ROTAVIRUS MRNA VACCINE
20220313813 · 2022-10-06
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2720/12322
CHEMISTRY; METALLURGY
C12N2720/12334
CHEMISTRY; METALLURGY
A61K2039/55555
HUMAN NECESSITIES
International classification
Abstract
The invention is directed to a coding RNA for a Rotavirus vaccine. The coding RNA comprises at least one coding region encoding at least one antigenic peptide or protein of a Rotavirus, in particular VPS* of a Rotavirus, or immunogenic fragment or immunogenic variant thereof. The present invention is also directed to compositions and vaccines comprising said coding RNA in association with a polymeric carrier, a polycationic protein or peptide, or a lipid nanoparticle (LNP). Further, the invention concerns a kit, particularly a kit of parts comprising the coding RNA, or the composition, or the vaccine. The invention is also directed to a kit or kit of parts, medical treatments, and the first and second medical uses.
Claims
1. A coding RNA for a Rotavirus vaccine comprising a) at least one heterologous 5′ untranslated region (5′-UTR) and/or at least one heterologous 3′ untranslated region (3′-UTR); and b) at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic protein of a Rotavirus, wherein said antigenic protein is or is derived from VP8* or an immunogenic fragment or immunogenic variant thereof.
2. Coding RNA of claim 1, wherein the Rotavirus is selected from species A, B or C, preferably wherein the Rotavirus is Rotavirus A.
3. Coding RNA of claim 1 or 2, wherein the Rotavirus is selected from the G-serotypes or P-serotypes G1, G2, G3, G4, G9, G12, P[4], P[6] or P[8].
4. Coding RNA of any one of the preceding claims, wherein the Rotavirus is a Rotavirus A selected from the P-serotypes P[4], P[6] or P[8].
5. Coding RNA of any one of the preceding claims, wherein the Rotavirus is a Rotavirus A selected from Human rotavirus A BE1058 (RVA/Human-wt/BEL/BE1058/2008/G2P[4], G2P[4], JN849123.1, GI:371455744, AEX30665.1, acronym: RVA/BE1058/P[4]), Human rotavirus A F01322 (Hu/BEL/F01322/2009/G3P[6], G3P[6], JF460826.1, GI: 37531451, AFA51886.1, acronym: RVA/F01322/P[6]), Human rotavirus A BE1128 (RVA/Human-wt/BEL/BE1128/2009/G1P[8], G1P[8], JN849135.1. GI: 371455756, AEX30671, acronym: RVA/BE1128/P[8]), or Human rotavirus A WA-VirWa (Wa variant VirWa, G1P[8], ACR22783.1, GI: 237846292, FJ423116, acronym: RVA/Wa-VirWa/P[8]).
6. Coding RNA of any one of the preceding claims, wherein the VP8* is a full length VP8* protein having an amino acid sequence comprising or consisting of amino acid 1 to amino acid 240, or a fragment of a VP8* protein.
7. Coding RNA of any one of claim 6, wherein the fragment of a VP8* comprises the lectin domain and lacks the N-terminal alpha helix-domain.
8. Coding RNA of any one of the preceding claims, wherein the amino acid sequences of the at least one antigenic protein derived from VP8* is mutated to delete at least one predicted or potential glycosylation site.
9. Coding RNA of any one of the preceding claims, wherein the amino acid sequences of the at least one antigenic protein derived from VP8* is mutated to delete all predicted or potential glycosylation sites.
10. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 19-45, or an immunogenic fragment or immunogenic variant of any of these.
11. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence additionally encodes one or more heterologous peptide or protein elements selected from a signal peptide, a linker, a helper epitope, an antigen clustering domain, or a transmembrane domain.
12. Coding RNA of claim 11, wherein the signal peptide is or is derived from HsPLAT, HsALB, IgE, wherein the amino acid sequences of said heterologous signal peptides is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of amino acid sequences SEQ ID NOs: 1738-1740, or fragment or variant of any of these.
13. Coding RNA of claim 11, wherein the helper epitope is or is derived from P2, wherein the amino acid sequences of said helper epitopes is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequence SEQ ID NOs: 1750, or fragment or variant thereof.
14. Coding RNA of claim 11, wherein the antigen clustering domain is or is derived from ferritin or lumazine-synthase, wherein the amino acid sequences of said antigen clustering domain is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of amino acid sequences SEQ ID NOs1759, 1764, or fragment or variant of any of these.
15. Coding RNA of claim 11, wherein the transmembrane domain is or is derived from an influenza HA transmembrane domain, preferably derived from an influenza A HA H1N1, more preferably from H1N1/A/Netherlands/602/2009, TM domain_HA, aa521-566, NCBI Acc. No.: ACQ45338.1, CY039527.1), or fragment or variant thereof.
16. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence encodes the following elements preferably in N-terminal to C-terminal direction: a) helper epitope, VP8*protein or VP8*fragment; or b) helper epitope, VP8*protein or VP8*fragment; antigen clustering domain; or c) Signal peptide, helper epitope, VP8*protein or fragment thereof; or d) Signal peptide, helper epitope, VP8*protein or VP8*fragment, antigen clustering domain; or e) Signal peptide, helper epitope, VP8*protein or VP8*fragment, transmembrane domain; or f) antigen clustering domain, helper epitope; VP8*protein or VP8*fragment.
17. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-6, 46-117, 1899, 1900, or an immunogenic fragment or immunogenic variant of any of these.
18. Coding RNA of claim 17, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-3, 4-6, 46-54, 64-72, 91-99, 109-117, or an immunogenic fragment or immunogenic variant of any of these.
19. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence comprises a codon modified coding sequence comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 190-261, 298-369, 406-477, 514-585, 1901-1906, or a fragment or variant of any of these sequences.
20. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence comprises at least one modified nucleotide selected from pseudouridine (LP) and N1-methylpseudouridine (ml P), preferably wherein all uracil nucleotides are replaced by pseudouridine (V) nucleotides and/or N1-methylpseudouridine (ml 4) nucleotides.
21. Coding RNA of any one of the preceding claims, wherein the at least one coding sequence is a codon modified coding sequence, wherein the amino acid sequence encoded by the at least one codon modified coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.
22. Coding RNA according to claim 21, wherein the at least one codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.
23. Coding RNA of claim 21 or 22, wherein the at least one coding sequence comprises or consists of a codon modified coding sequence comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 154-585, 1901-1906 or a fragment or variant of any of these sequences.
24. Coding RNA of any one of claims 21 to 23, wherein the at least one coding sequence comprises or consists of a codon modified coding sequence comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 190-198, 208-216, 235-243, 253-261, 298-306, 316-324, 343-351, 361-369, 1901-1906 or a fragment or variant of any of these sequences.
25. Coding RNA of any one of the preceding claims, wherein the coding RNA is an mRNA, a self-replicating RNA, a circular RNA, a viral RNA, or a replicon RNA.
26. Coding RNA of any one of the preceding claims, wherein the coding RNA is an mRNA.
27. Coding RNA of any one of the preceding claims, wherein the coding RNA comprises a 5′-cap structure, preferably cap0, cap1, cap2, a modified cap0 or a modified cap1 structure.
28. Coding RNA of claim 27, wherein the a 5′-cap structure is a cap1 structure,
29. Coding RNA of any one of the preceding claims, wherein the coding RNA comprises a cap1 structure, wherein said cap1 structure is obtainable by co-transcriptional capping preferably using a trinucleotide cap1 analogue.
30. Coding RNA of any one of claim 27 to 29, wherein about 70%, 75%, 80%, 85%, 90%, 95% of the coding RNA (species) comprises a cap1 structure as determined using a capping assay.
31. Coding RNA of any one of the preceding claims, wherein the coding RNA comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides, preferably comprising about 100 adenosine nucleotides.
32. Coding RNA of claim 31, wherein the at least one poly(A) sequence is located at the 3′ terminus, preferably wherein the 3′ terminal nucleotide of the coding RNA is the 3′ terminal A nucleotide of the poly(A) sequence.
33. Coding RNA of any one of the preceding claims, wherein the coding RNA comprises a cap1 structure as defined in claims 27 to 30 and at least one poly(A) sequence as defined in claims 31 to 32.
34. Coding RNA of any one of the preceding claims, wherein the RNA comprises at least one histone stem-loop, wherein the histone stem-loop preferably comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1819 or 1820, or fragments or variants thereof.
35. Coding RNA of any one of the preceding claims, wherein the RNA comprises at least one 3′ terminal sequence element comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1825-1856, or a fragment or variant thereof.
36. Coding RNA of any one of the preceding claims, wherein the at least one heterologous 3′-UTR comprises or consisting of a nucleic acid sequence derived from a 3′-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes.
37. Coding RNA of any one of the preceding claims, wherein the at least one heterologous 5′-UTR comprises or consisting of a nucleic acid sequence derived from a 5′-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.
38. Coding RNA of any one of the preceding claims, wherein the at least one heterologous 5′-UTR is derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and the at least one 3-UTR is derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof, preferably wherein said 5′-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1781 or 1782 or a fragment or a variant thereof, and wherein said 3′-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1803 or 1804 or a fragment or a variant thereof; or the at least one heterologous 3′-UTR is derived from a 3′-UTR of a alpha-globin gene gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof, preferably wherein said 3′-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1817 or 1818 or a fragment or a variant thereof.
39. Coding RNA of any one of the preceding claims, wherein the coding RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 586-1737, 1862-1882, 1885-1898, 1907-1930 or a fragment or variant of any of these sequences.
40. Coding RNA of claim 39, wherein the coding RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 586-594, 604-612, 631-639, 649-666, 676-684, 703-711, 721-738, 748-756, 775-783, 793-810, 820-828, 847-855, 865-882, 892-900, 919-927, 937-954, 964-972, 991-999, 1009-1026, 1036-1044, 1063-1071, 1081-1098, 1108-1116, 1135-1143, 1153-1170, 1180-1188, 1207-1215, 1225-1242, 1252-1260, 1279-1287, 1297-1314, 1324-1332, 1351-1359, 1369-1386, 1396-1404, 1423-1431, 1441-1458, 1468-1476, 1495-1503, 1513-1530, 1540-1548, 1567-1575, 1585-1602, 1612-1620, 1639-1647, 1657-1674, 1684-1692, 1711-1719, 1729-1737, 1862-1870, 1872-1877, 1885, 1898, 1907-1930 or a fragment or variant of any of these sequences.
