HENIPAVIRUS VACCINE
20230226167 · 2023-07-20
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
A61K2039/64
HUMAN NECESSITIES
A61K2039/6093
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
C12N2760/18222
CHEMISTRY; METALLURGY
C12N2760/18234
CHEMISTRY; METALLURGY
International classification
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 Henipavirus, particularly Hendra virus and/or Nipah virus or a disorder related to such an infection. In particular, the present invention concerns a Hendra virus and/or Nipah virus 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 sequence encoding at least one antigenic peptide or protein derived from glycoprotein and/or fusion protein of a Henipavirus or a fragment or variant thereof.
2. The artificial nucleic acid according to claim 1, wherein the Henipavirus is selected from Hendra virus and Nipah virus.
3. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one encoded antigenic peptide or protein comprises at least one of the amino acid sequences derived from Henipavirus being 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 any one of SEQ ID NOs: 1-26, 573-598, 807-832, 1041-1066, 1513-1515 or a fragment or variant or orthologue or paralogue of any of these.
4. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one antigenic peptide or protein comprises at least one of the amino acid sequences derived from Hendra virus being 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 any one of SEQ ID NOs: 8-11, 19-26, 580-583, 591-598, 814-817, 825-832, 1048-1051, 1059-1066, or a fragment or variant or orthologue or paralogue of any of these.
5. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one encoded antigenic peptide or protein comprises at least one of the amino acid sequences derived from Nipah virus being 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 any one of SEQ ID NOs: 1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047, 1052-1058, 1513-1515 or a fragment or variant or orthologue or paralogue of any of these.
6. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence additionally encodes at least one further peptide or protein element selected from a secretory signal peptide, a transmembrane domain, a VLP forming domain, a peptide linker, a self-cleaving peptide, an immunologic adjuvant sequence, and/or a dendritic cell targeting sequence.
7. The artificial nucleic acid according claim 6, wherein the secretory signal peptide comprises an amino acid sequence being 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 any one of SEQ ID NOs: 258-282, 310-316, or a fragment or variant of any of these sequences.
8. The artificial nucleic acid according to to any one of the preceding claims, wherein the at least one coding sequence encodes a heterologuous secretory signal peptide and a Henipavirus antigenic peptide or protein being 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 the amino acid sequences according to SEQ ID NOs: 807-832, 1041-1066, 1513-1515 or a fragment or variant of any of these sequences.
9. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence comprises at least one of the RNA sequences being 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 SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548 or at least one of the RNA sequences which are capable of hybridizing with a complement sequence derived from SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548 or a fragment or variant or orthologue or paralogue of any of these.
10. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence comprises at least one of the RNA sequences being 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 SEQ ID NOs: 34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130, 138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219, 227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739, 762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728, 747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274, 1282-1285, 1293-1300, 1308-1311, 1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378, 1386-1389, 1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467, 1490-1493, 1449-1456, 1475-1482, 1501-1508 or at least one of the RNA sequences which are capable of hybridizing with a complement sequence derived from SEQ ID NOs: 34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130, 138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219, 227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739, 762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728, 747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274, 1282-1285, 1293-1300, 1308-1311, 1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378, 1386-1389, 1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467, 1490-1493, 1449-1456, 1475-1482, 1501-1508 or a fragment or variant or orthologue or paralogue of any of these.
11. The artificial nucleic acid according to any one of the preceding claims, wherein the at least one coding sequence comprises at least one of the RNA sequences being 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 SEQ ID NOs: 27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137, 142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226, 599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761, 781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746, 766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099, 1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255, 1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266, 1275-1281, 1286-1292, 1301-1307, 1312-1318, 1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385, 1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489, 1442-1448, 1468-1474, 1494-1500, 1516-1539, 1540-1548 or at least one of the RNA sequences which are capable of hybridizing with a complement sequence derived from SEQ ID NOs: 27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137, 142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226, 599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761, 781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746, 766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099, 1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255, 1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266, 1275-1281, 1286-1292, 1301-1307, 1312-1318, 1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385, 1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489, 1442-1448, 1468-1474, 1494-1500, 1516-1539, 1540-1548 or a fragment or variant or orthologue or paralogue of any of these.
12. The artificial nucleic acid according to any one of the preceding claims, wherein the artificial nucleic acid is monocistronic, bicistronic or multicistronic.
