein VP1, or a variant thereof, wherein the C-terminal fragment consists of 3 to 20 amino acid residues, b) an amino acid sequence derived from a signal sequence of Norovirus capsid protein VP1, or a fragment or variant thereof, and c) an amino acid sequence derived from an N-terminal fragment from mature Norovirus non-structural protein NS1/NS2, NS3, NS4, NS5, NS6, or NS7, or a variant thereof, wherein the N-terminal fragment consists of 3 to 20 amino acid residues.
59. The polypeptide according to any one of claims 57 to 58 comprising a molecular tag, wherein the molecular tag is selected from the group consisting of a FLAG tag, a glutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, a Strep tag, a green fluorescent protein (GFP) tag and an HA tag.
60. Composition comprising the polypeptide according to any one of claims 57 to 59, and a pharmaceutically acceptable carrier.
61. Vaccine comprising the artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 5B, the polypeptide according to any one of claims 57 to 59, and/or the composition according to claim 60.
62. The vaccine according to claim 61, wherein the artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, or the composition according to claim 60 elicits an adaptive immune response.
63. The vaccine according to claim 61 to 62, wherein the vaccine further comprises a pharmaceutically acceptable carrier.
64. The vaccine according to any one of claims 61 to 63 further comprising an adjuvant.
65. The vaccine according to any one of claims 61 to 64, wherein the vaccine is multivalent and comprises (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial nucleic acids as defined in claims 1 to 42; or (ii) at least 10, 15, 20 or 50 artificial nucleic acids as defined in claims 1 to 42; or (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial nucleic acids as defined in claims 1 to 43.
66. The vaccine according to any one of claims 61 to 65, wherein (i) the artificial nucleic acids are derived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI Noroviruses; or (ii) the artificial nucleic acids are derived from a single GII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII Noroviruses; or (iii) the artificial nucleic acids are derived from a single GIII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GIII Noroviruses; or (iv) the artificial nucleic acids are derived from a single GIV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GIV Noroviruses; or (v) the artificial nucleic acids are derived from a single GV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GV Noroviruses; or (vi) the artificial nucleic acids are derived from a single GI Norovirus and additionally from a single GII Norovirus, GIII Norovirus, GIV Norovirus and/or GV Norovirus; or (vii) the artificial nucleic acids are derived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI Noroviruses and additionally from a single GII, GIII, GIV and/or GV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII, GIII, GIV and/or GV Noroviruses.
67. The vaccine according to any one of claims 61 to 66, wherein (i) the artificial nucleic acids are derived from a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1 Noroviruses; or (ii) the artificial nucleic acids are derived from a single GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII.4 Noroviruses; or (iii) the artificial nucleic acids are derived from a single GI.1 Norovirus and additionally from a single GII.4 Norovirus; or (iv) the artificial nucleic acids are derived from a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1 Noroviruses and additionally from a single GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII.4 Noroviruses.
68. Kit or kit of parts comprising the artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim 60 or the vaccine according to any one of claims 61 to 67, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.
69. The kit or kit of parts according to claim 68 comprising Ringer lactate solution.
70. The artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim 60, the vaccine according to any one of claims 61 to 67, or the kit or kit of parts according to claim 68 to 69 for use as a medicament.
71. The artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim 60, the vaccine according to any one of claims 61 to 67, or the kit or kit of parts according to claim 68 to 69 for use in the treatment or prophylaxis of an infection with Norovirus or a disorder related to an infection with Norovirus.
72. The artificial nucleic acid according to any one of according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim 60, the vaccine according to any one of claims 61 to 67, or the kit or kit of parts according to claim 68 to 69, wherein the artificial nucleic acid, the composition, the vaccine or the active component of the kit or kit of parts is administered by injection, preferably by needleless injection, more preferably by jet injection.
73. The artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim Blithe vaccine according to any one of claims 61 to 67, or the kit or kit of parts according to claim 68 to 69 for use according to any one of claims 70 to 72, wherein the treatment or prophylaxis comprises the administration of a further active pharmaceutical ingredient.
