U.S. patent application number 16/471541 was filed with the patent office on 2019-11-21 for henipavirus vaccine.
The applicant listed for this patent is CureVac AG. Invention is credited to Edith JASNY, Benjamin PETSCH.
Application Number | 20190351047 16/471541 |
Document ID | / |
Family ID | 61002985 |
Filed Date | 2019-11-21 |
United States Patent
Application |
20190351047 |
Kind Code |
A1 |
JASNY; Edith ; et
al. |
November 21, 2019 |
HENIPAVIRUS VACCINE
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.
Inventors: |
JASNY; Edith; (Stuttgart,
DE) ; PETSCH; Benjamin; (Tubingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac AG |
Tubingen |
|
DE |
|
|
Family ID: |
61002985 |
Appl. No.: |
16/471541 |
Filed: |
December 22, 2017 |
PCT Filed: |
December 22, 2017 |
PCT NO: |
PCT/EP2017/084525 |
371 Date: |
June 19, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2760/18234 20130101; C07K 14/11 20130101; A61K 2039/6093
20130101; A61K 2039/70 20130101; C07K 14/115 20130101; A61K
2039/6031 20130101; A61K 2039/53 20130101; A61K 2039/55555
20130101; C07K 14/08 20130101; C12N 7/00 20130101; A61K 2039/51
20130101; A61K 2039/622 20130101; A61K 2039/64 20130101; A61K
39/145 20130101; C12N 2760/18271 20130101; A61K 39/155 20130101;
C12N 2760/18222 20130101 |
International
Class: |
A61K 39/155 20060101
A61K039/155; C07K 14/005 20060101 C07K014/005; C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2016 |
EP |
PCT/EP2016/082672 |
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 claim 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 .alpha.-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 of the
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 570P 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 claim 27; f) optionally, poly(C) sequence, preferably as
defined by any one of claim 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 claim 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 claims 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 claims 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 claims 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
[0001] The present invention is directed to an artificial nucleic
acid and to polypeptides suitable for use in treatment or
prophylaxis of an infection with 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 associated
with a Hendra virus and/or Nipah virus infection, 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.
[0002] Henipavirus is a genus of negative sense single stranded RNA
viruses belonging to the Paramyxovirinae virus superfamily. The
Henipavirus genome is about 18 kb in size, encoding for nine
proteins, comprising RNA-directed RNA polymerase (L), fusion
protein (F), non-structural protein (V), glycoprotein (G),
nucleoprotein (N), matrix protein (M), phosphoprotein (P), protein
C, and protein W. The genus currently contains five described and
established species, including the pathogenic viruses Hendra virus
and Nipah virus.
[0003] Hendra virus is the source of a recently emerging disease in
animals and human. Hendra virus was first recognized in September
1994 after an outbreak of respiratory illness among twenty horses
and two humans in Hendra, Queensland, Australia. In 1995, a second
unrelated outbreak was identified that had occurred in August 1994
in Mackay, Queensland, in which two horses died and one human
became. Four of the seven people who contracted the virus from
infected horses have died since the disease first emerged in 1994.
The fatality rate has been reported at more than 70% in horses and
50% in humans.
[0004] The Nipah virus was initially isolated in 1999 upon
examining samples from an outbreak of encephalitis and respiratory
illness among adult men in Malaysia and Singapore. The host for
Nipah virus is still unknown, but flying foxes (bats of the
Pteropus genus) are suspected to be the natural host. Infection
with Nipah virus in humans has been associated with encephalitis
characterized by fever and drowsiness and more serious central
nerve system disease, such as coma, seizures and inability to
maintain breathing. Illness with Nipah virus begins with 3-14 days
of fever and headache, followed by drowsiness and disorientation
characterized by mental confusion. These signs and symptoms can
progress to coma within 24-48 hours. Some patients have had a
respiratory illness during the early part of their infections.
Serious nerve disease with Nipah virus encephalitis has been marked
by some sequelae, such as persistent convulsions and personality
changes. During a Nipah virus disease outbreak in 1998-1999, about
40% of the patients with serious nerve disease who entered
hospitals died from the illness.
[0005] Hendra virus and Nipah virus, like the majority of other
paramyxoviruses, possess two surface glycoproteins, a fusion
protein (F) and a glycoprotein protein (G), both involved in
promotion of fusion between the viral membrane and the membrane of
the target host cell. Hendra viruses and Nipah viruses require both
their attachment and fusion proteins to initiate membrane fusion.
Various studies were conducted to understand the functions of the G
and F proteins in virus infection.
[0006] Current vaccine approaches for protection from Nipah virus
infection have focused on the use of Nipah virus glycoprotein (G)
and/or fusion protein (F) as immunogens in various platforms,
including DNA vaccines, subunit vaccines, non-replicating vectors,
as well as replicating vectors.