13. The artificial nucleic acid according to claim 12, wherein the artificial nucleic acid is monocistronic and wherein the coding sequence encodes at least two different Hendra virus and/or Nipah virus antigenic peptides or proteins, or a fragment or variant thereof.
14. The artificial nucleic acid according to claim 12, wherein the artificial nucleic acid is bi- or multicistronic and comprises at least two coding sequences, wherein the at least two coding sequences encode at least two different Hendra virus and/or Nipah virus antigenic peptides or proteins, or a fragment or variant of any of these.
15. The artificial nucleic acid according to any one of the preceding claims, wherein the artificial nucleic acid is an RNA, preferably an mRNA.
16. The artificial nucleic acid according to any one of the preceding claims, wherein the RNA is a modified RNA, preferably a stabilized RNA.
17. The artificial nucleic acid according to any one of the preceding claims, wherein the G/C content of the at least one coding sequence is increased compared to the G/C content of the corresponding wild type coding sequence, and/or wherein the C content of the at least one coding sequence is increased compared to the C content of the corresponding wild type coding sequence and/or wherein the codons in the at least one coding sequence are adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the at least one coding sequence, wherein the amino acid sequence encoded by the at least one coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.
18. 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 NOs: 53-234, 625-806, 859-1040, 1093-1274, 1275-1508, 1519-1539 or a fragment or variant of any of these sequences.
19. 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 NOs: 53-78, 625-635, 859-869, 1093-1103, 636-650, 870-884, 1104-1118, 1275-1508, 1519-1521 or a fragment or variant of any of these sequences.
20. The artificial nucleic acid according to any one of the preceding claims, wherein the artificial nucleic acid comprises a 5′-cap structure.
21. The artificial nucleic acid according to any one of the preceding claims, wherein the artificial nucleic acid comprises at least one histone stem-loop.
22. The artificial nucleic acid according to claims 21, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NOs: 253 or 254, or a fragment or variant thereof.
23. The artificial nucleic acid according to any one of claims 1 to 22, wherein the artificial nucleic acid comprises an untranslated region (UTR).
24. The artificial nucleic acid according to claim 23, wherein the artificial nucleic acid comprises a 3′-UTR.
25. The artificial nucleic acid according to claim 24, wherein the 3′-UTR comprises at least one heterologous 3′-UTR element.
26. The artificial nucleic acid according to claim 24 or 25, wherein the 3′-UTR comprises a poly(A) sequence and/or a poly(C) sequence.
27. The artificial nucleic acid according to claim 26, wherein the poly(A) sequence comprises 10 to 200, 10 to 100, 40 to 200, 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.
28. The artificial nucleic acid according to claims 24 to 27, wherein the 3′-UTR comprises a 3′-terminal sequence element according to SEQ ID NOs: 1509, 1510, 1511 or 1512 or a fragment or variant thereof.
29. The artificial nucleic acid according to any one of claims 25 to 28, 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.
30. The artificial nucleic acid according to claim 29, 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 alpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof.
31. The artificial nucleic acid according to claim 30, 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 NOs: 245 or 246, a homolog, a fragment, or a variant thereof.
32. The artificial nucleic acid according to claim 30, 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 homolog, fragment or variant thereof.
33. The artificial nucleic acid according to claim 32, wherein the at least one heterologous 3′-UTR element comprises a nucleic acid sequence according to SEQ ID NOs: 249-252, or a homolog, a fragment or a variant thereof.
34. The artificial nucleic acid according to any one ofthe preceding claims, wherein the artificial nucleic acid comprises a 5′-UTR.
35. The artificial nucleic acid according to claim 34, wherein the 5′-UTR comprises at least one heterologous 5′-UTR element.
36. The artificial nucleic acid according to claim 35, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from the 5′-UTR of a TOP gene, preferably from a corresponding RNA sequence, or a homolog, a fragment, or a variant thereof, preferably lacking the 5′TOP motif.
37. The artificial nucleic acid according to claim 36, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from a 5′-UTR of a TOP gene encoding a ribosomal protein, preferably from a corresponding RNA sequence, or from a homolog, a fragment or a variant thereof, preferably lacking the 5′TOP motif.
38. The artificial nucleic acid according to claim 36 or 37, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from a 5′-UTR of a TOP gene encoding a ribosomal Large protein (RPL), preferably RPL32 or RPL35A, or from a gene selected from the group consisting of HSD17B4, ATP5A1, AIG1, ASAH1, COX6C or ABCB7 (MDR), or from a homolog, a fragment or variant of any one of these genes, preferably lacking the 5′TOP motif.