74. Method of treating or preventing a disorder, wherein the method comprises administering to a subject in need thereof the artificial nucleic acid according to any one of claims 1 to 42, the composition according to any one of claims 43 to 56, the polypeptide according to any one of claims 57 to 59, the composition according to claim Blithe vaccine according to any one of claims 61 to 67, or the kit or kit of parts according to claim 68 to 69.
75. The method according to claim 74, wherein the disorder is an infection with Norovirus or a disorder related to an infection with Norovirus.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0944]
[0945]
[0946]
[0947]
[0948]
[0949]
EXAMPLES
[0950] 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 for In Vitro and In Vivo Experiments
[0951] 1.1. Preparation of DNA and mRNA Constructs:
[0952] For the present examples, DNA sequences encoding Noravirus antigenic proteins, derived from three or more different Noravirus strains were prepared and used for subsequent RNA in vitro transcription reactions. The prepared RNA constructs (coding sequences (cds) and mRNA sequences) are listed in Table 4 below.
[0953] Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a codon modified sequence or GC-optimized sequence for stabilization, using three or more different in silica algorithms that e.g. increase the GC content of the respective coding sequence (indicated as “GC opt 1”, “GC opt 2”, “GC opt 3”, “GC opt 4”, “opt 5”, “opt 6”, “opt 7” in Table 4; further details relating to sequence modifications are provided in the specifications of the invention). Some DNA sequences were used as a wild type coding sequence, without altering the GC content and without altering the codon usage of the coding sequence (indicated as “wt” in Table 4).
[0954] DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in sika algorithms that increase the GC content of the respective coding sequence (e.g., indicated as “opt1” in Table 4, see explanation in the paragraph above).
[0955] 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 “design 1” in Table 4. Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L4 5′-UTR ribosomal 5′TDP 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 “design 2” in Table 4. Further details are relating mRNA construct design are provided in the specifications of the invention)
[0956] The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia call) using common protocols known in the art.
TABLE-US-00003 TABLE 4 VPI coding sequences, protein sequences and mRNA constructs RNA Construct ID description Norovirus strain RNA design SEQ ID NO R1 mRNA VPI_(X124V) GII.4-031693-USA-2003 design 1, wt 39713 R2 mRNA VPI_(X124V) GII.4-031693-USA-2003 design 2, wt 39714 R3 mRNA VPI_(X124V) GII.4-031693-USA-2003 design 1, GC opt 1 39715 R4 mRNA VPI_(X124V) GII.4-031B93-USA-2003; C1N1 design 2, GC opt 1 39716 R5 protein VPI_(X124V) GII.4-031693-USA-2003 Protein* 2358 R6 cds VPI_(X124V) GII.4-031693-USA-2003 wild type, wt 6768 R7 cds VPI_(X124V) GII.4-031693-USA-2003 GC opt 1 39717 R8 cds VPI_(X124V) GII.4-031693-USA-2003 GC opt 2 11178 R9 cds VPI_(X124V) GII.4-031693-USA-2003 opt 5 15588 R10 cds VPI_(X124V) GII.4-031693-USA-2003 opt 6 19998 R11 cds VPI_(X124V) GII.4-031693-USA-2003 opt 7 24408 R12 cds VPI_(X124V) GII.4-031693-USA-2003 GC opt 3 28818 R13 cds VPI_(X124V) GII.4-031693-USA-2003 GC opt 4 33228 R14 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 1 39718 R15 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 2 39719 R16 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 1, GC opt 1 39720 R17 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 2, GC opt 1 39721 R18 protein Capsidprotein GII.4 Farmington Hills-2002-USA Protein* 1487 R19 cds Capsidprotein GII.4 Farmington Hills-2002-USA wild type 5897 R20 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 2 10307 R21 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 5 10307 R22 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 6 19127 R23 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 7 23537 R24 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 3 27947 R25 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 4 32357 R26 mRNA VPI GII.4-2006b 092895-USA-2008 design 2, GC opt 1 39729 R27 mRNA VPI GII.4-GZ2010-L87-Guangzhou-2011 design 2, GC opt 1 39734 R28 mRNA VPI GII.4-USA-1997 design 2, GC opt 1 39738 R29 mRNA VPI GI.1-USA-1968-Capsidprotein design 2, GC opt 1 39725 *protein sequence is back translated into RNA according to the above paragraph “G/C content modification”
[0957] 1.2. RNA in vitro Transcription:
[0958] The DNA plasmids prepared according to paragraph 1.1 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. The obtained mRNAs were purified using PureMessenger® (CureVac, Tubingen, Germany; WO 2008/077592 A1) and used for in vitro and in viva experiments.