41. A composition comprising at least one coding RNA as defined in any one of claims 1 to 40, wherein the composition optionally comprises at least one pharmaceutically acceptable carrier or excipient.
42. Composition of claim 41, wherein the composition comprises more than one or a plurality, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 different coding RNAs each defined in any one of claims 1 to 40.
43. Composition of claim 42, wherein the composition comprises (i) at least one coding RNA encoding at least one antigenic protein that is or is derived from VP8* of a Rotavirus A from a P[4] serotype, preferably according to SEQ ID Nos: 586-588, 595-597, 604-606, 613-615, 622-624, 631-633, 640-642, 649-651, 658-660, 667-669, 676-678, 685-687, 694-696, 703-705, 712-714, 721-723, 730-732, 739-741, 748-750, 757-759, 766-768, 775-777, 784-786, 793-795, 802-804, 811-813, 820-822, 829-831, 838-840, 847-849, 856-858, 865-867, 874-876, 883-885, 892-894, 901-903, 910-912, 919-921, 928-930, 937-939, 946-948, 955-957, 964-966, 973-975, 982-984, 991-993, 1000-1002, 1009-1011, 1018-1020, 1027-1029, 1036-1038, 1045-1047, 1054-1056, 1063-1065, 1072-1074, 1081-1083, 1090-1092, 1099-1101, 1108-1110, 1117-1119, 1126-1128, 1135-1137, 1144-1146, 1153-1155, 1162-1164, 1171-1173, 1180-1182, 1189-1191, 1198-1200, 1207-1209, 1216-1218, 1225-1227, 1234-1236, 1243-1245, 1252-1254, 1261-1263, 1270-1272, 1279-1281, 1288-1290, 1297-1299, 1306-1308, 1315-1317, 1324-1326, 1333-1335, 1342-1344, 1351-1353, 1360-1362, 1369-1371, 1378-1380, 1387-1389, 1396-1398, 1405-1407, 1414-1416, 1423-1425, 1432-1434, 1441-1443, 1450-1452, 1459-1461, 1468-1470, 1477-1479, 1486-1488, 1495-1497, 1504-1506, 1513-1515, 1522-1524, 1531-1533, 1540-1542, 1549-1551, 1558-1560, 1567-1569, 1576-1578, 1585-1587, 1594-1596, 1603-1605, 1612-1614, 1621-1623, 1630-1632, 1639-1641, 1648-1650, 1657-1659, 1666-1668, 1675-1677, 1684-1686, 1693-1695, 1702-1704, 1711-1713, 1720-1722, 1729-1731, 1886, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929 or fragments or variants thereof; and (ii) at least one coding RNA encoding at least one antigenic protein that is or is derived from VP8* of a Rotavirus A from a P[6] serotype, preferably according to SEQ ID Nos: 589, 590, 598, 599, 607, 608, 616, 617, 625, 626, 634, 635, 643, 644, 652, 653, 661, 662, 670, 671, 679, 680, 688, 689, 697, 698, 706, 707, 715, 716, 724, 725, 733, 734, 742, 743, 751, 752, 760, 761, 769, 770, 778, 779, 787, 788, 796, 797, 805, 806, 814, 815, 823, 824, 832, 833, 841, 842, 850, 851, 859, 860, 868, 869, 877, 878, 886, 887, 895, 896, 904, 905, 913, 914, 922, 923, 931, 932, 940, 941, 949, 950, 958, 959, 967, 968, 976, 977, 985, 986, 994, 995, 1003, 1004, 1012, 1013, 1021, 1022, 1030, 1031, 1039, 1040, 1048, 1049, 1057, 1058, 1066, 1067, 1075, 1076, 1084, 1085, 1093, 1094, 1102, 1103, 1111, 1112, 1120, 1121, 1129, 1130, 1138, 1139, 1147, 1148, 1156, 1157, 1165, 1166, 1174, 1175, 1183, 1184, 1192, 1193, 1201, 1202, 1210, 1211, 1219, 1220, 1228, 1229, 1237, 1238, 1246, 1247, 1255, 1256, 1264, 1265, 1273, 1274, 1282, 1283, 1291, 1292, 1300, 1301, 1309, 1310, 1318, 1319, 1327, 1328, 1336, 1337, 1345, 1346, 1354, 1355, 1363, 1364, 1372, 1373, 1381, 1382, 1390, 1391, 1399, 1400, 1408, 1409, 1417, 1418, 1426, 1427, 1435, 1436, 1444, 1445, 1453, 1454, 1462, 1463, 1471, 1472, 1480, 1481, 1489, 1490, 1498, 1499, 1507, 1508, 1516, 1517, 1525, 1526, 1534, 1535, 1543, 1544, 1552, 1553, 1561, 1562, 1570, 1571, 1579, 1580, 1588, 1589, 1597, 1598, 1606, 1607, 1615, 1616, 1624, 1625, 1633, 1634, 1642, 1643, 1651, 1652, 1660, 1661, 1669, 1670, 1678, 1679, 1687, 1688, 1696, 1697, 1705, 1706, 1714, 1715, 1723, 1724, 1732, 1733, 1887, 1890, 1895-1897, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930 or fragments or variants thereof; and (iii) at least one coding RNA encoding at least one antigenic protein that is or is derived from VP8* of a Rotavirus A from a P[8] serotype, preferably according to SEQ ID Nos: 591-594, 600-603, 609-612, 618-621, 627-630, 636-639, 645-648, 654-657, 663-666, 672-675, 681-684, 690-693, 699-702, 708-711, 717-720, 726-729, 735-738, 744-747, 753-756, 762-765, 771-774, 780-783, 789-792, 798-801, 807-810, 816-819, 825-828, 834-837, 843-846, 852-855, 861-864, 870-873, 879-882, 888-891, 897-900, 906-909, 915-918, 924-927, 933-936, 942-945, 951-954, 960-963, 969-972, 978-981, 987-990, 996-999, 1005-1008, 1014-1017, 1023-1026, 1032-1035, 1041-1044, 1050-1053, 1059-1062, 1068-1071, 1077-1080, 1086-1089, 1095-1098, 1104-1107, 1113-1116, 1122-1125, 1131-1134, 1140-1143, 1149-1152, 1158-1161, 1167-1170, 1176-1179, 1185-1188, 1194-1197, 1203-1206, 1212-1215, 1221-1224, 1230-1233, 1239-1242, 1248-1251, 1257-1260, 1266-1269, 1275-1278, 1284-1287, 1293-1296, 1302-1305, 1311-1314, 1320-1323, 1329-1332, 1338-1341, 1347-1350, 1356-1359, 1365-1368, 1374-1377, 1383-1386, 1392-1395, 1401-1404, 1410-1413, 1419-1422, 1428-1431, 1437-1440, 1446-1449, 1455-1458, 1464-1467, 1473-1476, 1482-1485, 1491-1494, 1500-1503, 1509-1512, 1518-1521, 1527-1530, 1536-1539, 1545-1548, 1554-1557, 1563-1566, 1572-1575, 1581-1584, 1590-1593, 1599-1602, 1608-1611, 1617-1620, 1626-1629, 1635-1638, 1644-1647, 1653-1656, 1662-1665, 1671-1674, 1680-1683, 1689-1692, 1698-1701, 1707-1710, 1716-1719, 1725-1728, 1734-1737, 1862-1882, 1885, 1888, 1889, 1891-1894, 1898 or fragments or variants thereof, wherein preferably the at least one antigenic protein comprises a heterologous element selected from a signal peptide, a linker, a helper epitope, an antigen clustering domain, or a transmembrane domain.
44. Composition of any one of claims 41 to 43, wherein the at least one coding RNA or the plurality of coding RNAs is complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound, preferably cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.
45. Composition of claim 44, wherein the at least one coding RNA or the plurality of coding RNAs is complexed, encapsulated, partially encapsulated, or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes, and/or nanoliposomes.
46. Composition of claim 45, wherein the at least one coding RNA or the plurality of coding RNAs is complexed with one or more lipids thereby forming lipid nanoparticles (LNP).
47. Composition of claim 46, wherein the LNP comprises a cationic lipid according to formula III-3: ##STR00008##
48. Composition of any one of claims 46 to 47, wherein the LNP comprises a PEG lipid, wherein the PEG-lipid is of formula (IVa): ##STR00009## wherein n has a mean value ranging from 30 to 60, preferably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most preferably wherein n has a mean value of 49.
49. Composition of any one of claims 46 to 48, wherein the LNP comprises one or more neutral lipids and/or one or more steroid or steroid analogues.
50. Composition of claim 49, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), preferably wherein the molar ratio of the cationic lipid to DSPC is in the range from about 2:1 to about 8:1.
51. Composition of claim 49, wherein the steroid is cholesterol, preferably wherein the molar ratio of the cationic lipid to cholesterol is in the range from about 2:1 to about 1:1
52. Composition of any one of claims 44 to 49, wherein the LNP comprises or consisting of (i) at least one cationic lipid, preferably as defined in claim 47; (ii) a neutral lipid, preferably as defined in claim 50; (iii) a steroid or steroid analogue, preferably as defined in claim 51; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, preferably as defined in claim 48.
53. Composition according to any one of claim 52, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.
54. Composition of claim 46, wherein the LNP comprises COATSOME® SS-EC.
55. Composition of any one of claims 46 and 54, wherein the LNP comprises a PEG lipid, wherein the PEG-lipid is DMG-PEG 2000.
56. Composition of any one of claims 46 and 54 to 55, wherein the LNP further comprises 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) and cholesterol.
57. Composition of claims 46 to 56, wherein the LNPs are preferably selected from GN01-LNP or LNP-III-3.
58. A vaccine comprising at least one coding RNA as defined in any one of claims 1 to 40, or the composition as defined in any one of claims 41 to 57.