[0007] To date, no effective antiviral therapies have been approved
for either the prevention or treatment of diseases caused by Hendra
virus infections and/or Nipah virus infections. Thus, there is a
significant unmet medical need to find agents that can prevent
Hendra virus and/or Nipah virus infection, shorten the duration of
Hendra virus and/or Nipah virus-induced illness, lessen the
severity of symptoms, minimize secondary bacterial infections and
exacerbations of underlying disease, and reduce virus transmission.
A prophylactic Hendra virus and Nipah virus vaccine should be
protective against a wide variety of serotypes to reduce the number
of Hendra virus and Nipah virus infections, hence, reducing the
risk of a global pandemic threat.
[0008] The underlying object of the present invention is therefore
to provide a Hendra virus and/or Nipah virus vaccine. It is a
further preferred object of the invention to provide a Hendra virus
and/or Nipah virus vaccine, which may be produced in a fast manner
at an industrial scale in a potential pandemic scenario. A further
object of the present invention is the provision of a
storage-stable Hendra virus and/or Nipah virus vaccine. Further
object of the underlying invention is to provide nucleic acid
sequences, particularly mRNA sequences coding for antigenic
peptides or proteins derived from a protein of a Hendra virus
and/or Nipah virus or a fragment or variant thereof for the use as
a vaccine for prophylaxis or treatment of Hendra virus and/or Nipah
virus infections. Furthermore, it is the object of the present
invention to provide an effective Hendra virus and/or Nipah virus
vaccine which can be stored without cold chain and which enables
rapid and scalable vaccine production which is of major importance
in the context of pandemic Hendra virus and/or Nipah virus
outbreaks.
[0009] The object underlying the present invention is solved by the
claimed subject-matter.
Definitions
[0010] For the sake of clarity and readability the following
definitions are provided. Any technical feature mentioned for these
definitions may be read on each and every embodiment of the
invention. Additional definitions and explanations may be
specifically provided in the context of these embodiments.
[0011] Adaptive immune response: The adaptive immune response is
typically understood to be an antigen-specific response of the
immune system. Antigen specificity allows for the generation of
responses that are tailored to specific pathogens or
pathogen-infected cells. The ability to mount these tailored
responses is usually maintained in the body by "memory cells".
Should a pathogen infect the body more than once, these specific
memory cells are used to quickly eliminate it. In this context, the
first step of an adaptive immune response is the activation of
naive antigen-specific T cells or different immune cells able to
induce an antigen-specific immune response by antigen-presenting
cells. This occurs in the lymphoid tissues and organs through which
naive T cells are constantly passing. The three cell types that may
serve as antigen-presenting cells are dendritic cells, macrophages,
and B cells. Each of these cells has a distinct function in
eliciting immune responses. Dendritic cells may take up antigens by
phagocytosis and macropinocytosis and may become stimulated by
contact with e.g. a foreign antigen to migrate to the local
lymphoid tissue, where they differentiate into mature dendritic
cells. Macrophages ingest particulate antigens such as bacteria and
are induced by infectious agents or other appropriate stimuli to
express MHC molecules. The unique ability of B cells to bind and
internalize soluble protein antigens via their receptors may also
be important to induce T cells. MHC-molecules are, typically,
responsible for presentation of an antigen to T-cells. Therein,
presenting the antigen on MHC molecules leads to activation of T
cells which induces their proliferation and differentiation into
armed effector T cells. The most important function of effector T
cells is the killing of infected cells by CD8+ cytotoxic T cells
and the activation of macrophages by Th1 cells which together make
up cell-mediated immunity, and the activation of B cells by both
Th2 and Th1 cells to produce different classes of antibody, thus
driving the humoral immune response. T cells recognize an antigen
by their T cell receptors which do not recognize and bind the
antigen directly, but instead recognize short peptide fragments
e.g. of pathogen-derived protein antigens, e.g. so-called epitopes,
which are bound to MHC molecules on the surfaces of other
cells.
[0012] Adaptive immune system: The adaptive immune system is
essentially dedicated to eliminate or prevent pathogenic growth. It
typically regulates the adaptive immune response by providing the
vertebrate immune system with the ability to recognize and remember
specific pathogens (to generate immunity), and to mount stronger
attacks each time the pathogen is encountered. The system is highly
adaptable because of somatic hyper mutation (a process of
accelerated somatic mutations), and V(D)J recombination (an
irreversible genetic recombination of antigen receptor gene
segments). This mechanism allows a small number of genes to
generate a vast number of different antigen receptors, which are
then uniquely expressed on each individual lymphocyte. Because the
gene rearrangement leads to an irreversible change in the DNA of
each cell, all of the progeny (offspring) of such a cell will then
inherit genes encoding the same receptor specificity, including the
Memory B cells and Memory T cells that are the keys to long-lived
specific immunity.
[0013] Adjuvant/adjuvant component: An adjuvant or an adjuvant
component in the broadest sense is typically a pharmacological
and/or immunological agent that may modify, e.g. enhance, the
effect of other agents, such as a drug or vaccine. It is to be
interpreted in a broad sense and refers to a broad spectrum of
substances. Typically, these substances are able to increase the
immunogenicity of antigens. For example, adjuvants may be
recognized by the innate immune systems and, e.g., may elicit an
innate immune response. "Adjuvants" typically do not elicit an
adaptive immune response. Insofar, "adjuvants" do not qualify as
antigens. Their mode of action is distinct from the effects
triggered by antigens resulting in an adaptive immune response.