39. The artificial nucleic acid according to any one of claims 36 to 38, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence according to SEQ ID NOs: 235-238, or a homolog, a fragment or a variant thereof.
40. The artificial nucleic acid according to any one of the preceding claims comprising, preferably in 5′ to 3′ direction, the following elements a)-h): a) 5′-cap structure, preferably as defined by claim 20; b) optionally, 5′-UTR element, preferably as defined by any one of claims 34 to 39; c) at least one coding sequence, preferably as defined by any one of claims 1 to 19; d) a 3′-UTR element, preferably as defined by any one of claims 24 to 33; e) optionally, poly(A) sequence, preferably as defined by any one of claims 27; f) optionally, poly(C) sequence, preferably as defined by any one of claims 27; g) optionally, a histone stem-loop, preferably as defined by any one of claims 21 to 22; and h) optionally, a 3′-terminal sequence element as defined by claim 28.
41. The artificial nucleic acid according to any one of the preceding items, wherein the nucleic acid sequence comprises or consists of a nucleic acid sequence selected from sequences being 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 the mRNA sequences according to SEQ ID NOs: 1275-1508, 1540-1548 or a fragment or variant thereof.
42. The artificial nucleic acid according to any one of the preceding items, wherein the nucleic acid sequence comprises or consists of a nucleic acid sequence selected from sequences being 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 the mRNA sequences according to SEQ ID NOs: 1282-1285, 1293-1300, 1308-1311, 1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378, 1386-1389, 1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467, 1490-1493, 1449-1456, 1475-1482, 1501-1508 or a fragment or variant thereof.
43. The artificial nucleic acid according to claims 41, wherein the nucleic acid sequence comprises or consists of a nucleic acid sequence selected from sequences being 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 the mRNA sequences according to SEQ ID NOs: 1275-1281,1286-1292,1301 -1307,1312-1318,1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385, 1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489, 1442-1448, 1468-1474, 1494-1500, 1540-1548 or a fragment or variant thereof.
44. Composition comprising at least one artificial nucleic acid as defined by any one of claims 1 to 43 and at least one pharmaceutically acceptable carrier.
45. The composition according to claim 44, comprising a plurality or at least more than one of the artificial nucleic acids as defined by any one of claims 1 to 43.
46. The composition according to claim 45, wherein each of the artificial nucleic acids encodes a different antigenic peptide or protein derived from a Henipavirus and/or Hendra virus and/or Nipah virus or from a homolog, fragment or variant thereof.
47. The composition according to claim 45, wherein each of the artificial nucleic acids encodes a different antigenic peptide or protein derived from genetically the same Henipavirus and/or Hendra virus and/or Nipah virus or from a homolog, fragment or variant thereof.
48. The composition according to claim 45, wherein each of the artificial nucleic acids encodes a different antigenic peptide or protein derived from a genetically different Henipavirus and/or Hendra virus and/or Nipah virus or from a homolog, fragment or variant thereof.
49. The composition according to any one of claims 44 to 48, wherein the at least one artificial nucleic acid is complexed with one or more cationic or polycationic component, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g. protamine, cationic or polycationic lipid.
50. The composition according to claim 49, wherein the N/P ratio of the at least one artificial nucleic acid to the one or more cationic or polycationic components 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.
51. The composition according to claim 49 or 50, wherein the at least one artificial nucleic acid 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 nucleic acid to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of nucleic acid to cationic or polycationic component 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.
52. The composition according to any one of claims 49 to 51 comprising the at least one artificial nucleic acid, which is complexed with one or more cationic or polycationic compounds, and at least one free artificial nucleic acid.
53. The composition according to claim 52, wherein the at least one complexed artificial nucleic acid is identical to the at least one free artificial nucleic acid.
54. The composition according to claim 52 or 53, wherein the molar ratio of the complexed nucleic acid to the free nucleic acid is selected from a molar ratio of about 0.001 :1 to about 1 :0.001, including a ratio of about 1:1.
55. The composition according to any one of claims 52 to 54, wherein 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).
56. The composition according to any one of claims 44 to 55, wherein the artificial nucleic acid is complexed with one or more polysaccharides.
57. The composition according to any one of claims 44 to 55, wherein the artificial nucleic acid is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.