[0959] 1.3. Preparation of Protamine Formulated RNA Vaccine:
[0960] The obtained mRNA, e.g. HPLC purified RNA, was complexed with protamine by addition of protamine-trehalose solution to RNA solution at a RNA:protamine weight to weight ratio of 2:1. Then, complexed RNA was mixed with non-complexed RNA in a ratio of 50% free RNA and 50% complexed RNA to obtain formulated RNA. Formulated RNA was used for in vivovaccination experiments.
[0961] 1.4. Preparation of LNP Formulated RNA Vaccine:21
[0962] RNA is encapsulated in lipid nanoparticle (LNP) using established protocols known in the art. Briefly, LNP-encapsulated RNA is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. RNA is diluted to a total concentration of about 0.05 mg/mL in 50 mM citrate buffer pH 4. Syringe pumps are used to mix the ethanolic lipid solution with RNA at a ratio of about 1:6 to 1:2 (vol/vol). Ethanol is then removed and the external buffer replaced with PBS by dialysis. Lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size may be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).
Example 2: Expression of Norovirus VP1 Antigens in HeLa Cells and Analysis by FADS
[0963] To determine in vitraprotein expression of the inventive Norovirus mRNA constructs, HeLa cells were transfected with mRNA constructs encoding Norovirus VP1 antigens and analyzed by intracellular FADS staining. For cell transfection, an mRNA comprising VP1_X124V (GII.4-031693-USA-2003) wild type coding sequence (SEQ ID NO: 39714; construct ID R2) an mRNA comprising VP1_X124V (GII.4-031693-USA-2003) GC-optimized coding sequence (SEQ ID NO: 39716; construct ID R4), an mRNA comprising VP1 (GII.4-2006b 092895-USA-2008) GC-optimized coding sequence (SEQ ID NO: 39729; construct ID R26), an mRNA comprising VP1 (GII.4-GZ2010-L87-Guangzhou-2011) GC-optimized coding sequence (SEQ ID NO: 39734; construct R27) and an mRNA comprising VP1 NOV(GII.4-USA-1997)-Capsidprotein GC-optimized coding sequence (SEQ ID NO: 39738; construct ID R28) were used. The detailed description of the performed experiment is provided below.
[0964] 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. Cells were transfected with 1 μg and 2 μg mRNA per construct using Lipofectamine 2000 (Invitrogen) as transfection reagent. As a negative control, water for injection (WFI) was used.
[0965] 24 hours post transfection, transfected HeLa cells were stained with a commercial mouse anti-Norovirus GII.4 antibody [2000-G5] (Abcam; 1:500) and an anti-mouse FITC labelled secondary antibody (F5262 from Sigma; 1:500) after Cytofix/Cytoperm (BD Biosciences) treatment according to manufacturer's protocol. Subsequently, cells were analyzed by flow cytometry (FADS) on a BD FADS Canto II using the FADS Diva software. Duantitative analysis of the fluorescent FITC signal was performed using the FlowJo software package (Tree Star, Inc.). The results of the FADS expression analysis are shown in
[0966] Results:
[0967]
Example 3: Analysis of Protein Expression using Western Blot
[0968] To determine in vitro protein expression upon HeLa cell transfection with the inventive mRNA constructs, HeLa cells were transiently transfected with an mRNA constructs comprising VP1_X124V coding sequences. Cell lysates were prepared and analyzed using western blot. The detailed description of the performed experiment is provided below.