[0014] Antigen: In the context of the present invention "antigen"
refers typically to a substance which may be recognized by the
immune system, preferably by the adaptive immune system, and is
capable of triggering an antigen-specific immune response, e.g. by
formation of antibodies and/or antigen-specific T cells as part of
an adaptive immune response. Typically, an antigen may be or may
comprise a peptide or protein which may be presented by the MHC to
T-cells. In the sense of the present invention an antigen may be
the product of translation of a provided nucleic acid molecule,
preferably an mRNA as defined herein. In this context, also
fragments, variants and derivatives of peptides and proteins
comprising at least one epitope are understood as antigens.
[0015] Antigenic peptide or protein: An antigenic peptide or
protein is a peptide or protein derived from a protein which may
stimulate the body's adaptive immune system to provide an adaptive
immune response. Therefore an antigenic peptide or protein
comprises at least one epitope of the protein it is derived
from.
[0016] Artificial nucleic acid molecule: The terms "artificial
nucleic acid molecule" and "artificial nucleic acid" may typically
be understood to be a nucleic acid molecule, e.g. a DNA or an RNA
that does not occur naturally. In other words, an artificial
nucleic acid molecule may be understood as a non-natural nucleic
acid molecule. Such nucleic acid molecule may be non-natural due to
its individual sequence (which does not occur naturally) and/or due
to other modifications, e.g. structural modifications of
nucleotides which do not occur naturally. An artificial nucleic
acid molecule may be a DNA molecule, an RNA molecule or a
hybrid-molecule comprising DNA and RNA portions. Typically,
artificial nucleic acid molecules may be designed and/or generated
by genetic engineering methods to correspond to a desired
artificial sequence of nucleotides (heterologous sequence). In this
context an artificial sequence is usually a sequence that may not
occur naturally, i.e. it differs from the wild type sequence by at
least one nucleotide. The term "wild type" may be understood as a
sequence occurring in nature. Further, the term "artificial nucleic
acid molecule" is not restricted to mean "one single molecule" but
is, typically, understood to comprise an ensemble of identical
molecules. Accordingly, it may relate to a plurality of identical
molecules contained in an aliquot.
[0017] Bicistronic nucleic acid, multicistronic nucleic acid: A
bicistronic or multicistronic nucleic acid is typically an RNA or
DNA, preferably an mRNA that typically may have two (bicistronic)
or more (multicistronic) coding sequences. A coding sequence in
this context is a sequence of codons that is translatable into a
peptide or protein.
[0018] Carrier/polymeric carrier: A carrier in the context of the
invention may typically be a compound that facilitates transport
and/or complexation of another compound (cargo). A polymeric
carrier is typically a carrier that is formed of a polymer. A
carrier may be associated to its cargo by covalent or non-covalent
interaction. A carrier may transport nucleic acids, e.g. RNA or
DNA, to the target cells. The carrier may--for some embodiments--be
a cationic component.
[0019] Cationic component: The term "cationic component" or
"cationic compound" typically refers to a charged molecule, which
is positively charged (cation) at a pH value typically from 1 to 9,
preferably at a pH value of or below 9 (e.g. from 5 to 9), of or
below 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to 7), most
preferably at a physiological pH, e.g. from 7.3 to 7.4.
Accordingly, a cationic component, e.g. a cationic peptide,
cationic polysaccharide, a cationic lipid may be any positively
charged compound or polymer, preferably a cationic peptide or
protein which is positively charged under physiological conditions,
particularly under physiological conditions in vivo. A "cationic
peptide or protein" may contain at least one positively charged
amino acid, or more than one positively charged amino acid, e.g.
selected from Arg, His, Lys or Orn. Accordingly, "polycationic"
components are also within the scope exhibiting more than one
positive charge under the conditions given.
[0020] Cap analogue: A cap analogue refers to a non-polymerizable
di-nucleotide that has cap functionality in that it facilitates
translation or localization, and/or prevents degradation of a
nucleic acid molecule, particularly of an RNA molecule, when
incorporated at the 5'-end of the nucleic acid molecule.
Non-polymerizable means that the cap analogue will be incorporated
only at the 5' terminus because it does not have a 5' triphosphate
and therefore cannot be extended in the 3' direction by a
template-dependent polymerase, particularly, by template-dependent
RNA polymerase. Cap analogues include, but are not limited to, a
chemical structure selected from the group consisting of m7GpppG,
m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG);
dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap
analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap
analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g.,
ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and
their tetraphosphate derivatives) (Stepinski et al., 2001. RNA
7(10):1486-95). Further cap analogues have been described
previously (U.S. Pat. No. 7,074,596, WO 2008/016473, WO
2008/157688, WO 2009/149253, WO 2011/015347, and WO 2013/059475).