59. Vaccine of claim 58, wherein the vaccine elicits an adaptive immune response.
60. Vaccine of claims 58 to 59, wherein the vaccine is a polyvalent vaccine, preferably a trivalent vaccine.
61. A Kit or kit of parts, comprising at least one coding RNA as defined in any one of claims 1 to 40, at least one composition as defined in any one of claims 41 to 57, and/or at least one vaccine as defined in any one of claims 58 to 60, optionally comprising a liquid vehicle for solubilising, and, optionally, technical instructions providing information on administration and dosage of the components.
62. Coding RNA as defined in any one of claims 1 to 40, the composition as defined in any one of claims 41 to 57, the vaccine as defined in any one of claims 58 to 60, or the kit or kit of parts as defined in claim 57, for use as a medicament.
63. Coding RNA as defined in any one of claims 1 to 40, the composition as defined in any one of claims 41 to 53, the vaccine as defined in any one of claims 54 to 56, or the kit or kit of parts as defined in claim 61, for use in the treatment or prophylaxis of a Rotavirus infection, or of a disorder related to such an infection.
64. Use according to claim 63, wherein the Rotavirus infection is a Rotavirus A infection, in particular a Rotavirus A infection of serotypes [P4], [P6], and/or [P8].
65. A method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof at least one coding RNA as defined in any one of claims 1 to 40, at least one composition as defined in any one of claims 41 to 57, at least one vaccine as defined in any one of claims 58 to 60, or at least one kit or kit of parts as defined in claim 61.
66. Method of claim 65 wherein the disorder is an infection with a Rotavirus, or a disorder related to such an infection, preferably a Rotavirus A, or a disorder related to such an infection.
67. Method of claims 65 to 66, wherein the subject in need is a mammalian subject, preferably a human subject.
68. Method of any one of claims 65 to 67, wherein applying or administering to a subject is performed using intramuscular administration, preferably intramuscular injection.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0846]
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EXAMPLES
[0874] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.
Example 1: Preparation of DNA and RNA Constructs, Compositions, and Vaccines
[0875] The present Example provides methods of obtaining the RNA of the invention as well as methods of generating a composition or a vaccine of the invention.
[0876] 1.1. Preparation of DNA and RNA Constructs:
[0877] DNA sequences encoding different Rotavirus antigenic proteins were prepared and used for subsequent RNA in vitro transcription. Said DNA sequences were prepared by modifying the wild type CDS sequences by introducing an optimized CDS. Sequences were introduced into a plasmid vector to comprise optionally (i) advantageous 3′-UTR sequences derived from PSMB3, ALB7 or alpha-globin (“muag”) and (ii) advantageous 5′-UTR sequences selected from HSD17B4 or RPL32, additionally comprising (iii) a stretch of adenosines, and optionally a histone-stem-loop structure, and optionally a stretch of 30 cytosines (Table 5).
[0878] Obtained plasmid DNA was transformed and propagated in bacteria using common protocols and plasmid DNA was extracted, purified, and used for subsequent RNA in vitro transcription (see section 1.2.). Alternatively, DNA plasmids were used as DNA template for PCR-based amplification. The generated PCR products were purified and used for subsequent RNA in vitro transcription (see section 1.3.).
[0879] 1.2. RNA In Vitro Transcription from Plasmid DNA Templates:
[0880] DNA plasmids prepared according to section 1.1 were enzymatically linearized using a restriction enzyme and used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analogue (e.g., m7GpppG or m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG)) under suitable buffer conditions. m7G(5′)ppp(5′)(2′OMeA)pG cap analog was used for preparation of some RNA constructs to generate co-transcriptionally a cap1 structure. The obtained RNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. DNA templates may also be generated using PCR. Such PCR templates were used for DNA dependent RNA in vitro transcription using an RNA polymerase as outlined herein.
[0881] To obtain modified mRNA RNA in vitro transcription was performed in the presence of a modified nucleotide mixture (ATP, GTP, CTP, pseudouridine (Ψ) and cap analogue (m7GpppG or m7G(5′)ppp(5′)(2′OMeA)pG) under suitable buffer conditions. The obtained Ψ-modified mRNAs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tübingen, Germany; WO2008/077592) and used for further experiments. Some RNA constructs were in vitro transcribed in the absence of a cap analog. The cap-structure (cap0 or cap1) was then added enzymatically using capping enzymes as commonly known in the art. In short, in vitro transcribed RNA was capped using a capping kit to obtain cap0-RNA. cap0-RNA was additionally modified using cap specific 2′-O-methyltransferase to obtain cap1-RNA. cap1-RNA was purified e.g. as explained above and used for further experiments.
[0882] RNA for clinical development is produced under current good manufacturing practice e.g. according to WO2016/180430, implementing various quality control steps on DNA and RNA level.
[0883] The generated RNA sequences/constructs are provided in Table 5 with the encoded antigenic protein and the respective UTR elements indicated therein.
[0884] 1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates:
[0885] Purified PCR amplified DNA templates prepared according to paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analogue (m7GpppG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions. Alternatively, PCR amplified DNA is transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N1-methylpseudouridine (m1Ψ) or pseudouridine (Ψ) and cap analogue (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. Some RNA constructs are in vitro transcribed in the absence of a cap analogue and the cap-structure (cap0 or cap1) is added enzymatically using capping enzymes as commonly known in the art. The obtained RNA is purified e.g. as explained above and used for further experiments.
TABLE-US-00007 TABLE 5 RNA constructs encoding different Rotavirus antigen design used in the present examples mRNA Design Poly(A) UTR sequence, 5′-cap design located at 3′ RNA Construct Rotavirus structure 5′-UTR/ terminus Modified SEQ ID SEQ ID ID design Serotype (Description) 3′-UTR (Description) nucleotides NO: RNA NO: PRT R5470 P2-Linker- P[8] cap0 RPL32/ — — 1865 4, 51 VP8*(65-223) ALB7 R6326 P2-Linker- P[8] cap0 RPL32/ — — 1874 69 VP8*(65- ALB7 223)-Linker- Ferritin R5488 SP(IgE)-P2- P[8] cap0 RPL32/ — — 1880 114 Linker- ALB7 VP8*(41-223) R6322 SP(IgE)-P2- P[8] cap0 RPL32/ — — 1881 — Linker- ALB7 VP8*(41- 223)-Linker- Ferritin R6324 SP(IgE)-P2- P[8] cap0 RPL32/ — — 1882 — Linker- ALB7 VP8*(41- 223)-Linker- TM(HA) R6328 LumSynt.- P[8] cap0 RPL32/ — — 1877 1, 96 Linker-P2- ALB7 Linker- VP8*(41-223) R5433 SP(HsPLAT)- P[6] cap0 RPL32/ — — 1895 — VP8*(41-223) ALB7 R5434 SP(HsPLAT)- P[6] cap0 RPL32/ — — 1896 — P2-Linker- ALB7 VP8*(41-223) R5435 SP(HsPLAT)- P[6] cap0 RPL32/ — — 1897 — VP8*(2-230) ALB7 R5436 SP(HsPLAT)- P[6] cap0 RPL32/ — — 1890 — VP8*(21-240) ALB7 (N => Q mutation) R5480 SP(HSA)- P[8] cap0 RPL32/ — — 1891 — VP8*(2-230) ALB7 R5482 SP(HSA)- P[8] cap0 RPL32/ — — 1892 — VP8*(11-223) ALB7 R5484 SP(HSA)- P[8] cap0 RPL32/ — — 1893 — VP8*(41-223) ALB7 R5486 SP(HSA)-P2- P[8] cap0 RPL32/ — — 1894 — Linker- ALB7 VP8*(41-223) R8044 P2-Linker- P[8] cap1 (co- HSD17B4/ — — 1864 4, 51 VP8*(65-223) trans. cap) PSMB3 R8046 P2-Linker- P[8] cap1 (enzym. RPL32/ + (enzym. — 1865 4, 51 VP8*(65-223) cap) ALB7 poly(A)) R8047 P2-Linker- P[8] cap0 RPL32/ — m1Ψ 1870 4, 51 VP8*(65-223) ALB7 R8049 P2-Linker- P[8] cap1 (co- HSD17B4/ — m1Ψ 1868 4, 51 VP8*(65-223) trans. cap) PSMB3 R7411 P2-Linker- P[8] cap1 (enzym. —/muag + (enzym. m1Ψ 1869 4, 51 VP8*(65-223) cap) poly(A)) R8134 P2-Linker- P[8] cap1 (co- HSD17B4/ + (enzym. — 1864 4, 51 VP8*(65-223) trans. cap) PSMB3 poly(A)) R8131 P2-Linker- P{8] cap1 (co- HSD17B4/ + (A100) — 1862, 4, 51 VP8*(65-223) trans. cap) PSMB3 591 R8135 P2-Linker- P[8] cap1 (enzym. RPL32/ — — 1865 4, 51 VP8*(65-223) cap) ALB7 R8138 P2-Linker- P[8] cap1 (enzym. RPL32/ + (A100) — 1898 4, 51 VP8*(65-223) cap) ALB7 R8136 P2-Linker- P[8] cap1 (co- HSD17B4/ + (enzym. m1Ψ 1868 4, 51 VP8*(65-223) trans. cap) PSMB3 poly(A)) R8133 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) m1Ψ 1866 4, 51 VP8*(65-223) trans. cap) PSMB3 R8137 P2-Linker- P[8] cap1 (enzym. —/muag — m1Ψ 1869 4, 51 VP8*(65-223) cap) R8628 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) — 1863, 4. 51 VP8*(65-223) trans. cap) PSMB3 1167 R8575 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) m1Ψ 1863, 4, 51 VP8*(65-223) trans. cap) PSMB3 1167 R8576 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) Ψ 1863, 4, 51 VP8*(65-223) trans. cap) PSMB3 1167 R8629 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) Ψ 1867 4, 51 VP8*(65-223) trans. cap) PSMB3 R8577 P2-Linker- P[8] cap1 (co- HSD17B4/ + (A100) — 1873, 69 VP8*(65- trans. cap) PSMB3 1185 223)-Linker- Ferritin R8578 LumSynt.- P[8] cap1 (co- HSD17B4/ + (A100) — 1876, 1, 96 Linker-P2- trans. cap) PSMB3 1212 Linker- VP8*(41-223) R8579 SP(IgE)-P2- P[8] cap1 (co- HSD17B4/ + (A100) — 1879, 114 Linker- trans. cap) PSMB3 1230 VP8*(41-223) R9247 P2-Linker- P[4] cap1 (co- HSD17B4/ + (A100) — 1919 1899 VP8*(64-223) trans. cap) PSMB3 R9246 P2-Linker- P[6] cap1 (co- HSD17B4/ + (A100) — 1920 1900 VP8*(64-223) trans. cap) PSMB3 R9078 P2-Linker- P[4] cap1 (co- HSD17B4/ + (A100) — 1921 6, 46 VP8*(65-223) trans. cap) PSMB3 R9077 P2-Linker- P[6] cap1 (co- HSD17B4/ + (A100) — 1922 5, 49 VP8*(65-223) trans. cap) PSMB3 R9092 LumSynt.- P[4] cap1 (co- HSD17B4/ + (A100) — 1923 3, 91 Linker-P2- trans. cap) PSMB3 Linker- VP8*(41-223) R9091 LumSynt.- P[6] cap1 (co- HSD17B4/ + (A100) — 1924 2, 94 Linker-P2- trans. cap) PSMB3 Linker- VP8*(41-223)
[0886] Brief Description of the Table 5:
[0887] P2: T cell helper epitope from tetanus toxin; VP8*: Virus protein 8*, cleavage product of rotavirus VP4 protein (preferably having a length of 65-223, 41-223, 1-223, 20-240, 1-230, 2-230, 10-223 or 11-223); SP: signal peptide (preferably derived from secretory signal peptides, preferably human serum albumin (HSA), tissue plasminogen activator (HsPLAT) or immunoglobulin IgE (IgE)); Ferritin: iron storage protein ferritin, heterologous antigen-clustering element; LumSynt: Lumazine synthase, heterologous antigen-clustering element; TM: Transmembrane domain (preferably derived from an influenza HA); P[X]: Rotavirus P serotypes; m1Ψ: modified nucleotide (N1-methylpseudouridine); Ψ: modified nucleotide (pseudouridine); poly(A) sequence, located at 3′ terminus: poly(A) sequence obtained by enzymatic polyadenylation (enzym. poly(A)) or a DNA template (A100); cap0: methylation of the first nucleobase, e.g. m7GpppN; cap1: additional methylation of the ribose of the adjacent nucleotide of m7GpppN; co trans. cap: co-transcriptional capping (preferably CleanCap); enzym. cap: enzymatically capping (preferably ScriptCap); N=>Q mutation: mutation in position N32Q, N56Q, N97Q, N111Q, N114Q, N132Q, N171Q and/or N182Q.