58. The composition according to any one of claims 44 to 57, wherein the composition comprises at least one adjuvant component.
59. Polypeptide encoded by the artificial nucleic acid according to any one of claims 1 to 43.
60. Vaccine comprising the artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, or the polypeptide as defined in claim 59.
61. The vaccine according to claim 60, wherein the artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, or the polypeptide as defined in claim 59, elicits an adaptive immune response.
62. The vaccine according to claim 60 or 61, wherein the vaccine further comprises a pharmaceutically acceptable carrier and optionally at least one adjuvant as defined in claim 58.
63. Kit or kit of parts comprising the artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, the polypeptide as defined in claim 59, or the vaccine as defined in any one of claims 60 to 62, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.
64. The kit or kit of parts according to claim 63 further comprising Ringer lactate solution.
65. The artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, the polypeptide as defined in claim 59, the vaccine as defined in any one of claims 60 to 62, or the kit or kit of parts as defined in claim 63 or 64 for use as a medicament.
66. The artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, the polypeptide as defined in claim 59, the vaccine as defined in any one of claims 60 to 62, or the kit or kit of parts as defined in claim 63 or 64 for use in the treatment or prophylaxis of an infection with Henipavirus or a disorder related to an infection with Henipavirus.
67. The use according to claim 66, wherein the Henipavirus is Hendra virus and/or Nipah virus.
68. The artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, the polypeptide as defined in claim 59, or the vaccine as defined in any one of claims 60 to 62, or the kit or kit of parts as defined in claim 63 or 64 for use as defined according to claim 65 to 67, wherein the artificial nucleic acid, the composition, the polypeptide, the composition comprising the polypeptide, the vaccine or the active component of the kit or kit of parts is administered by injection, preferably by needle-less injection, more preferably by jet injection.
69. Method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof the artificial nucleic acid as defined in any one of claims 1 to 43, the composition as defined in any one of claims 44 to 58, the polypeptide as defined in claim 59, or the vaccine as defined in any one of claims 60 to 62, or the kit or kit of parts as defined in claim 63 or 64.
70. The method according to claim 69, wherein the disorder is an infection with Henipavirus or a disorder related to an infection with Henipavirus.
71. The method according to claim 70, wherein the Henipavirus is Hendra virus and/or Nipah virus.
72. The method according to claim 69 to 71, wherein the applying or administering to a subject in need is carried out by injection, wherein injection is carried out by using conventional needle injection or jet injection.
73. The method according to claim 69 to 72, wherein the subject in need is a mammalian subject or an avian subject.
74. The method according to claim 73, wherein the mammal subject is a human subject.
Description
BRIEF DECRIPTION OF THE DRAWINGS
[0709]
[0710]
EXAMPLES
[0711] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.
Example 1: Preparation of mRNA Constructs for In Vitro and In Vivo Experiments
[0712] For the present examples, DNA sequences encoding Nipah virus proteins as well as DNA sequences encoding Hendra virus proteins are prepared and used for subsequent RNA in vitro transcription reactions. The generated coding sequences (RNA sequences) are provided in the sequence listing (SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274, 1275-1508). DNA sequences are prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithms that increase the GC content of the respective coding sequence. Moreover, sequences are introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 1” in Table 5, Table 6, Table 7). Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L4 5′-UTR ribosomal 5′TOP UTR and 3′-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 2” in Table 5, Table 6, Table 7). The obtained plasmid DNA constructs are transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.
RNA In Vitro Transcription on Linearized pDNA
[0713] The DNA plasmids prepared according to paragraph 1 are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. RNA production is performed under current good manufacturing practice according to WO2016180430. The obtained mRNAs are purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008077592) and used for in vitro and in vivo experiments.
RNA In Vitro Transcription on PCR Amplified DNA Templates
[0714] DNA plasmids prepared according to paragraph 1, or synthic DNA constructs are used for PCR-amplification. The generated PCR templates are used for subsequent RNA in vitro transcription using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. The obtained mRNA constructs are purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008077592) and used for in vitro and in vivo experiments. The generated mRNA constructs are indicated as “mRNA design 3” Table 5 and Table 6.