The synthesis of N7-(4-chlorophenoxyethyl) substituted dinucleotide
cap analogues has been described recently (Kore et al. (2013)
Bioorg. Med. Chem. 21(15): 4570-4).
[0021] 5'-cap-Structure: A 5'-cap is typically a modified
nucleotide (cap analogue), particularly a guanine nucleotide, added
to the 5'-end of a nucleic acid molecule, particularly of an RNA
molecule, e.g. an mRNA molecule. Preferably, the 5'-cap is added
using a 5'-5'-triphosphate linkage (also named m7GpppN). Further
examples of 5'-cap structures include glyceryl, inverted deoxy
abasic residue (moiety), 4',5' methylene nucleotide,
1-(beta-D-erythrofuranosyl) nucleotide, 4 `-thio nucleotide,
carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,
L-nucleotides, alpha-nucleotide, modified base nucleotide,
threo-pentofuranosyl nucleotide, acyclic 3`,4''-seco nucleotide,
acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl
nucleotide, 3''-3'-inverted nucleotide moiety, 3'-3'-inverted
abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted
abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate,
hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'
phosphorothioate, phosphorodithioate, or bridging or non-bridging
methylphosphonate moiety. These modified 5'-cap structures may be
used in the context of the present invention to modify the mRNA
sequence of the inventive composition. Further modified 5'-cap
structures which may be used in the context of the present
invention are cap 1 (additional methylation of the ribose of the
adjacent nucleotide of m7GpppN), cap2 (additional methylation of
the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3
(additional methylation of the ribose of the 3rd nucleotide
downstream of the m7GpppN), cap4 (additional methylation of the
ribose of the 4th nucleotide downstream of the m7GpppN), ARCA
(anti-reverse cap analogue), modified ARCA (e.g. phosphothioate
modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine. In the context of the present
invention, a 5'-cap (cap0 or cap1) structure may also be formed in
chemical RNA synthesis or RNA in vitro transcription
(co-transcriptional capping) using cap analogues, or a cap
structure may be formed in vitro using capping enzymes (e.g.,
commercially available capping kits) or using immobilized capping
enzymes, e.g. in a capping reactor (WO 2016/193226).
[0022] Cellular immunity/cellular immune response: Cellular
immunity relates typically to the activation of macrophages,
natural killer cells (NK), antigen-specific cytotoxic
T-lymphocytes, and the release of various cytokines in response to
an antigen. In more general terms, cellular immunity is not based
on antibodies, but on the activation of cells of the immune system.
Typically, a cellular immune response may be characterized e.g. by
activating antigen-specific cytotoxic T-lymphocytes that are able
to induce apoptosis in cells, e.g. specific immune cells like
dendritic cells or other cells, displaying epitopes of foreign
antigens on their surface. Such cells may be virus-infected or
infected with intracellular bacteria, or cancer cells displaying
tumor antigens. Further characteristics may be activation of
macrophages and natural killer cells, enabling them to destroy
pathogens and stimulation of cells to secrete a variety of
cytokines that influence the function of other cells involved in
adaptive immune responses and innate immune responses.
[0023] Chemical synthesis of nucleic acids: Chemical synthesis of
relatively short fragments of oligonucleotides with defined
chemical structure provides a rapid and inexpensive access to
custom-made oligonucleotides of any desired sequence. Whereas
enzymes synthesize DNA and RNA only in the 5' to 3' direction,
chemical oligonucleotide synthesis does not have this limitation,
although it is most often carried out in the opposite, i.e. the 3'
to 5' direction. Currently, the process is implemented as
solid-phase synthesis using the phosphoramidite method and
phosphoramidite building blocks derived from protected nucleosides
(A, C, G, and U), or chemically modified nucleosides. To obtain the
desired oligonucleotide, the building blocks are sequentially
coupled to the growing oligonucleotide chain on a solid phase in
the order required by the sequence of the product in a fully
automated process. Upon the completion of the chain assembly, the
product is released from the solid phase to the solution,
deprotected, and collected. The occurrence of side reactions sets
practical limits for the length of synthetic oligonucleotides (up
to about 200 nucleotide residues), because the number of errors
increases with the length of the oligonucleotide being synthesized.
Products are often isolated by HPLC to obtain the desired
oligonucleotides in high purity. Chemically synthesized
oligonucleotides find a variety of applications in molecular
biology and medicine. They are most commonly used as antisense
oligonucleotides, small interfering RNA, primers for DNA sequencing
and amplification, probes for detecting complementary DNA or RNA
via molecular hybridization, tools for the targeted introduction of
mutations and restriction sites, and for the synthesis of
artificial genes. Moreover, long-chain DNA molecules and long-chain
RNA molecules may be chemically synthetized and used in the context
of the present invention.