[0969] HeLa cells are transfected with 2 μg mRNA comprising wild type VP1_X124V coding sequence (SEQ ID NO: 39714; construct ID R2) and 2 μg mRNA comprising GC-optimized VP1_X124V coding sequence (SEQ ID NO: 39716; construct ID R4). As a negative control water for injection (WFI) was used. After 24 hours post transfection lysis buffer was added to the culture to prepare cellular lysates. Cellular lysates as well as a commercial Norovirus virus like particle (VLP; obtained from Medigen) were reduced by heating the samples to 95° C. for 10 minute. Subsequently, samples were subjected to SDS-PAGE under denaturating/reducing conditions followed by western blot detection. For the detection of Norovirus proteins, a commercial mouse anti-Norovirus GII.4 antibody [2002-G5] (1:250; Abcam) was used as primary antibody followed by secondary goat anti mouse antibody coupled to IRDye 800CW (1:10000; Licor Biosciences). The results of the experiment are shown in
[0970] Results:
[0971]
Example 4: Immunization of Mice and Evaluation of Norovirus Specific Immune Responses
[0972] Female BALB/c mice were immunized intradermally (i.d.) with protamine formulated mRNA vaccine (construct ID R4) with doses, application routes and vaccination schedules as indicated in Table 5. As a negative control, one group of mice was injected with buffer (ringer lactate, RiLa). All animals were vaccinated on day 0, 21 and 35. Blood samples were collected on day 49 for the determination of binding antibody titers (using a homologous and heterologous ELISA assay), blocking antibody titers (using a heterologous HGBA assay) and T-cell responses (intracellular cytokine assay). Detailed descriptions of the performed experiments are provided below.
TABLE-US-00004 TABLE 5 Vaccination regimen (Example 4) No of Route/ Vaccination Group mice Treatment Dose Volume schedule 1 6 Novovirus GC-optimized 80 μg i.d. d0, d21, d35 VP1_X124V 2 × 50 μl SEQ ID NO: 39716; R4 Protamine formulated 2 6 100% RiLa Control i.m. d0, d21, d35 1 × 25 μl
[0973] 4.1. Determination of Homologous and Heterologous Immune Responses by ELISA:
[0974] ELISA was performed using synthetically produced norovirus Virus like particles (VLP) as coating material. For the analysis of homologous immune responses, plates were coated with VLP of the same strain of genotype GII.4 (GII.4 CIN1). For the analysis of heterologous immune responses, plates were coated with VLPs of another strain of genotype GII.4 (GII.4 2011). Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the Norovirus coating material was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate.
[0975] Endpoint titers of antibodies were measured by ELISA on day 49 after three vaccinations (see Table 5). The results are shown in
[0976] 4.2. Determination of Blocking Antibody Titers using a HBGA Blocking Assay:
[0977] Respective sera (day 49 after three vaccinations) were pre-incubated with synthetic norovirus VLPs (VLP (GII.4 2011)) and subsequently added to HBGA coated plates. VLP binding to Histo-Blood Group Antigen (HBGA) was detected by norovirus specific antibodies. In the presence of functional blocking antibodies in serum of immunized animals, VLP binding to HBGA was blocked which results in a reduction of the detected antibody signal. The respective blocking index was calculated as commonly known in the art. The results of the assay are shown in
[0978] 4.3. Determination of Specific CDB T-Cell Responses using ICS:
[0979] Splenocytes from vaccinated mice were isolated according to a standard protocol known in the art. Briefly, isolated spleens were 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 were seeded into 96-well plates (2×10.sup.6 cells per well). The cells were stimulated with ten Norovirus CD8 peptide epitopes (1 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) and anti-CD107α-PE-Cy7 antibody, after one hour at 37° C. After stimulation, cells were washed and stained and for staining of intracellular cytokines Cytofix/Cytoperm reagent (BD Biosciences) was used according to the manufacturer's instructions. The following antibodies were 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 was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software package (Tree Star, Inc.). Results for CD8+ T-cells are shown in
[0980] Results:
[0981]
[0982]
[0983]
[0984] Overall, the results of the immunization experiments in mice show that the inventive Norovirus mRNA vaccine induced a broad immune response engaging both the humoral-secretory and cellular immunity effector arms. Notably, heterologous immune responses were also observed (ELISA, HGBA). The data suggests that analogous mRNA constructs encoding other Norovirus antigens (as defined in the specifications or listed in Table 1 or Table 3) may also induce board immune responses in a similar manner.