[0888] 1.4. Preparation of Vaccine:
[0889] 1.4.1 LNP Formulation
[0890] Lipid nanoparticles (LNP), cationic lipids, and polymer conjugated lipids (PEG-lipid) were prepared and tested essentially according to the general procedures described in WO2015/199952, WO2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein by reference. LNP formulated RNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Briefly, cationic lipid compound of formula III-3, DSPC, cholesterol, and PEG-lipid of formula IVa were solubilized in ethanol at a molar ratio (%) of approximately 50:10:38.5:1.5 or 47.4:10:40.9:1.7. LNPs comprising cationic lipid compound of formula III-3 and PEG-lipid compound of formula IVa were prepared at a ratio of RNA to total Lipid of 0.03-0.04 w/w. The RNA was diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM citrate buffer, pH4. Syringe pumps were used to mix the ethanolic lipid solution with the RNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol was then removed and the external buffer replaced with a PBS buffer comprising Sucrose by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 um pore sterile filter and the LNP-formulated RNA composition was adjusted to about 1 mg/ml total RNA. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is essentially similar.
[0891] In general, LNPs were prepared using cationic lipids, structural lipids, a PEG-lipids, and cholesterol. Lipid solution (in ethanol) was mixed with RNA solution (aqueous buffer) using a microfluidic mixing device or using T-piece formulation. Obtained LNPs were re-buffered in a carbohydrate buffer via dialysis, and up-concentrated to a target concentration using ultracentrifugation tubes. LNP-formulated mRNA can be stored at −80° C. prior to use in in vitro or in vivo experiments.
[0892] The obtained LNP-formulated RNA composition (1 mg/ml total RNA) was diluted to the desired target concentration using Saline before in vivo application.
[0893] 1.4.2 Protamine Complexation:
[0894] The mRNA vaccine 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.
Example 2: Analysis of Rotavirus Antigen Designs
[0895] 2.1 In Vitro Analysis of Expression and Secretion of Rotavirus Antigen Designs
[0896] The present example shows that RNA constructs encoding different Rotavirus antigen designs were expressed and secreted in mammalian cells.
[0897] To determine in vitro protein expression of some of the RNA constructs, HEK 293T cells were transfected with unformulated mRNA encoding different Rotavirus antigen designs using Lipofectamine 2000. 24 h-48 h after transfection, cell lysates and cell culture supernatants were subjected to SDS-PAGE and Western blot analysis using rabbit anti-VP8* P[4 and 8] antibody (1:1000; Aldevron) or mouse anti-alpha-tubulin antibody (1:1000; Abcam) as primary antibodies as well as goat anti-rabbit IgG IRDye®800CW or 680RD antibody (1:10000; Li-Cor) or goat anti-mouse IgG IRDye® 680RD or 800CW antibody (1:10000; Li-Cor) as secondary antibodies. Detection and quantification was performed using a Li-Cor detection system (Odyssey CLx image system) in combination with Image Studio Lite software. Table 6 contains mRNA constructs that were used in the experiment:
TABLE-US-00008 TABLE 6 RNA constructs used for Western blot analysis (Example 2) RNA Size SEQ ID Group ID Construct design kDa NO: RNA 1 R5470 P2-Linker-VP8*(65-223) 19 1865 2 R6326 P2-Linker-VP8*(65-223)-Linker-Ferritin 38 1874 3 R5488 SP(IgE)-P2-Linker-VP8*(41-223) 24 1880 4 R6322 SP(IgE)-P2-Linker-VP8*(41-223)-Linker-Ferritin 41 1881 5 R6324 SP(IgE)-P2-Linker-VP8*(41-223)-Linker-TM 30 1882 6 R6328 LumSynt-Linker-P2-Linker-VP8*(41-223) 43 1877 7 — Negative control (WFI = water for infusion) — 1883
[0898] Results:
[0899] Expression of all six RNA constructs was demonstrated in the corresponding cell lysates (see
[0900] 2.2: In Vivo Analysis of Immunogenicity of Rotavirus Antigen Designs
[0901] The present example shows that Rotavirus mRNA vaccines encoding different antigen designs induce humoral immune responses in mice (Balb/c).
[0902] mRNA constructs encoding different Rotavirus antigen designs (see Table 7) were prepared according to Example 1. The mRNA was formulated with protamine (see Example 1.4.2 Protamine complexation). The different mRNA vaccine candidates were applied on day 0, 21, and 42 and administered intradermal (i.d.) with 80 ug of RNA as shown in Table 7. One negative control group (1) received an irrelevant mRNA. Blood samples were taken at day 21, 42, and 56 for determination of humoral immune responses.
[0903] ELISA was performed using recombinant Rotavirus protein P2-VP8*P[8] for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the respective recombinant Rotavirus protein P2-VP8*P[8] was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG1, IgG2a) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 21, 42, and 56 post vaccinations.
TABLE-US-00009 TABLE 7 Vaccination scheme of Example 2 No. of RNA SEQ ID Group mice Dose Volume ID Construct design NO: RNA 1 7 80 μg 2 × 50 μl — Irrelevant RNA 1883 2 7 80 μg 2 × 50 μl R5470 P2-Linker-VP8*(65-223) 1865 3 7 80 μg 2 × 50 μl R6326 P2-Linker-VP8*(65-223)-Linker-Ferritin 1874 4 7 80 μg 2 × 50 μl R5488 SP(IgE)-P2-Linker-VP8*(41-223) 1880 5 7 80 μg 2 × 50 μl R6324 SP(IgE)-P2-Linker-VP8*(41-223)-Linker-TM 1882 6 7 80 μg 2 × 50 μl R6328 LumSynt-Linker-P2-Linker-VP8*(41-223) 1877
Example 3: (Cross) Reactivity of Rotavirus Antigen Designs
[0904] 3.1 Cross Reactivity of Rotavirus Serotype P[6] Antigen Designs
[0905] 3.1.1 Determination of Specific and Cross Reactive Humoral Immune Responses by ELISA
[0906] Balb/c mice were immunized with RNA vaccines (as prepared in Example 1) encoding different VP8* antigen designs from serotype P[6], recombinant Rotavirus protein P2-VP8*P[8] as a positive control or RiLa (Ringer lactate) as a negative control as indicated in Table X4 below. The mRNA was formulated with protamine (see Example 1.4.2 Protamine complexation). Vaccinations were performed on day 0, 21 and 42. Blood samples taken on day 21, 42, 57 and 71 were analyzed for the presence of VP8* specific IgG1 and IgG2a antibodies by ELISA using recombinant Rotavirus protein P2-VP8*P[6] (
[0907] 3.1.2 Intracellular Cytokine Staining (ICS):
[0908] Splenocytes from vaccinated mice were isolated on day 71 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). Cells were stimulated with a mixture of Rotavirus VP8* peptides (see Table 9) (5 ug/ml each) in the presence of 2.5 ug/ml each of an anti-CD28 antibody (BD Biosciences) and a protein transport inhibitor for 6 h at 37° C. After stimulation, cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-H7 (1:100), 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 BD FACS Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software (Tree Star, Inc.). Results are shown in
TABLE-US-00010 TABLE 8 Vaccination scheme of Example 3.1 No. of RNA SEQ ID Group mice Dose Volume ID Construct design NO: RNA 1 6 — 2 × 50 μl — RiLa - negative control — 2 6 6 μg 4 × 25 μl — Recombinant Rotavirus protein — VP8*P[8] + Alum 3 12 80 μg 2 × 50 μl R5433 SP(HsPLAT)-VP8*[P6](41-223) 1895 4 12 80 μg 2 × 50 μl R5434 SP(HsPLAT)-P2-VP8*[P6](41-223) 1896 5 12 80 μg 2 × 50 μl R5435 SP(HsPLAT)-VP8*[P6](1-223) 1897 6 12 80 μg 2 × 50 μl R5436 SP(HsPLAT)-VP8*[P6](20-240) (N.fwdarw.Q mutation) 1890
TABLE-US-00011 TABLE 9 Rotavirus VP8* peptide mix for ICS MHC class Serotype Peptide sequence MHCI P[4]/P[6]/P[8] FYIIPRSQE MHCI P[4]/P[8] KYGGRVWTF MHCI P[4]/[P8] VYESTNNSD MHCI P[6] FYNSVWTFH MHCI P[6] GFMKFYNSV MHCII P[4]/P[8] SDFWTAVIAVEPHVN MHCII P[6] TNKTDIWVALLLVEP MHCII P[6] HKRTLTSDTKLAGFM
[0909] Results:
[0910] As shown in
[0911]
[0912] CD4 positive T cells play an important role in the immune system, particularly in the adaptive immune system. They help the activity of other immune cells by releasing T cell cytokines and are essential in B cell antibody class switching, in the activation and growth of cytotoxic T cells. An effective Rotavirus vaccine should induce CD4+ T cell responses. CD8+ T cells are a major protective immune mechanism against intracellular infections, like Rotavirus virus infections. An effective Rotavirus vaccine should also induce CD8+ T cells responses.