[0024] Cloning site: A cloning site is typically understood to be a
segment of a nucleic acid molecule, which is suitable for insertion
of a nucleic acid sequence, e.g., a nucleic acid sequence
comprising a coding sequence. Insertion may be performed by any
molecular biological method known to the one skilled in the art,
e.g. by restriction and ligation. A cloning site typically
comprises one or more restriction enzyme recognition sites
(restriction sites). These one or more restrictions sites may be
recognized by restriction enzymes which cleave the DNA at these
sites. A cloning site which comprises more than one restriction
site may also be termed a multiple cloning site (MCS) or a
polylinker.
[0025] Coding sequence: A coding sequence (cds) in the context of
the invention is typically a sequence of several nucleotide
triplets, which may be translated into a peptide or protein. A
coding sequence preferably contains a start codon, i.e. a
combination of three subsequent nucleotides coding usually for the
amino acid methionine (ATG), at its 5''-end and a subsequent region
which usually exhibits a length which is a multiple of 3
nucleotides. A coding sequence is preferably terminated by a
stop-codon (e.g., TAA, TAG, and TGA). Typically, this is the only
stop-codon of the coding sequence. Thus, a coding sequence in the
context of the present invention is preferably a nucleotide
sequence, consisting of a number of nucleotides that may be divided
by three, which starts with a start codon (e.g. ATG) and which
preferably terminates with a stop codon (e.g., TAA, TGA, or TAG).
The coding sequence may be isolated or it may be incorporated in a
longer nucleic acid sequence, for example in a vector or an mRNA.
In the context of the present invention, a coding sequence may also
be termed "protein coding region", "coding sequence", "cds", "open
reading frame" or "ORF".
[0026] Derived from: The phrase "derived from" as used throughout
the present specification in the context of a nucleic acid, i.e.
for a nucleic acid "derived from" (another) nucleic acid, means
that the nucleic acid, which is derived from (another) nucleic
acid, shares at least 50%, preferably at least 55%, preferably at
least 60%, preferably at least 65%, preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, 81%, 82%,
83%, 84%, more preferably at least 85%, 86%, 87%, 88%, 89% even
more preferably at least 90%, 91%, 92%, 93%, 94%, even more
preferably at least 95%, 96%, 97%, and particularly preferably at
least 98%, 99% sequence identity with the nucleic acid from which
it is derived. The skilled person is aware that sequence identity
is typically calculated for the same types of nucleic acids, i.e.
for DNA sequences or for RNA sequences. Thus, it is understood, if
a DNA is "derived from" an RNA or if an RNA is "derived from" a
DNA, in a first step the RNA sequence is converted into the
corresponding DNA sequence (in particular by replacing the uracils
(U) by thymidines (T) throughout the sequence) or, vice versa, the
DNA sequence is converted into the corresponding RNA sequence (in
particular by replacing the thymidines (T) by uracils (U)
throughout the sequence). Thereafter, the sequence identity of the
DNA sequences or the sequence identity of the RNA sequences is
determined. Preferably, a nucleic acid "derived from" a nucleic
acid also refers to nucleic acid, which is modified in comparison
to the nucleic acid from which it is derived, e.g. in order to
increase RNA stability even further and/or to prolong and/or
increase protein production. It goes without saying that such
modifications are preferred, which do not impair RNA stability,
e.g. in comparison to the nucleic acid from which it is
derived.
[0027] "Different Hendra virus", "different Nipah virus",
"different Henipavirus": The terms "different Hendra virus",
[0028] "different Nipah virus", "different Henipavirus" in the
context of the invention has to be understood as the difference
between at least two respective viruses, wherein the difference is
manifested on the RNA genome of the respective different virus. In
the broadest sense, "different Nipah virus" has to be understood as
genetically "different Nipah virus". Similarly, "different Hendra
virus" has to be understood as genetically "different Hendra
virus". Particularly, said (genetically) different viruses express
at least one different protein or peptide, wherein the at least one
different protein or peptide preferably differs in at least one
amino acid.
[0029] "Same Henipavirus", "same Nipah virus", "same Hendra virus":
In the broadest sense, "same Henipavirus", "same Nipah virus", or
"same Hendra virus" has to be understood as genetically.
Particularly, said (genetically) same virus expresses the same
proteins or peptides (e.g., at least one structural and/or
non-structural protein), wherein all proteins or peptides have the
same amino acid sequence.
[0030] DNA: DNA is the usual abbreviation for deoxy-ribonucleic
acid. It is a nucleic acid molecule, i.e. a polymer consisting of
nucleotides. These nucleotides are usually
deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate,
deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate
monomers which are--by themselves--composed of a sugar moiety
(deoxyribose), a base moiety and a phosphate moiety, and polymerize
by a characteristic backbone structure. The backbone structure is,
typically, formed by phosphodiester bonds between the sugar moiety
of the nucleotide, i.e. deoxyribose, of a first and a phosphate
moiety of a second, adjacent monomer. The specific order of the
monomers, i.e. the order of the bases linked to the
sugar/phosphate-backbone, is called the DNA sequence. DNA may be
single stranded or double stranded. In the double stranded form,
the nucleotides of the first strand typically hybridize with the
nucleotides of the second strand, e.g. by NT-base-pairing and
G/C-base-pairing.