TABLE-US-00010 mRNA constructs used in the Example section: Name SEQ ID NO: Protein mRNA NIPAV(Malaysia) 1 SEQ ID NO: 1353 mRNA design 2; opt1 NIPAV(Malaysia) 12 SEQ ID NO: 1364 mRNA design 2; opt1 NIPAV(Bangladesh2004) 3 SEQ ID NO: 1355 mRNA design 2; opt1 NIPAV(Bangladesh2004) 13 SEQ ID NO: 1365 mRNA design 2; opt1 HeV(Horse-Autralia-Hendra-1994)-F 8 SEQ ID NO: 1360 mRNA design 2; opt1 HeV(Horse-Autralia-Hendra-1994)-G 19 SEQ ID NO: 1371 mRNA design 2; opt1 IgE-leader(GC)_HeV(Horse-Autralia-Hendra-1994)-G(71-604) 825 SEQ ID NO: 1397 mRNA design 2; opt1 IgE-leader_Nipha(Bangladesh2004)-F 809 SEQ ID NO: 1381 mRNA design 2; opt1 SP-Influenza-HA_Nipha(Bangladesh2004)-F 1043 SEQ ID NO: 1407 mRNA design 2; opt1 SP-Osteonectin BM40_Nipha(Bangladesh2004)-F 1513 SEQ ID NO: 1543 mRNA design 2; opt1 SP-HsChemotripsinogen_Nipha(Bangladesh2004) 1514 SEQ ID NO: 1544 mRNA design 2; opt1 SP-Nipha(Malaysia1999)- F(1-26)_Nipha(Bangladesh2004)-F(27-546) 1515 SEQ ID NO: 1545 mRNA design 2; opt1
Example 2: Vaccination of Mice With mRNA Encoding Nipah
[0715] The results of the present Example shows that mRNA encoding Nipah virus F protein (NIV F Malaysia 1999; R6311) is expressed in mice after intradermal injection. In addition, the expressed Nipah virus F protein provided by the inventive mRNA of the invention induces specific humoral immune responses after immunization in mice.
Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”)
[0716] Nipah virus mRNA construct (SEQ ID NO: 1353) was prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA was complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer’s lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer’s lactate solution.
Immunization:
[0717] Female BALB/c mice were injected intradermally (i.d.) with mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table A. As a negative control, one group of mice was vaccinated with buffer (ringer lactate). All animals were vaccinated on day 0, 21 and 42. Blood samples were collected on day 21, 35, and 56 for the determination of antibody titers.
TABLE-US-00011 Vaccination regimen (Example 2): Number of mice Vaccine composition Dose Route/Volume 10 NIV F (Malaysia 1999) R6311 ; Vaccine composition 1 80 .Math.g i.d. 2×50 .Math.l 10 100% RiLa Control i.d. 2×25 .Math.l
Detection of Specific Humoral Immune Responses
[0718] Hela cells were transfected with 2 .Math.g of either R6311 vaccine composition using lipofectamine. The cells were harvested 20 h post transfection, and seeded at 1×10.sup.5 per well into an 96 well plate. The cells were incubated with sera of R6311 vaccinated mice (diluted 1:50) followed by aFITC-conjugated anti-mouse IgG antibody. Cells were aquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.
Results
[0719] As shown in
[0720] The results exemplify that the inventive mRNA-based Nipah virus vaccine works and that similar mRNA vaccines comprising alternative mRNA constructs according to the invention may also be suitably used.
Example 3: Expression Analysis of Nipah Virus and Hendra Virus G Proteins Using Western Blot
[0721] The results of the present Example shows that mRNA encoding Nipah virus G protein and Hendra virus G protein are expressed in HeLa cells after transfection.
[0722] For the analysis of Nipah virus protein and Hendra virus G protein expression, HeLa cells were transfected with 2 .Math.g unformulated mRNA (wfi as negative control) using Lipofectamine as the transfection agent 20 hours post transfection, HeLa cells were detached by trypsin-free/EDTA buffer, harvested, and cell lysates were prepared. Cell lysates were subjected to SDS-PAGE followed by western blot detection. Western Blot analysis was performed using an anti-NIV G protein polyclonal IgG serum fraction (custom made by through immunization of rabbits with peptides from NIV G (with x-reactivity to HeV G protein)) used in a 1:200 dilution in combination with secondary anti rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin was analyzed (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti rabbit antibody coupled to IRDye 680RD (Licor Biosciences). Inactivated Nipah virus was used as positive control for the western blot (indicated as “ctr” in
TABLE-US-00012 Expression analysis experiment (Example 2): Lane SEQ ID NO Transfected composition 1 1364 Nipah virus G (Malaysia) R6003 2 1365 Nipah virus G (Bangladesh) R6007 3 1371 Hendra virus G R6011 4 -- wfi
Results
[0723] As shown in
Example 4: Expression of Nipah Virus and Hendra Virus Proteins in HeLa Cells and Analysis by FACS
[0724] To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Nipah virus (NiV) and Hendra virus (HeV) antigens and stained using suitable customized anti-NiV antibodies (raised in mouse) and anti-HeV antibodies, counterstained with a FITC-coupled secondary antibody (F5262 from Sigma). HeLa cells are seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1 % Pen/Strep), 24 h prior to transfection. HeLa cells are transfected with 1 and 2 .Math.g unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs according to Example 1 are used in the experiment, including a negative control encoding an irrelevant protein. 24 hours post transfection, HeLa cells are stained with suitable anti anti-NiV or anti-HeV antibodies (raised in mouse; 1 :500) and anti-mouse FITC labelled secondary antibody (1 :500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).