[0913] As shown in
[0914] Additional improvements to the mRNA design or formulation could lead to an enhanced immune response after vaccination (see Example 5).
[0915] Accordingly, these findings highlight one of the advantageous features of the inventive mRNA-based Rotavirus vaccine designs.
[0916] 3.2: Cross Reactivity of Rotavirus Serotype P[8] Antigen Designs
[0917] 3.2.1 Determination of Specific and Cross Reactive Humoral Immune Responses by ELISA
[0918] Balb/c mice were immunized with RNA vaccines (as prepared in Example 1) encoding different VP8* antigen designs from serotype P[8], recombinant protein VP8*P[8] as a positive control or RiLa (Ringer lactate) as a negative control as indicated in Table 10 below. The mRNA was formulated with protamine (see Example 1.4.2 Protamine complexation). Vaccinations were performed on day 0, 21 and 42. Blood samples taken on day 21, 42, 56 and 71 were analyzed for the presence of VP8* specific IgG1 and IgG2a antibodies by ELISA using VP8*P[8] protein (
[0919] 3.2.2: Intracellular Cytokine Staining
[0920] Splenocytes from vaccinated mice were isolated on day 71 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). Cells were stimulated with a mixture of Rotavirus VP8* peptides (see Table 9) (5 ug/ml each) in the presence of 2.5 ug/ml each of an anti-CD28 antibody (BD Biosciences) and a protein transport inhibitor for 6 h at 37° C. After stimulation, cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-H7 (1:100), 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 BD FACS Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software (Tree Star, Inc.). Results are shown in
TABLE-US-00012 TABLE 10 Vaccination scheme of Example 3.2 No. of RNA SEQ ID Group mice Route Dose Volume ID Construct Design NO: RNA 1 6 i.d. — 2 × 50 μl — RiLa (negative control) 2 6 i.m. 6 μg 4 × 25 μl — Recombinant Rotavirus protein VP8*P[8] + Alum 3 12 i.d. 80 μg 2 × 50 μl R5480 SP(HSA)-VP8*P[8](2-230) 1891 4 12 i.d. 80 μg 2 × 50 μl R5482 SP(HSA)-VP8*P[8](11-223) 1892 5 12 i.d. 80 μg 2 × 50 μl R5484 SP(HSA)-VP8*P[8](41-223) 1893 6 12 i.d. 80 μg 2 × 50 μl R5486 SP(HSA)-P2-VP8*P[8](41-223) 1894 7 12 i.d. 80 μg 2 × 50 μl R5488 SP(IgE)-P2-VP8*P[8](41-223) 1880 8 12 i.d. 80 μg 2 × 50 μl R5470 P2-VP8*[P8](65-223) 1865
[0921] Results:
[0922] As shown in
[0923]
[0924] CD4 positive T cells play an important role in the immune system, particularly in the adaptive immune system. They help the activity of other immune cells by releasing T cell cytokines and are essential in B cell antibody class switching, in the activation and growth of cytotoxic T cells. An effective Rotavirus vaccine should induce CD4+ T cell responses. CD8+ T cells are a major protective immune mechanism against intracellular infections, like Rotavirus virus infections. An effective Rotavirus vaccine should also induce CD8+ T cells responses.
[0925] As shown in
Example 4: In Vitro Analysis of Expression of Different mRNA Design Encoding a Rotavirus Antigen
[0926] The present example shows that different mRNA constructs encoding a Rotavirus antigen were expressed in mammalian cells.
[0927] To determine in vitro protein expression of the RNA constructs, HEK 293T cells were transfected with unformulated mRNA encoding a Rotavirus antigen using Lipofectamine 2000. 24 h-48 h after transfection, cell lysates and cell culture supernatants were subjected to SDS-PAGE and Western blot analysis using rabbit anti-VP8*P[4] or anti-VP8*P[8] or antibody (1:1000; Aldevron) or mouse anti-alpha-tubulin antibody (1:1000; Abcam) as primary antibodies as well as goat anti-rabbit IgG IRDye®800CW or 680RD antibody (1:10000; Li-Cor) or goat anti-mouse IgG IRDye® 680RD or 800CW antibody (1:10000; Li-Cor) as secondary antibodies. Detection and quantification was performed using a Li-Cor detection system (Odyssey CLx image system) in combination with Image Studio Lite software. Table 11 contains mRNA constructs that were used in the experiment:
TABLE-US-00013 TABLE 11 RNA constructs used for Western blot analysis (Example 4) RNA SEQ ID Group ID mRNA design Serotype Construct design NO: 1 R5470 cap0 P[8] P2-Linker-VP8*(65-223) 1865 2 R8044 cap1 P[8] P2-Linker-VP8*(65-223) 1864 3 R8046 cap1 + poly(A) sequence, P[8] P2-Linker-VP8*(65-223) 1865 located at the 3′ terminus
[0928] Results:
[0929] Expression of all three mRNA constructs was demonstrated in the corresponding cell lysates (see
Example 5: Analysis of Different mRNA Designs Encoding a Rotavirus Antigen
[0930] Different mRNA constructs encoding a Rotavirus antigen (see Table X8) were prepared according to Example 1. The mRNA was formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccine candidates were applied on days 0 and 21 and administered intramuscular (i.m.) with 4 ug of RNA in mice as shown in Table 12. One negative control group (1) received an irrelevant mRNA. Blood samples were taken at day 21, 42, and 56 for determination of humoral immune responses.
[0931] ELISA was performed using recombinant Rotavirus protein P2-VP8*P[8] for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the recombinant Rotavirus protein P2-VP8*P[8] was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG1, IgG2a) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 21, 42, and 56 post vaccinations.
[0932] 5.1: In Vivo Analysis of Immunogenicity
[0933] Experimental setting described before in Example 5. Table 12 contains mRNA constructs that were used in the experiment.
TABLE-US-00014 TABLE 12 Vaccination scheme of Example 5 No. of RNA mRNA Modified SEQ ID Group mice Dose Volume ID design nucleotides Construct design NO: 1 7 4 μg 25 μl — — — Irrelevant RNA 1884 2 7 4 μg 25 μl R5470 cap0 — P2-Linker-VP8*(65-223) 1865 3 7 4 μg 25 μl R8047 cap0 m1Ψ P2-Linker-VP8*(65-223) 1870 4 7 4 μg 25 μl R8044 cap1(co- — P2-Linker-VP8*(65-223) 1864 trans . . . cap) 5 7 4 μg 25 μl R8049 cap1(co-trans. m1Ψ P2-Linker-VP8*(65-223) 1868 cap) 6 7 4 μg 25 μl R8046 cap1(enzym. — P2-Linker-VP8*(65-223) 1865 cap) + poly(A) sequence, located at the 3′ terminus 7 7 4 μg 25 μl R7411 cap1 (enzym. m1Ψ P2-Linker-VP8*(65-223) 1869 cap) + poly(A) sequence, located at the 3′ terminus
[0934] Results:
[0935] As shown in
[0936] In addition mRNA designs comprising modified nucleotides (grey bars,
[0937] Improvements of the mRNA design enhanced the immune responses to mRNA vaccine.
[0938] 5.2 Determination of Rotavirus Virus Neutralization Titers (VNTs)
[0939] Serum was collected on day 56 after vaccination described in Example 5 and Rotavirus neutralization titers were measured as described below. Table 12 contains mRNA constructs that were used in the experiment.
[0940] Diluted serum samples were incubated with a constant amount of Rotavirus virus strain Wa (G1P[8]) for 1 hour at 37° C., before transfer to a 96 well plate containing confluent MA104 cells. After 1 hour the monolayers were washed and incubated with the serum-virus mixture for 14-16 hours, frozen and thawed. VNTs were determined by measuring Rotavirus virus antigen in the lysed monolayers in wells receiving serum compared to viral control wells, using a standard enzyme-linked immunosorbent format. The neutralization titer represents a 60% reduction in the amount of virus.
[0941] Results:
[0942] As can be seen from
Example 6: Analysis of Different mRNA Designs Encoding a Rotavirus Antigen
[0943] 6.1: In Vitro Analysis of Expression
[0944] The present example shows that different mRNA constructs encoding a Rotavirus antigen were expressed in mammalian cells.