[0031] Epitope: An "epitope" (also called "antigen determinant")
can be distinguished in T cell epitopes and B cell epitopes. T cell
epitopes or parts of the proteins in the context of the present
invention may comprise fragments preferably having a length of
about 6 to about 20 or even more amino acids, e.g. fragments as
processed and presented by MHC class I molecules, preferably having
a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or
even 11, or 12 amino acids), or fragments as processed and
presented by MHC class II molecules, preferably having a length of
about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20
or even more amino acids, wherein these fragments may be selected
from any part of the amino acid sequence. These fragments are
typically recognized by T cells in form of a complex consisting of
the peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form. B cell epitopes are
typically fragments located on the outer surface of (native)
protein or peptide antigens as defined herein, preferably having 5
to 15 amino acids, more preferably having 5 to 12 amino acids, even
more preferably having 6 to 9 amino acids, which may be recognized
by antibodies, i.e. in their native form. Such epitopes of proteins
or peptides may furthermore be selected from any of the herein
mentioned variants of such proteins or peptides. In this context
antigenic determinants can be conformational or discontinuous
epitopes which are composed of segments of the proteins or peptides
as defined herein that are discontinuous in the amino acid sequence
of the proteins or peptides as defined herein but are brought
together in the three-dimensional structure or continuous or linear
epitopes which are composed of a single polypeptide chain.
[0032] Fragment of a sequence: A fragment of a sequence may
typically be a shorter portion of a full-length sequence of e.g. a
nucleic acid sequence or an amino acid sequence. Accordingly, a
fragment, typically, consists of a sequence that is identical to
the corresponding stretch within the full-length sequence. A
preferred fragment of a sequence in the context of the present
invention, consists of a continuous stretch of entities, such as
nucleotides or amino acids corresponding to a continuous stretch of
entities in the molecule the fragment is derived from, which
represents at least 5%, 10%, 20%, preferably at least 30%, more
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, even more preferably at least 70%, and
most preferably at least 80% of the total (i.e. full-length)
molecule from which the fragment is derived.
[0033] Fragments of proteins: "Fragments" of proteins or peptides
in the context of the present invention may, typically, comprise a
sequence of a protein or peptide as defined herein, which is, with
regard to its amino acid sequence (or its encoded nucleic acid
molecule), N-terminally and/or C-terminally truncated compared to
the amino acid sequence of the original (native) protein (or its
encoded nucleic acid molecule). Such truncation may thus occur
either on the amino acid level or correspondingly on the nucleic
acid level. A sequence identity with respect to such a fragment as
defined herein may therefore preferably refer to the entire protein
or peptide as defined herein or to the entire (coding) nucleic acid
molecule of such a protein or peptide. In the context of antigens
such fragment may have a length of about 6 to about 20 or even more
amino acids, e.g. fragments as processed and presented by MHC class
I molecules, preferably having a length of about 8 to about 10
amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino
acids), or fragments as processed and presented by MHC class II
molecules, preferably having a length of about 13 or more amino
acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino
acids, wherein these fragments may be selected from any part of the
amino acid sequence. These fragments are typically recognized by
T-cells in form of a complex consisting of the peptide fragment and
an MHC molecule, i.e. the fragments are typically not recognized in
their native form. Fragments of proteins or peptides (e.g. in the
context of antigens) may comprise at least one epitope of those
proteins or peptides. Furthermore also domains of a protein, like
the extracellular domain, the intracellular domain or the
transmembrane domain and shortened or truncated versions of a
protein may be understood to comprise a fragment of a protein.
[0034] G/C modified: The terms "G/C modified" or "G/C content
modification" may typically be a nucleic acid, preferably an
artificial nucleic acid molecule as defined herein, based on a
modified wild type sequence comprising a preferably increased
number of guanosine and/or cytosine nucleotides as compared to the
wild type sequence.
[0035] Such an increased number may be generated by substitution of
codons containing adenosine or thymidine nucleotides by codons
containing guanosine or cytosine nucleotides. If the enriched G/C
content occurs in a coding sequence of DNA or RNA, it makes use of
the degeneracy of the genetic code. Accordingly, the codon
substitutions preferably do not alter the encoded amino acid
residues, but exclusively increase the G/C content of the nucleic
acid molecule.
[0036] Gene therapy: Gene therapy may typically be understood to
mean a treatment of a patient's body or isolated elements of a
patient's body, for example isolated tissues/cells, by nucleic
acids encoding a peptide or protein. It typically may comprise at
least one of the steps of a) administration of a nucleic acid,
preferably an artificial nucleic acid molecule as defined herein,
directly to the patient--by whatever administration route--or in
vitro to isolated cells/tissues of the patient, which results in
transfection of the patient's cells either in vivo/ex vivo or in
vitro; b) transcription and/or translation of the introduced
nucleic acid molecule; and optionally c) re-administration of
isolated, transfected cells to the patient, if the nucleic acid has
not been administered directly to the patient.