Example 5: Immunization of Mice and Further Evaluation of Heterologous Immune Responses
[0985] Female BALB/c mice are immunized intradermally (i.d.) and intramuscularily (i.m.) with protamine formulated or LNP formulated mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 6. As a negative control, one group of mice was injected with buffer (ringer lactate). All animals were vaccinated on day 0, 21 and 35. Blood samples are collected on day 49 for the determination of binding antibody titers (using a homologous and heterologous ELISA assay), blocking antibody titers (using a homologous and heterologous HGBA assay). Detailed descriptions of the performed experiments are provided below.
TABLE-US-00005 TABLE 6 Vaccination regimen of mice (Example 5) No. of Route/ Vaccination Group mice Treatment Dose Volume schedule 1 6 Norovirus GC-optimized VP1 80 μg i.d. d0, d21, d35 GII.4-USA-1997 2 × 50 μl SEQ ID NO: 39738; R28 Protamine formulated 2 6 Norovirus GC-optimized VP1 80 μg i.d. d0, d21, d35 GII.4-2006b 092895-USA-2008 2 × 50 μl SEQ ID NO: 39729; R26 Protamine formulated 3 6 Norovirus GC-optimized VP1 80 μg i.d. d0, d21, d35 GII.4-GZ2010-L87-Guangzhou-2011 2 × 50 μl SEQ ID NO: 39734; R27 Protamine formulated 4 6 Norovirus GC-optimized VP1_X124V 80 μg i.d. d0, d21, d35 SEQ ID NO: 39716; R4 2 × 50 μl Protamine formulated 5 6 Norovirus GC-optimized VP1 20 μg i.m. d0, d21, d35 GII.4-USA-1997 2 × 25 μl SEQ ID NO: 39738; R28 LNP formulated 6 6 Norovirus GC-optimized VP1 20 μg i.m. d0, d21, d35 GII.4-2006b 092895-USA-2008 2 × 25 μl SEQ ID NO: 39729; R26 LNP formulated 7 6 Norovirus GC-optimized VP1 20 μg i.m. d0, d21, d35 GII.4-GZ2010-L87-Guangzhou-2011 2 × 25 μl SEQ ID NO: 39734; R27 LNP formulated 8 6 Norovirus GC-optimized VP1_X124V 20 μg i.m. d0, d21, d35 SEQ ID NO: 39716; R4 2 × 25 μl LNP formulated 9 6 100% RiLa Control i.m. d0, d21, d35 1 × 25 μl
[0986] 5.1. Determination of homologous and Heterologous Immune Responses by ELISA:
[0987] ELISA is performed essentially as described in Example 4.1. Plates are coated with VLP GII.4 CIN1 and VLP GII.4 2011 to determine homologous and heterologous immune responses.
[0988] 5.2. Determination of Blocking Antibody Titers using a Heterologous HBGA Blocking Assay:
[0989] The HGBA assay is performed essentially as described in Example 4.2. The respective blocking index are calculated as commonly known in the art to evaluate homologous and heterologous cross neutralizing capacities of the used mRNA vaccines.
[0990] 5.3. Determination of Specific CDB T-Cell Responses using ICS:
[0991] Multifunctional CD8 T-cell responses are analyzed as described in Example 4.3.
Example 6: Norovirus mRNA Vaccine Challenge Study in Gnotobiotic Pigs
[0992] 6.1. Immunization of Gnotobiotic Pigs:
[0993] Gnotobiotic pigs are derived by hysterectomy from near-term sows and maintained in germ-free isolator units. Pigs are fed commercial ultra-high-temperature-treated sterile food. All pigs are confirmed as seronegative for Norovirus and germ-free prior to immunization experiments. Gnotobiotic pigs are immunized with protamine formulated or LNP formulated mRNA vaccines (monovalent, bivalent, or tetravalent) with doses, application routes and vaccination schedules as indicated in Table 7. Analysis of immune responses is performed essentially as described in Example 4 (ELISA, HGBA, and ICS).