[0945] To determine in vitro protein expression of the RNA constructs, HEK 293T cells were transfected with unformulated mRNA encoding a Rotavirus antigen using Lipofectamine 2000. 24 h-48 h after transfection, cell lysates and cell culture supernatants were subjected to SDS-PAGE and Western blot analysis using rabbit anti-VP8* P[4 and 8] antibody (1:1000; Aldevron) or mouse anti-alpha-tubulin antibody (1:1000; Abcam) as primary antibodies as well as goat anti-rabbit IgG IRDye®800CW or 680RD antibody (1:10000; Li-Cor) or goat anti-mouse IgG IRDye® 680RD or 8000W antibody (1:10000; Li-Cor) as secondary antibodies. Detection and quantification was performed using a Li-Cor detection system (Odyssey CLx image system) in combination with Image Studio Lite software. Table 13 contains mRNA constructs that were used in the experiment.
TABLE-US-00015 TABLE 13 RNA constructs used for Western blot analysis (Example 6) 5′-cap structure/ poly(A) sequence, UTRs No. of RNA located at 3′ 5′-UTR/ SEQ ID Group mice ID Construct design terminus 3′-UTR NO: 1 7 R8044 P2-Linker-VP8*(65-223) co-trans. cap/− HSD17B4/PSMB3 1864 2 7 R8134 P2-Linker-VP8*(65-223) co-trans. cap/+ HSD17B4/PSMB3 1864 (enzym. Poly(A)) 3 7 R8131 P2-Linker-VP8*(65-223) co-trans. cap/+ HSD17B4/PSMB3 1862 or (A100) 591 4 7 R8135 P2-Linker-VP8*(65-223) enzym. cap/− RPL32/ALB7 1865 5 7 R8046 P2-Linker-VP8*(65-223) enzym. cap/+ RPL32/ALB7 1865 (enzym. Poly(A)) 6 7 R8138 P2-Linker-VP8*(65-223) enzym. cap/+ (A100) RPL32/ALB7 1865
[0946] Results:
[0947] Expression of all six mRNA constructs was demonstrated in the corresponding cell lysates (see
[0948] 6.2: In Vivo Analysis of Immunogenicity
[0949] Different mRNA constructs encoding a Rotavirus antigen (see Table 14) were prepared according to Example 1. The mRNA was formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccine candidates were applied on days 0 and 21 and administered intramuscular (i.m.) with 4 ug of RNA as shown in Table 14. One negative control group (1) received Buffer and one positive control group (2) received recombinant Rotavirus protein P2-VP8*P[8]. Blood samples were taken at day 21, 42, and 56 for determination of humoral immune responses.
[0950] ELISA was performed using recombinant Rotavirus protein P2-VP8*P[8] for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the recombinant Rotavirus protein P2-VP8*P[8] was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG1, IgG2a) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 21, 42, and 56 post vaccinations.
TABLE-US-00016 TABLE 14 Vaccination scheme of Example 6.2 5′-cap structure/ poly(A) sequence, UTRs No. of RNA Construct located at 3′ 5′-UTR/ Modified SEQ ID Group mice Dose Volume ID design terminus 3′-UTR nucleotides NO: 1 6 — 25 μl — 0.9% NaCl — — − buffer, negative control 2 6 6 μg 4 × — Recombinant — — − 25 μl Rotavirus protein P2- VP8*P[8] + Alum 3 7 4 μg 25 μl R8044 P2-Linker- co-trans. cap/− HSD17B4/ − 1864 VP8*(65-223) PSMB3 4 7 4 μg 25 μl R8134 P2-Linker- co-trans. cap/+ HSD17B4/ − 1864 VP8*(65-223) (enzym. Poly(A)) PSMB3 5 7 4 μg 25 μl R8131 P2-Linker- co-trans. cap/+ HSD17B4/ − 1862 or VP8*(65-223) (A100) PSMB3 591 6 7 4 μg 25 μl R8135 P2-Linker- enzym. cap/− RPL32/ − 1865 VP8*(65-223) ALB7 7 7 4 μg 25 μl R8046 P2-Linker- enzym. cap/+ RPL32/ − 1865 VP8*(65-223) (enzym. Poly(A)) ALB7 8 7 4 μg 25 μl R8138 P2-Linker- enzym. cap/+ RPL32/ − 1865 VP8*(65-223) (A100) ALB7 9 7 4 μg 25 μl R8049 P2-Linker- co-trans. cap/− HSD17B4/ + 1868 VP8*(65-223) PSMB3 10 7 4 μg 25 μl R8136 P2-Linker- co-trans. cap/+ HSD17B4/ + 1868 VP8*(65-223) (enzym. Poly(A)) PSMB3 11 7 4 μg 25 μl R8133 P2-Linker- co-trans. cap/+ HSD17B4/ + 1866 VP8*(65-223) (A100) PSMB3 12 7 4 μg 25 μl R8137 P2-Linker- enzym. cap/− —/muag + 1869 VP8*(65-223) 13 7 4 μg 25 μl R7411 P2-Linker- enzym. cap/+ —/muag + 1869 VP8*(65-223) (enzym. Poly(A))
[0951] Results:
[0952] As shown in
[0953] Early immune responses are very important for a fast and robust protection against Rotavirus virus infections. The high titers in later time points show that this immune response resulting from mRNA vaccination can be boosted.
[0954] 6.3 Determination of Rotavirus Virus Neutralization Titers (VNTs)
[0955] Serum was collected on day 56 after vaccination described in Example 6.2 and Rotavirus neutralization titers were measured as described below. Table 14 contains mRNA constructs that were used in the experiment.
[0956] Diluted serum samples were incubated with a constant amount of Rotavirus virus strain Wa (G1P[8]) for 1 hour at 37° C., before transfer to a 96 well plate containing confluent MA104 cells. After 1 hour the monolayers were washed and incubated with the serum-virus mixture for 14-16 hours, frozen and thawed. VNTs were determined by measuring Rotavirus virus antigen in the lysed monolayers in wells receiving serum compared to viral control wells, using a standard enzyme-linked immunosorbent format. The neutralization titer represents a 60% reduction in the amount of virus.
[0957] Results:
[0958]
[0959] 6.4: In Vivo Analysis of Cytokines
[0960] Appropriate dilutions of sera collected 14 hours after prime immunization (see Example 6.2) were analyzed by a mouse IFN-alpha ELISA kit according to the manufacturer's protocol (PBL, cat.: 42115-1). Table 14 contains mRNA constructs that were used in the experiment.
[0961] Results:
[0962] The recombinant Rotavirus protein P2-VP8*P[8] (Group 2, see
[0963] INFalpha has a main role in the immune response against viruses. It activates immune cells, such as natural killer cells and macrophages, increases host defenses by up-regulating antigen presentation by increasing the expression of major histocompatibility complex (MHC) antigens. This activation of the innate immune system can be seen as supportive for the subsequent development of a strong adaptive immune response. However, high levels of INFalpha can lead to fever, muscle pain and flu like symptoms. Therefore a moderate increasing of INFalpha could be marker for an optimal immune response to a vaccine against a Rotavirus virus infection.
[0964] 6.5 Intracellular Cytokine Staining (ICS):
[0965] Splenocytes from vaccinated mice (see Example 6.2) were isolated on day 71 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). Cells were stimulated with a mixture of Rotavirus VP8* peptides (see Table 9) (5 ug/ml each) in the presence of 2.5 ug/ml each of an anti-CD28 antibody (BD Biosciences) and a protein transport inhibitor for 6 h at 37° C. After stimulation, cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-H7 (1:100), 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 BD FACS Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software (Tree Star, Inc.). Results are shown in
[0966] Results:
[0967] Groups contained mRNA designs with co-transcriptional capping, a poly(A) sequence, located at 3′ terminus and a UTR combination HSD17B4/PSMB3 (Group 4, 5, 10 and 11, see
Example 7: Clinical Development of a Rotavirus mRNA Vaccine
[0968] To demonstrate safety and efficacy of the Rotavirus mRNA vaccine, a double blind, randomised, placebo controlled, dose escalation clinical trial will be initiated.
[0969] For clinical development, mRNA produced under GMP conditions (e.g. using a procedure as described in WO2016/180430) will be used.
[0970] First in Human exposure would most likely be performed in healthy young adults, to establish safety/tolerability and immunogenicity of the candidate vaccine. In an example of a subsequent clinical trial a cohort of healthy HIV uninfected toddlers (aged 1 to <3 years) and infants (aged 6 to <8 weeks) without previous rotavirus vaccination, is intramuscularly injected with the Rotavirus mRNA vaccine. Exclusion criteria can include acute illness at time of enrolment, presence of malnutrition or any systemic disorder, congenital defects, known or suspected impaired immunological function and immunoglobulin therapy or chronic immunosuppressant medications.
[0971] The dose escalation phase can be designed to test 2 ug to 200 ug dose levels of vaccine, depending on non-clinical data and clinical data with related vaccines at the time, first in toddlers and then in infants. Toddlers and infants are randomly assigned to receive vaccine or placebo, beginning with the lowest dose. Toddlers in the dose escalation phase of the trial receive up to three intramuscular injections of vaccine or placebo in the anterolateral thigh in a 2 to 8 weeks interval. Infants in the dose escalation phase and the expanded cohort receive three intramuscular injections at 2 to 8-week intervals of vaccine or placebo.
[0972] Infants also receive three doses of the oral Rotarix rotavirus vaccine (GlaxoSmithKline, Rixensart, Belgium) as part of this study, at 4, 8, and 12 weeks after the third study injection. Participants are observed for at least 30 min after administration of each injection.
[0973] The primary objectives are to assess the safety and reactogenicity of the Rotavirus mRNA vaccine at escalating doses in toddlers and infants, and to investigate the immunogenicity at different doses.
[0974] Primary safety endpoints are local and systemic reactions within 7 days after each injection, adverse events within 28 days after each injection, and all serious adverse events, assessed in toddlers and infants who receive at least one dose. Primary immunogenicity endpoints are IgA and IgG titers against P2-VP8 and neutralising antibody sera responses and geometric mean titers 4 weeks after third injection. Therefore serum is collected at baseline, after each vaccination and after the final study injection.