[0037] Genetic vaccination: Genetic vaccination may typically be
understood to be vaccination by administration of a nucleic acid
molecule encoding an antigen or an immunogen or fragments thereof.
The nucleic acid molecule may be administered to a subject's body
or to isolated cells of a subject. Upon transfection of certain
cells of the body or upon transfection of the isolated cells, the
antigen or immunogen may be expressed by those cells and
subsequently presented to the immune system, eliciting an adaptive,
i.e. antigen-specific immune response. Accordingly, genetic
vaccination typically comprises at least one of the steps of a)
administration of a nucleic acid, preferably an artificial nucleic
acid molecule as defined herein, to a subject, preferably a
patient, or to isolated cells of a subject, preferably a patient,
which usually results in transfection of the subject's cells either
in vivo or in vitro; b) transcription and/or translation of the
introduced nucleic acid molecule; and optionally c)
re-administration of isolated, transfected cells to the subject,
preferably the patient, if the nucleic acid has not been
administered directly to the patient.
[0038] Genotype, genotype of a virus: The genetic constitution of
an individual or a group or class of organisms having the same
genetically consistent structure. Genotyping means determining
differences in the genetic of an individual. In the context of the
invention, Nipah virus genotype has to be understood as a Nipah
virus having the same genetically consistent structure and Hendra
virus genotype has to be understood as a Hendra virus having the
same genetically consistent structure.
[0039] Heteroloqous sequence: Two sequences are typically
understood to be "heterologous" if they are not derivable from the
same gene or in the same allele. I.e., although heterologous
sequences may be derivable from the same organism, they naturally
(in nature) do not occur in the same nucleic acid molecule, such as
in the same mRNA.
[0040] Homoloq of a nucleic acid sequence: The term "homolog" of a
nucleic acid sequence refers to sequences of other species than the
particular sequence. E.g., in the context of the invention, it is
particularly preferred that the nucleic acid sequence is derived
from a Nipah virus and/or Hendra virus; therefore it is preferred
that the homolog is a homolog of a respective Nipah virus or
respective Hendra virus nucleic acid sequence.
[0041] Humoral immunity/humoral immune response: Humoral immunity
refers typically to B-cell mediated antibody production and
optionally to accessory processes accompanying antibody production.
A humoral immune response may be typically characterized, e.g., by
Th2 activation and cytokine production, germinal center formation
and isotype switching, affinity maturation and memory cell
generation. Humoral immunity also typically may refer to the
effector functions of antibodies, which include pathogen and toxin
neutralization, classical complement activation, and opsonin
promotion of phagocytosis and pathogen elimination.
[0042] "Hybridizing" or "Hybridizing with a complement sequence":
Nucleic acid molecules which are advantageously for the process
according to the invention can be isolated based on their homology
to the nucleic acid molecules or a complement sequence of the
nucleic acid molecules disclosed herein using the sequences or part
thereof as hybridization probe and following standard hybridization
techniques under stringent hybridization conditions. In this
context, it is possible to use, for example, isolated nucleic acid
molecules of at least 15, 20, 25, 30, 35, 40, 50, 60 or more
nucleotides, preferably of at least 15, 20 or 25 nucleotides in
length which hybridize under stringent conditions with the
above-described nucleic acid molecules, in particular with those
which encompass a nucleotide sequence of the nucleic acid molecule
used in the invention or encoding a protein used in the invention
or of the nucleic acid molecule of the invention. Nucleic acid
molecules with 30, 50, 100, 250 or more nucleotides may also be
used. The term "homology" means that the respective nucleic acid
molecules or encoded proteins are functionally and/or structurally
equivalent. The nucleic acid molecules that are homologous to the
nucleic acid molecules described above and that are derivatives of
said nucleic acid molecules are, for example, variations of said
nucleic acid molecules which represent modifications having the
same biological function, in particular encoding proteins with the
same or substantially the same biological function. They may be
naturally occurring variations, such as sequences from other
species, strains, or mutations. These mutations may occur naturally
or may be obtained by mutagenesis techniques. The allelic
variations may be naturally occurring allelic variants as well as
synthetically produced or genetically engineered variants.