TABLE-US-00006 TABLE 7 Vaccination regimen of pigs (Example 6) No. of Dose/ Vaccination Group pigs Treatment Route schedule 1 6 Monovalent vaccine; protamine formulated 240 μg d0, d21 GII.4-2006b 092895-USA-2008 i.d. SEQ ID NO: 39729; R26 2 × 200 μl 2 6 Bivalent vaccine; protamine formulated 240 μg d0, d21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.d. GII.4-GZ2010-L87-Guangzhou-2011 2 × 200 μl SEQ ID NO: 39734; R27 3 6 Tetravalent vaccine, protamine formulated 240 μg d0, d21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.d. GII.4-031693-USA-2003 2 × 200 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Monovalent vaccine; LNP formulated 60 μg d0, d21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729; R26 2 × 100 μl 5 6 Bivalent vaccine; LNP formulated 60 μg d0, d21 GII-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m. GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6 Tetravalent vaccine; LNP formulated 60 μg d0, d21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.m. GII.4-031693-USA-2003 2 × 100 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100% RiLa Control — d0, d21
[0994] 6.2. Norovirus Challenge Experiment:
[0995] At day 3D days post immunization, the vaccinated and buffer-injected control pigs are challenged orally with Norovirus GII.4 (isolated from human stool samples) to assess the protection against Norovirus-induced diarrhea and fecal virus shedding. After virus challenge, rectal swaps and feces samples are collected at day 1, 3,5,7 and 10. Norovirus loads in rectal swaps and feces samples are determined using quantitative PER. In addition, pigs are monitored for Norovirus-associated symptoms and fecal consistence scores are recorded to assess severity of the Norovirus infection.
Example 7: Immunization of Non-Human Primates and Evaluation of Immune Responses
[0996] Non-human primates (NHPs) are immunized with protamine or LNP formulated mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 8. Analysis of immune responses is performed essentially as described in Example 4 (ELISA, HGBA, and ICS).
TABLE-US-00007 TABLE 8 Vaccination regimen of NHPs (Example 7) Number Dose/ Vaccination Group of NHPs Treatment Route schedule 1 6 Monovalent vaccine 240 μg d0, d21 protamine formulated i.d. GII.4-2006b 092895-USA-2008 2 × 200 μl SEQ ID NO: 39729; R26 2 6 Bivalent vaccine 240 μg d0, d21 protamine formulated (total) GI.1-USA-1968-Capsidprotein i.d. SEQ ID NO: 39725; R29 + 2 × 200 μl GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 3 6 Tetravalent vaccine; 240 μg d0, d21 protamine formulated (total) GII.4-USA-1997 i.d. SEQ ID NO: 39738; R28 + 2 × 200 μl GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Monovalent vaccine; LNP formulated 60 μg d0, d21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729; R26 2 × 100 μl 5 6 Bivalent vaccine; MP formulated 60 μg d0, d21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m. GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6 Tetravalent vaccine; LNP formulated 60 μg d0, d21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.m. GII.4-031593-USA-2003 2 × 100 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100% RiLa Control i.m. d0, d21 1 × 100 μl
Example 8: Development of a Multivalent Norovirus mRNA Vaccine
[0997] 8.1. Generation of Bivalent, Tetravalent and Multivalent Norovirus mRNA Vaccines
[0998] For bivalent and tetravalent Norovirus mRNA vaccines, each mRNA construct is individually produced (as described in Example 1). Multivalent Norovirus vaccine compositions are produced according to procedures as disclosed in the PST application PST/EP2016/082487. In short, Norovirus DNA constructs (each of which comprising different norovirus coding sequences and a T7 promotor; e.g. synthetic DNA templates immobilized on a chip) are used as a matrix for simultaneous KR amplification. The obtained KR product mixture is purified and used as a template for simultaneous RNA in vitrotranscription to generate a mixture of Norovirus mRNA constructs. The obtained Norovirus mRNA mixture is subjected to quantitative and qualitative measurements (e.g., RNA AGE, RT-qPCR, NGS, and Spectrometry). Following that, purification and formulation is performed (protamine formulation and LNP formulation). For the preparation of multivalent mRNA mixtures, Norovirus sequences as provided in Table 3 (see specifications) are used.
[0999] The produced bivalent, tetravalent and multivalent Norovirus mRNA vaccines are used for ill vitraand in viva experiments.