[0975] The secondary objective is to assess the effect of Rotavirus mRNA vaccination on shedding of Rotarix vaccine virus subsequently administered in infants, with the endpoint being the proportion of infants shedding rotavirus (determined by ELISA) at 5, 7, or 9 days after administration of the first dose of Rotarix (4 weeks after the third Rotavirus mRNA or placebo injection). Therefore stool samples are collected from infants at 5, 7, and 9 days after the first dose of Rotarix and tested for the presence of rotavirus using e.g. the commercially available ProsPecT Rotavirus Microplate Assay (Oxoid Ltd, Ely, UK), according to the manufacturer's instructions.
[0976] Subsequent phase 2b/3 trials would follow to assess efficacy in larger populations, also including specific at-risk populations such as, e.g., HIV positive and malnourished infants.
Example 8: Analysis of Different mRNA Designs Encoding a Rotavirus Antigen
[0977] The present example shows that Rotavirus VP8* mRNA vaccines encoding different antigen designs with modified or unmodified nucleotides induce humoral immune responses in guinea pigs. Binding antibodies were measured using ELISA and virus-neutralizing antibodies to a homologous strain (Wa(G1P[8]), the vaccine strain were analyzed.
[0978] mRNA constructs encoding a P2-linker-VP8* (65-223) construct containing either natural nucleotides or modified nucleotides (m1Ψ or Ψ). The constructs were prepared according to Example 1. The mRNAs were formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccine candidates (see Table 15) were applied on day 0 and 21 and administered intramuscular (i.m.) with different doses of RNA. Blood samples were taken at day 1, 21, 42, and 56 for determination of humoral immune responses.
[0979] Groups 1, 2, 6, 8 and 10 were selected for longevity testing with additional serum sampling time points at day 84, 112 and 140.
TABLE-US-00017 TABLE 15 Vaccination scheme of Example 8 5′-cap structure/ Poly(A) No. of sequence, UTRs guinea RNA Construct located at 3′ 5′-UTR/ Modified SEQ ID Group pigs Dose Volume ID design terminus 3′-UTR nucleotides NO: 1 7 — 100 μl — 0.9% NaCl — — — — buffer, negative control 2 7 20 μg 2 × — Recombinant — — — — 167 μl Rotavirus protein P2-VP8* P[8] + Alum 3 7 8 μg 100 μl R8628 P2-Linker- co-trans. HSD17B4/ — 1863 or VP8*(65-223) cap/A100 PSMB3 1167 4 7 6 μg 100 μl R8575 P2-Linker- co-trans. HSD17B4/ m1Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167 5 7 6 μg 100 μl R8576 P2-Linker- co-trans. HSD17B4/ Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167 6 7 25 μg 100 μl R8628 P2-Linker- co-trans. HSD17B4/ — 1863 or VP8*(65-223) cap/A100 PSMB3 1167 7 7 25 μg 100 μl R8131 P2-Linker- co-trans. HSD17B4/ — 1862 or VP8*(65-223) cap/A100 PSMB3 591 8 7 25 μg 100 μl R8575 P2-Linker- co-trans. HSD17B4/ m1Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167 9 7 25 μg 100 μl R8629 P2-Linker- co-trans. HSD17B4/ m1Ψ 1867 VP8*(65-223) cap/A100 PSMB3 10 7 25 μg 100 μl R8576 P2-Linker- co-trans. HSD17B4/ Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167 11 7 100 μg 100 μl R8628 P2-Linker- co-trans. HSD17B4/ — 1863 or VP8*(65-223) cap/A100 PSMB3 1167 12 7 100 μg 100 μl R8575 P2-Linker- co-trans. HSD17B4/ m1Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167 13 7 100 μg 100 μl R8576 P2-Linker- co-trans. HSD17B4/ Ψ 1863 or VP8*(65-223) cap/A100 PSMB3 1167
[0980] 8.1. Determination of Specific Humoral Immune Responses by ELISA:
[0981] ELISA was performed using recombinant Rotavirus protein P2-VP8*P[8] for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the recombinant Rotavirus protein P2-VP8*P[8] was detected using biotinylated isotype-specific anti-guinea pig antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 21, 42, and 56 post prime vaccinations for all groups and for groups 1, 2, 6, 8 and 10 also at day 84, 112 and 140. Results are shown in
[0982] 8.2 Determination of Virus-Neutralizing Antibody Titers (VNTs) Against Rotavirus
[0983] Serum samples were collected on day 56 after prime vaccination described in Example 5 and virus-neutralizing antibody titers against Rotavirus were measured as described below.
[0984] Diluted serum samples were incubated with a constant amount of Rotavirus virus strain Wa (G1P[8]) for 1 hour at 37° C., before transfer to a 96 well plate containing confluent MA104 cells. After 1 hour the monolayers were washed and incubated with the serum-virus mixture for 14-16 hours, frozen and thawed. VNTs were determined by measuring Rotavirus virus antigen in the lysed monolayers in wells receiving serum compared to viral control wells, using a standard enzyme-linked immunosorbent format. The neutralization titer represents a 60% reduction in the amount of virus. Results are shown in
[0985] Results
[0986] As shown in
[0987] As shown in
[0988] As shown in
Example 9: Analysis of Different Rotavirus Antigen Designs in Combination with Different mRNA Designs
[0989] The present example shows different that Rotavirus mRNA vaccines encoding different antigen designs induce humoral immune responses in guinea pigs. Binding antibodies were measured using ELISA and virus-neutralizing antibodies to homolgous and heterologous strain were analyzed.
[0990] mRNA constructs encoding different VP8* constructs were prepared according to Example 1. The mRNAs were formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccine candidates (see Table 16) were applied on day 0 and 21 and administered intramuscular (i.m.) with different doses of RNA. Blood samples were taken at day 1, 21, 42, and 56 for determination of humoral immune responses.
[0991] Group 1, 2, 7, 8, 9 and 10 were selected for longevity testing with additional serum sampling time points at day 84, 112, 140, 168 and 196.
TABLE-US-00018 TABLE 16 Vaccination scheme of Example 9 5′-cap structure/ poly(A) No. of sequence, UTRs guinea RNA located at 3′ 5′-UTR/ SEQ ID Group pigs Dose Volume ID Construct design terminus 3′-UTR NO: 1 8 — 100 μl — 0.9% NaCl buffer, — — — negative control 2 8 20 μg 2 × — Recombinant Rotavirus — — — 167 μl protein P2-VP8*P(8] + Alum 3 8 6 μg 100 μl R8628 P2-Linker-VP8*(65- co-trans. HSD17B4/ 1863 or 223) cap/A100 PSMB3 1167 4 8 6 μg 100 μl R8577 P2-Linker-VP8*(65- co-trans. HSD17B4/ 1873 or 223)-Linker-Ferritin cap/A100 PSMB3 1185 5 8 6 μg 100 μl R8578 LumSynt-Linker-P2- co-trans. HSD17B4/ 1876 or Linker-VP8*(41-223) cap/A100 PSMB3 1212 6 8 6 μg 100 μl R8579 SP(IgE)-P2-Linker- co-trans. HSD17B4/ 1879 or VP8*(41-223) cap/A100 PSMB3 1230 7 8 25 μg 100 μl R8628 P2-Linker-VP8*(65- co-trans. HSD17B4/ 1863 or 223) cap/A100 PSMB3 1167 8 8 25 μg 100 μl R8577 P2-Linker-VP8*(65- co-trans. HSD17B4/ 1873 or 223)-Linker-Ferritin cap/A100 PSMB3 1185 9 8 25 μg 100 μl R8578 LumSynt-Linker-P2- co-trans. HSD17B4/ 1876 or Linker-VP8*(41-223) cap/A100 PSMB3 1212 10 8 25 μg 100 μl R8579 SP(IgE)-P2-Linker- co-trans. HSD17B4/ 1879 or VP8*(41-223) cap/A100 PSMB3 1230
[0992] 9.1. Determination of Specific Humoral Immune Responses by ELISA:
[0993] ELISA was performed using recombinant Rotavirus protein P2-VP8*P[8] for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the recombinant Rotavirus protein P2-VP8*P[8] was detected using biotinylated isotype specific anti-guinea pig antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 21, 42, and 56 post prime vaccinations for all groups and for group 1, 2, 7, 8, 9 and 10 also at day 84, 112, 140, 168 and 196. Results are shown in
[0994] Endpoint titers of antibodies (IgA) directed against the recombinant Rotavirus protein P2-VP8*P[8] were measured by ELISA on day 56 post prime vaccinations for all groups. Results are shown in
[0995] 9.2 Determination of Virus-Neutralizing Antibody Titers (VNTs) Against Rotavirus Serum samples were collected on day 56 after prime vaccination described in Example 5 and virus-neutralizing antibody titers against Rotavirus were measured as described below.
[0996] Diluted serum samples were incubated with a constant amount of Rotavirus virus strain Wa (G1P[8]), 1076 (G2P[6]) or DS-1 (G2P[4]) for 1 hour at 37° C., before transfer to a 96 well plate containing confluent MA104 cells. After 1 hour the monolayers were washed and incubated with the serum-virus mixture for 14-16 hours, frozen and thawed. VNTs were determined by measuring Rotavirus virus antigen in the lysed monolayers in wells receiving serum compared to viral control wells, using a standard enzyme-linked immunosorbent format. The neutralization titer represents a 60% reduction in the amount of virus. Results are shown in
[0997] Results
[0998] As shown in
[0999] As shown in
[1000] As shown in
[1001] As shown in
[1002] For the homologous VNT responses (
Example 10: Analysis of Multivalent Rotavirus Antigen Vaccine
[1003] The present example shows different mRNA vaccines encoding different Rotavirus serotypes and combinations that induce humoral immune responses in guinea pigs. Binding antibodies will be measured using ELISA and moreover virus neutralizing antibodies to the vaccine strain will be analysed.
[1004] mRNA constructs encoding VP8* of different Rotavirus serotypes are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccines (see Table 17) are applied on day 0 and 21 and administered intramuscular (i.m.) with different doses of RNA. Blood samples are taken at day 1, 21, 42, and 56 for determination of humoral immune responses.