Example 5: Analysis of Expression and Secretion of Nipah Virus and Hendra Virus Proteins Using Western Blot
[0725] For the analysis of Nipah virus protein and Hendra virus protein secretion, HeLa cells are transfected with 1 .Math.g and 2 .Math.g unformulated mRNA (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 .Math.m 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. Protein content is analyzed by Western Blot using anti-NiV and anti-HeV antibodies 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 IRDye 680RD (Licor Biosciences). For the analysis of NiV and HeV proteins in cell lysates, HeLa cells are transfected with 1 .Math.g and 2 .Math.g unformulated mRNAs (generated according to Example 1) 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 anti NiV and anti-HeV antibodies as primary antibody in combination with secondary anti mouse antibody coupled to IRDye 800CW (Licor Biosciences).
Example 6: Preparation of Nipah Virus and Hendra Virus Vaccine Compositions
[0726] For further in vivo vaccination experiments, different compositions of Nipah virus mRNA vaccine and Hendra virus mRNA vaccine are prepared using constructs obtained in Example 1. One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated without protamine (“naked”), one composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs), and one composition comprises polymer-lipidoid complexed mRNA.
[0727] Nipah virus and Hendra virus mRNA constructs are complexed as described in Example 2.
[0728] Nipah virus and Hendra virus mRNA constructs are formulated without protamine. The final concentration of the vaccine is adjusted with Ringer’s lactate solution.
Preparation of LNP Encapsulated mRNA (“Vaccine Composition 3”)
[0729] A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 .Math.m pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).
Preparation of Polymer-Lipidoid Complexed mRNA (“Vaccine Composition 4”)
[0730] 20 mg peptide (CHHHHHHRRRRHHHHHHC-NH2; SEQ ID NO: 309) TFA salt is 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-amino acid component: SEQ ID NO: 309) are used for further formulation and are hereinafter referred to as PB83.
[0731] Preparation of 3-C12-OH lipidoid: First, lipidoid 3-C12 was obtained by acylation of tris(2-aminoethyl)amine with an activated lauric (C12) acid derivative, followed by reduction of the amide. Alternatively, it may be prepared by reductive amination with the corresponding aldehyde. Lipidoid 3-C12-OH was prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5.
[0732] Preparation of compositions with nanoparticles of polymer-lipidoid complexed mRNA: First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of a polymer (PB83) are mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the carrier compositions are used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier and allowing an incubation period of 10 minutes at room temperature such as to enable the formation of a complex between the lipidoid, polymer and mRNA. In order to characterize the integrity of the obtained polymer-lipidoid complexed mRNA particles, RNA agarose gel shift assays are performed. In addition, size measurements are performed (gel shift assay, Zetasizer) to evaluate whether the obtained nanoparticles have a uniform size profile.
Example 7: Vaccination of Mice and Evaluation of Nipah Virus Specific Immune Response
[0733] Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 6) with doses, application routes and vaccination schedules as indicated in Table C. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below).