[1000] 8.2. Expression Analysis of Multivalent Norovirus mRNA Vaccines using Quantitative Mass Spectrometry
[1001] Hela cells are transfected with bivalent, tetravalent and multivalent mRNA mixtures (see Table 9) and protein expression is analyzed using quantitative mass spectrometry to show that every mRNA comprised in the respective mRNA mixture is efficiently translated into Norovirus protein/antigen.
[1002] 8.3. Immunization of Mice and Evaluation of Norovirus Specific Immune Responses
[1003] Female BALB/c mice are with protamine or LNP formulated monovalent, bivalent, tetravalent or multivalent mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 9. As a negative control, one group of mice is injected with buffer (ringer lactate, Rita). All animals are vaccinated on day 0, 21 and 35. Blood samples are collected on day 49 for the determination of binding antibody titers (using an ELISA assay), blocking antibody titers (using a HGBA assay) and cellular immune responses (ICS) performed essentially as described in Example 4.
TABLE-US-00008 TABLE 9 Vaccination regimen of mice (Example 8) Number Dose/ Vaccination Group of mice Treatment Route schedule 1 6 Monovalent vaccine; 40 μg d0, d21, d35 Protamine formulated i.d. GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 2 6 Bivalent vaccine; Protamine formulated 80 μg d0, d21, d35 R4 or R26 or R27 or R28 + (40 μg each) R4 or R26 or R27 or R28 i.d. 3 6 Tetravalent vaccine; 80 μg d0, d21, d35 Protamine formulated (20 μg each) GII.4-USA-1997 i.d. SEQ ID NO: 39738; R28 + GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Bivalent vaccine; Protamine formulated 80 μg d0, d21, d35 GI.1-USA-1968-Capsidprotein (40 μg each) SEQ ID NO: 39725; R29 + i.d. R4 or R26 or R27 or R28 5 6 Tetravalent vaccine; Protamine formulated 80 μg d0, d21, d35 GI.1-USA-1968-Capsidprotein (20 μg each) SEQ ID NO: 39725; R29 + i.d. GII.4-USA-1997 SEQ ID NO: 39738; R28 + GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 6 6 Multivalent; Protamine formulated 80 μg d0, d21, d35 20 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.d. (selected from Table 3) 7 6 Multivalent; Protamine formulated 80 μg d0, d21, d35 50 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.d. (selected from Table 3) 8 6 Multivalent; LNP formulated 80 μg d0, d21, d35 20 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.m. (selected from Table 3) 9 6 Multivalent; LNP formulated 80 μg d0, d21, d35 50 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.m. (selected from Table 3) 10 6 100% RiLa Control — d0, d21, d35
Example 9: Expression of Norovirus Proteins in HeLa Cells and Analysis by FADS
[1004] To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Norovirus antigens and stained using suitable customized anti Norovirus-protein antibodies (raised in mouse) and a FITC-coupled secondary antibody (F5262 from Sigma).
[1005] HeLa cells are seeded in a B-well plate at a density of 400000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. HeLa cells are transfected with 1 and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs are used in the experiment, including a negative control encoding an irrelevant protein. 24 hours post transfection, HeLa cells are stained with suitable anti Norovirus-protein antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FADS) on a BD FADS Canto II using the FADS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).
Example 10: Expression and Secretion of Norovirus Proteins using Western Blot
[1006] For the analysis of Norovirus protein secretion, HeLa cells are transfected with 1 μg and 2 μg unformulated mRNA (R1-R29, see Table 4) including a negative control encoding an irrelevant protein using Lipofectamine as the transfection agent. Supernatants, harvested 24 hours post transfection, are filtered through a 0.2 μm filter. Clarified supernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) and centrifuged at 14000 rcf (relative centrifugal force) for 2 hours at 4° C. Norovirus protein content is analyzed by Western Blot suitable customized anti Norovirus-protein antibodies (raised in mouse; 1:500 diluted) as primary antibody in combination with secondary anti mouse antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin is also analyzed as control for cellular contamination (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti rabbit antibody coupled to IRDγe 680RD (Licor Biosciences).