TABLE-US-00019 TABLE 17 Vaccination scheme of Example 10 No. of Modified Group guinea pigs Rotavirus Serotype nucleotides 1 8 0.9% NaCl buffer, negative control — 2 8 Recombinant Rotavirus protein — P2-VP8*P[8] + Alum 3 8 VP8* construct P[4] — 4 8 VP8* construct P[6] — 5 8 VP8* construct P[8] — 6 8 VP8* construct combination of P[4], — P[6], P[8] 7 8 VP8* construct P[4] m1Ψ 8 8 VP8* construct P[4] m1Ψ 9 8 VP8* construct P[4] m1Ψ 10 8 VP8* construct combination of P[4], m1Ψ P[6], P[8] 11 8 VP8* construct P[4] Ψ 12 8 VP8* construct P[4] Ψ 13 8 VP8* construct P[4] Ψ 14 8 VP8* construct combination of P[4], Ψ P[6], P[8]
Example 11: Analysis of Multivalent Rotavirus Antigen Vaccine with Different Ratios
[1005] The present example shows different mRNA vaccine combinations encoding different Rotavirus serotypes with different ratios that induce humoral immune responses in guinea pigs. Binding antibodies will be measured using ELISA and moreover virus neutralizing antibodies to the vaccine strain will be analyzed.
[1006] mRNA constructs encoding VP8* of different Rotavirus serotypes are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccines (see Table 18) are applied on day 0 and 56 and administered intramuscular (i.m.) with different doses of RNA. Blood samples are taken at day 1, 28, 56 and 84 for determination of humoral immune responses.
TABLE-US-00020 TABLE 18 Vaccination scheme of Example 11 No. of Group guinea pigs Rotavirus Serotype Ratios 1 8 0.9% NaCl buffer, negative control — 2 8 Recombinant Rotavirus protein P2-VP8*P[8] + Alum — 3 8 VP8* construct combination of P[4], P[6], P[8] Ratio1 4 8 VP8* construct combination of P[4], P[6], P[8] Ratio2 5 8 VP8* construct combination of P[4], P[6], P[8] Ratio3 6 8 VP8* construct combination of P[4], P[6], P[8] Ratio4 7 8 VP8* construct combination of P[4], P[6], P[8] Ratio5 8 8 VP8* construct combination of P[4], P[6], P[8] Ratio6 9 8 VP8* construct combination of P[4], P[6], P[8] Ratio7 10 8 VP8* construct combination of P[4], P[6], P[8] Ratio8
Example 12: Analysis and Challenge of Multivalent Rotavirus Antigen Vaccine
[1007] The present example shows different mRNA vaccine combinations encoding different Rotavirus serotypes with two different doses that induce protective immune responses in gnotobiotic pigs. Binding antibodies are measured using ELISA and moreover virus-neutralizing antibodies to the vaccine strain is analysed.
[1008] mRNA constructs encoding VP8* constructs of different Rotavirus P-serotypes are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccines (see Table 19) are applied on day 0, 14 and 28 and administered intramuscular (i.m.) with two different doses of RNA. Blood samples are taken at day 1, 14, 28, 35, and 42 for determination of humoral immune responses. To test the protective efficacy of the mRNA vaccines, an oral challenge with Rotavirus Wa (G1P[8]) will be performed at day 35. To assess the effect of Rotavirus mRNA vaccination on shedding of virus, stool samples are collected from pigs at day 35 to 42.
TABLE-US-00021 TABLE 19 Vaccination scheme of Example 12 No. of Group guinea pigs Dosis Rotavirus Serotype Challenge 1 7 — 0.9% NaCl buffer, negative control Rotavirus Wa (G1P[8] strain) 2 7 3 × 30 μg Trivalent recombinant Rotavirus protein P2-VP8* P[8], Rotavirus Wa P[6], P[4] + Alum (G1P[8] strain) 3 7 Dose 1 VP8* mRNA construct 1 combination of P[4], P[6], Rotavirus Wa P[8] (G1P[8] strain) 4 7 Dose 1 VP8* construct 2 combination of P[4], P[6], P[8] Rotavirus Wa (G1P[8] strain) 5 7 Dose 2 VP8* construct 2 combination of P[4], P[6], P[8] Rotavirus Wa (G1P[8] strain)
Example 13: Analysis of Trivalent Rotavirus Antigen Vaccine with Different Candidates
[1009] The present example shows trivalent mRNA vaccine combinations encoding different Rotavirus serotypes always with a 1:1:1 ratio that are tested for their capacity to induce humoral immune responses in guinea pigs. Binding antibodies are measured using ELISA and moreover virus-neutralizing antibodies to the vaccine strain are analyzed.
[1010] mRNA constructs encoding VP8* of different Rotavirus serotypes are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.1 LNP formulation). The different mRNA vaccines are applied at two or three time points and administered intramuscular (i.m.) to seven guinea pigs per group with different doses of RNA (see Table 20). Blood samples are taken at day 1, 21/22, 42 and 70 for determination of humoral immune responses.
TABLE-US-00022 TABLE 20 Vaccination scheme of Example 13 5′-cap structure/ poly(A) sequence, UTRs RNA Construct Serum located at 3′ 5′-UTR/ SEQ ID Group Dose ID design Immunization samples terminus 3′-UTR NO: 1 100 μl — 0.9% NaCl d 0, d 21, d 1, d 21, — — — buffer, negative d 42 d 42, d 70 control 2 1 × — Monovalent rec. d 0, d 21, d 1, d 21, — — — 20 μg Rotavirus d 42 d 42, d 70 protein P2- VP8*P[8] + Alum 3 3 × — Trivalent rec. d 21, d 42 d 22, d 42, — — — 6.7 μg Rotavirus d 70 protein P2- VP8*P[8], P[6], P[4] + Alum 4 3 × — Trivalent rec. d 0, d 21, d 1, d 21, — — — 6.7 μg Rotavirus d 42 d 42, d 70 protein P2- VP8*P[8], P[6], P[4] + Alum 5 1 × R8628 P2-Linker- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1863 or 25 μg VP8*(65-223) d 42 d 42, d 70 cap/A100 PSMB3 1167 P[8] 6 1 × R9077 P2-Linker- d 0, d21, d 1, d 21, co-trans. HSD17B4/ 1922 25 μg VP8*(65-223) d42 d 42, d 70 cap/A100 PSMB3 P[6] 7 1 × R9078 P2-Linker- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1921 25 μg VP8*(65-223) d 42 d 42, d 70 cap/A100 PSMB3 P[4] 8 3 × R8628 + Trivalent P2- d 21, d 42 d 22, d 42, co-trans. HSD17B4/ 1863 or 8.3 μg R9077 + Linker-VP8*(65-223) d 70 cap/A100 PSMB3 1167, R9078 P[8, 1922, P[6], P[4]] 1921 9 3 × R8628 + Trivalent P2- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1863 or 8.3 μg R9077 + Linker-VP8*(65-223) d 42 d 42, d 70 cap/A100 PSMB3 1167, R9078 P[8, 1922, P[6], P[4] 1921 10 1 × R8578 LumSynt-Linker- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1876 or 25 μg P2-Linker- d 42 d 42, d 70 cap/A100 PSMB3 1212 VP8*(41-223) P[8] 11 1 × R9091 LumSynt-Linker- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1924 25 μg P2-Linker- d 42 d 42, d 70 cap/A100 PSMB3 VP8*(41-223) P[6] 12 1 × R9092 LumSynt-Linker- d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1923 25 μg P2-Linker- d 42 d 42, d 70 cap/A100 PSMB3 VP8*(41-223) P[4] 13 3 × R8578, Trivalent d 21, d 42 d 22, d 42, co-trans. HSD17B4/ 1876 or 1 μg R9091, LumSynt-Linker- d 70 cap/A100 PSMB3 1212, R9092 P2-Linker- 1924, VP8*(41-223) 1923 P[8], P[6], P[4] 14 3 × R8578, Trivalent d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1876 or 1 μg R9091, LumSynt-Linker- d 42 d 42, d 70 cap/A100 PSMB3 1212, R9092 P2-Linker- 1924, VP8*(41-223) 1923 P[8], P[6], P[4] 15 3 × R8578, Trivalent d 21, d 42 d 22, d 42, co-trans. HSD17B4/ 1876 or 8.3 μg R9091, LumSynt-Linker- d 70 cap/A100 PSMB3 1212, R9092 P2-Linker- 1924, VP8*(41-223) 1923 P[8], P[6], P[4] 16 3 × R8578, Trivalent d 0, d 21, d 1, d 21, co-trans. HSD17B4/ 1876 or 8.3 μg R9091, LumSynt-Linker- d 42 d 42, d 70 cap/A100 PSMB3 1212, R9092 P2-Linker- 1924, VP8*(41-223) 1923 P[8], P[6], P[4]
[1011] 9.1. Determination of Specific Humoral Immune Responses by ELISA:
[1012] ELISA are performed using recombinant Rotavirus protein P2-VP8* P[8], P[6] or P[4] for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the recombinant Rotavirus protein P2-VP8* is detected using biotinylated isotype specific anti-guinea pig antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG) directed against the recombinant Rotavirus protein P2-VP8* P[8], P[6] or P[4] are measured by ELISA on day 21, 42, and 70 post prime immunization for groups 1, 2, 4-7, 9-12, 14 and 16 and on day 21 and 49 post prime immunization for groups 3, 8, 13, 15.
[1013] 9.2 Determination of Virus-Neutralizing Antibody Titers (VNTs) Against Rotavirus
[1014] Serum samples are collected on day 70 post prime for groups 1, 2, 4-7, 9-12, 14 and 16 and on day 49 post prime for groups 3, 8, 13, 15 as described in Example 5 and Rotavirus neutralization titers are measured as described below.
[1015] Diluted serum samples are incubated with a constant amount of Rotavirus virus strain Wa (G1P[8]), 1076 (G2P[6]), or DS-1 (G2P[4]) for 1 hour at 37° C., before transfer to a 96 well plate containing confluent MA104 cells. After 1 hour the monolayers are washed and incubated with the serum-virus mixture for 15-17 hours, frozen and thawed. VNTs are determined by measuring Rotavirus virus antigen in the lysed monolayers in wells receiving serum compared to viral control wells, using a standard enzyme-linked immunosorbent format. The neutralization titer represents a 60% reduction in the amount of virus.