TABLE-US-00013 Vaccination regimen - Nipah virus experiment (Example 7) Group Number of mice Vaccine composition Route/ Volume Vaccination Schedule (day) 1 10 40 .Math.g Nipah virus RNA Composition 1 i. d. 2 × 25 .Math.l 0/21/35 2 10 40 .Math.g Nipah virus RNA Composition 1 i. m. 2 × 25 .Math.l 0/21/35 3 10 20 .Math.g Nipah virus RNA Composition 2 i. d. 2 × 25 .Math.l 0/21/35 4 10 20 .Math.g Nipah virus RNA Composition 2 i. m. 2 × 25 .Math.l 0/21/35 5 10 10 .Math.g Nipah virus RNA Composition 3 i. d. 2 × 25 .Math.l 0/21/35 6 10 10 .Math.g Nipah virus RNA Composition 3 i. m. 2 × 25 .Math.l 0/21/35 7 10 100% RiLa Control i. d. 2 × 25 .Math.l 0/21/35
Determination of Anti Nipah Virus Protein Antibodies by ELISA
[0734] ELISA is performed using inactivated Nipah virus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Nipah virus 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 Nipah virus antigens are measured by ELISA on day 63 after three vaccinations.
Intracellular Cytokine Staining
[0735] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1%FBS followed by red blood cell lysis. After an extensive washing step with PBS/1%FBS splenocytes are seeded into 96-well plates (2 × 10.sup.6 cells per well). The cells are stimulated with a mixture of four Nipah virus protein specific peptide epitopes (5 .Math.g/ml of each peptide) in the presence of 2.5 .Math.g/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of a protein transport inhibitor. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer’s instructions. The following antibodies are used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1 :200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcy-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.)
Nipah Virus Plaque Reduction Neutralization Test (PRNT50)
[0736] 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 Nipah virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.
Example 8: Vaccination of Mice and Evaluation of Hendra Virus Specific Immune Response
[0737] Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 6) with doses, application routes and vaccination schedules as indicated in Table D. 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).
Determination of Anti Hendra Virus Protein Antibodies by ELISA
[0738] ELISA is performed using inactivated Hendra virus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Hendra virus 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 Hendra virus antigens are measured by ELISA on day 63 after three vaccinations.
TABLE-US-00014 Vaccination regimen – Hendra virus experiment (Example 8): Group Number of mice Vaccine composition Route/ Volume Vaccination Schedule (day) 1 10 40 .Math.g Hendra virus RNA Composition 1 i.d. 2 × 25 .Math.l 0/21/35 2 10 40 .Math.g Hendra virus RNA Composition 1 i.m. 2 × 25 .Math.l 0/21/35 3 10 20 .Math.g Hendra virus RNA Composition 2 i.d. 2 × 25 .Math.l 0/21/35 4 10 20 .Math.g Hendra virus RNA Composition 2 i.m. 2 × 25 .Math.l 0/21/35 5 10 10 .Math.g Hendra virus RNA Composition 3 i.d. 2 × 25 .Math.l 0/21/35 6 10 10 .Math.g Hendra virus RNA Composition 3 i.m. 2 × 25 .Math.l 0/21/35 7 10 100% RiLa Control i.d. 2 × 25 .Math.l 0/21/35
Intracellular Cytokine Staining
[0739] 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 Hendra virus protein specific peptide epitopes (5 .Math.g/ml of each peptide) in the presence of 2.5 .Math.g/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of a protein transport inhibitor. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer’s instructions. The following antibodies are used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNy-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcy-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.)
Nipah Virus Plaque Reduction Neutralization Test (PRNT50)
[0740] 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 Hendra virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.
Example 9: Vaccination of Mice Using Polymer-Lipidoid Complexed RNA and Evaluation of Immune Response
[0741] Female BALB/c mice are injected intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 6) with doses, application routes and vaccination schedules as indicated in Table F. 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).
Evaluation of Specific Immune Responses
[0742] ELISA is performed using inactivated Hendra virus or Nipah virus infected cell lysate for coating (as described above). Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art and intracellular cytokine staining is performed as described above. In addition, sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as described above.
TABLE-US-00015 Vaccination regimen (Example 9): Group Number of mice Vaccine composition Route/ Volume Vaccination Schedule (day) 1 10 40 .Math.g Hendra virus RNA Composition 4 i.m. 2 × 25 .Math.l 0/21/35 2 10 40 .Math.g Nipah virus RNA Composition 4 i.m. 2 × 25 .Math.l 0/21/35 3 10 100% RiLa Control i.d. 2 × 25 .Math.l 0/21/35
Example 10: Clinical Development of a Nipah Virus and Hendra Virus mRNA Vaccine Composition
[0743] To demonstrate safety and efficiency of the Nipah virus and Hendra virus mRNA vaccine composition, a clinical trial (phase I) is initiated. In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times. In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis). The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.