[1007] For the analysis of Norovirus proteins in cell lysates, HeLa cells are transfected with 1 μg and 2 μg unformulated mRNAs (R1-R29, see Table 4) including a negative control encoding an irrelevant protein using Lipofectamine as the transfection agent 24 hours post transfection, HeLa cells are detached by trypsin-free/EDTA buffer, harvested, and cell lysates are prepared. Cell lysates are subjected to SDS-PAGE under non-denaturating/non-reducting followed by western blot detection. Western Blot analysis is performed using a suitable customized anti Norovirus-protein antibodies antibody (raised in mouse; 1:500 diluted) as primary antibody in combination with secondary anti mouse antibody coupled to IRDγe 800CW (Licor Biosciences).
Example 11: Preparation of Norovirus Vaccine Compositions
[1008] For in vivo vaccination experiments, different compositions of Norovirus mRNA vaccine are prepared using Norovirus mRNA constructs (see Table 4). One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated with an aluminum phosphate adjuvant.
[1009] 11.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”; RNActive®):
[1010] Norovirus mRNA constructs are complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes are stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.
[1011] 11.2. Preparation of mRNA with Alum Phosphate (“Vaccine Composition 2”):
[1012] mRNA constructs are mixed with the desired amount of aluminum phosphate adjuvant in Ringer's lactate solution (“naked mRNA”).
Example 12: Vaccination of Mice and Evaluation of Norovirus Specific Immune Response
[1013] 12.1. Immunization
[1014] Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 11) with doses, application routes and vaccination schedules as indicated in Table 10. 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-00009 TABLE 10 Vaccination regimen (Example 12) Vaccination Number Route/ Schedule Group of mice Vaccine composition Volume (day) 1 10 80 μg Norovirus RNActive ® i. d. 0/21/35 Composition 1 2 × 50 μl 2 10 40 μg Norovirus RNActive ® i. d. 0/21/35 Composition 1 2 × 50 μl 3 10 20 μg Norovirus RNActive ® i. d. 0/21/35 Composition 1 2 × 50 μl 4 10 40 μg Norovirus naked RNA i. m. 0/21/35 Composition 2 2 × 25 μl 5 10 40 μg Norovirus naked RNA i. m. 0/21/35 Composition 2 2 × 25 μl 6 10 40 μg Norovirus naked RNA i. m. 0/21/35 Composition 2 2 × 25 μl 7 10 100% RiLa i. d. 0/21/35 Control 2 × 50 μl
[1015] 12.2. Determination of Anti-Norovirus Protein Antibodies by ELISA:
[1016] ELISA is performed using inactivated Norovirus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Norovirus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Norovirus antigens are measured by ELISA on day 63 after three vaccinations.
[1017] 12.3. Intracellular Cytokine Staining
[1018] 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 Norovirus 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), CDB-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.)
[1019] 12.4. Norovirus Plaque Reduction Neutralization Test (PRNT50)
[1020] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice are incubated with Norovirus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.
Example 13: Clinical Development of a Norovirus mRNA Vaccine Composition
[1021] To demonstrate safety and efficiency of the Norovirus mRNA vaccine composition, a randomized, double blind, placebo-controlled clinical trial (phase I) is initiated.
[1022] For clinical development, GMP-grade RNA is produced using an established GMP process, implementing various quality controls on DNA level and RNA level as described in detail in WO 2016/180430A1.
[1023] In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times with a monovalent, or a bivalent, or a tetravalent or a multivalent mRNA based Norovirus vaccine as specified herein.
[1024] In order to assess the safety profile of the Norovirus vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis).
[1025] The efficacy of the immunization is analysed by determination of virus neutralizing titers (VNT) or HBGA blocking titers 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 or HBGA blocking antibodies.
[1026] Furthermore, a subset of subjects is challenged with live GI.1 Norwalk virus or placebo by oral administration. Subjects are followed post-challenge for symptoms of Norovirus associated illness, infection and immune responses. There are multiple clinical assessments and collection of blood, emesis, saliva, and stool specimens.
TABLE-US-00010 Lengthy table referenced here US20210401966A1-20211230-T00001 Please refer to the end of the specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).