Structurally equivalents can, for example, be identified by testing
the binding of said polypeptide to antibodies or computer based
predictions. By "hybridizing" it is meant that such nucleic acid
molecules hybridize under conventional hybridization conditions,
preferably under stringent conditions such as described by, e.g.,
Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) or
in Current Protocols in Molecular Biology, John Wiley & Sons,
N. Y. (1989), 6.3.1-6.3.6. According to the invention, DNA as well
as RNA molecules of the nucleic acid of the invention can be used
as probes. Further, as template for the identification of
functional homologues Northern blot assays as well as Southern blot
assays can be performed. The Northern blot assay advantageously
provides further information about the expressed gene product: e.g.
expression pattern, occurrence of processing steps, like splicing
and capping, etc. The Southern blot assay provides additional
information about the chromosomal localization and organization of
the gene encoding the nucleic acid molecule of the invention. A
preferred, no limiting example of stringent hybridization
conditions are hybridizations in 6.times. sodium chloride/sodium
citrate (=SSC) at approximately 45.degree. C., followed by one or
more wash steps in 0.2.times.SSC, 0.1% SDS at 50 to 65.degree. C.,
for example at 50.degree. C., 55.degree. C. or 60.degree. C. The
skilled worker knows that these hybridization conditions differ as
a function of the type of the nucleic acid and, for example when
organic solvents are present, with regard to the temperature and
concentration of the buffer. The temperature under "standard
hybridization conditions" differs for example as a function of the
type of the nucleic acid between 42.degree. C. and 58.degree. C.,
preferably between 45.degree. C. and 50.degree. C. in an aqueous
buffer with a concentration of 0.1.times. 0.5.times., 1.times.,
2.times., 3.times., 4.times. or 5.times.SSC (pH 7.2). If organic
solvent(s) is/are present in the abovementioned buffer, for example
50% formamide, the temperature under standard conditions is
approximately 40.degree. C., 42.degree. C. or 45.degree. C. The
hybridization conditions for DNA:DNA hybrids are preferably for
example 0.1.times.SSC and 20.degree. C., 25.degree. C., 30.degree.
C., 35.degree. C., 40.degree. C. or 45.degree. C., preferably
between 30.degree. C. and 45.degree. C. The hybridization
conditions for DNA:RNA hybrids are preferably for example
0.1.times.SSC and 30.degree. C., 35.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C. or 55.degree. C., preferably between
45.degree. C. and 55.degree. C. The abovementioned hybridization
temperatures are determined for example for a nucleic acid
approximately 100 bp (=base pairs) in length and a G+C content of
50% in the absence of formamide. The skilled worker knows to
determine the hybridization conditions required with the aid of
textbooks, for example the ones mentioned above, or from the
following textbooks: Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985,
"Nucleic Acids Hybridization: A Practical Approach", IRL Press at
Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential
Molecular Biology: A Practical Approach", IRL Press at Oxford
University Press, Oxford. A further example of one such stringent
hybridization condition is hybridization at 4.times.SSC at
65.degree. C., followed by a washing in 0.1.times.SSC at 65.degree.
C. for one hour. Alternatively, an exemplary stringent
hybridization condition is in 50% formamide, 4.times.SSC at
42.degree. C. Further, the conditions during the wash step can be
selected from the range of conditions delimited by low-stringency
conditions (approximately 2.times.SSC at 50.degree. C.) and
high-stringency conditions (approximately 0.2.times.SSC at
50.degree. C., preferably at 65.degree. C.) (20.times.SSC: 0.3M
sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature
during the wash step can be raised from low-stringency conditions
at room temperature, approximately 22.degree. C., to
higher-stringency conditions at approximately 65.degree. C. Both of
the parameters salt concentration and temperature can be varied
simultaneously, or else one of the two parameters can be kept
constant while only the other is varied. Denaturants, for example
formamide or SDS, may also be employed during the hybridization. In
the presence of 50% formamide, hybridization is preferably effected
at 42.degree. C. Relevant factors like i) length of treatment, ii)
salt conditions, iii) detergent conditions, iv) competitor DNAs, v)
temperature and vi) probe selection can be combined case by case so
that not all possibilities can be mentioned herein.
[0043] Some examples of conditions for DNA hybridization (Southern
blot assays) and wash step are shown herein below:
(1) Hybridization conditions can be selected, for example, from the
following conditions:
a) 4.times.SSC at 65.degree. C.,
b) 6.times.SSC at 45.degree. C.,
[0044] c) 6.times.SSC, 100 mg/ml denatured fragmented fish sperm
DNA at 68.degree. C., d) 6.times.SSC, 0.5% SDS, 100 mg/ml denatured
salmon sperm DNA at 68.degree. C., e) 6.times.SSC, 0.5% SDS, 100
mg/mI denatured fragmented salmon sperm DNA, 50% formamide at
42.degree. C., f) 50% formamide, 4.times.SSC at 42.degree. C., g)
50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll,
0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5,
750 mM NaCl, 75 mM sodium citrate at 42.degree. C., h) 2.times. or
4.times.SSC at 50.degree. C. (low-stringency condition), or i) 30
to 40% formamide, 2.times. or 4.times.SSC at 42.degree. C.
(low-stringency condition). (2) Wash steps can be selected, for
example, from the following conditions: a) 0.015 M NaCl/0.0015 M
sodium citrate/0.1% SDS at 50.degree. C.
b) 0.1.times.SSC at 65.degree. C.
c) 0.1.times.SSC, 0.5% SDS at 68.degree. C.
[0045] d) 0.1.times.SSC, 0.5% SDS, 50% formamide at 42.degree.
C.
e) 0.2.times.SSC, 0.1% SDS at 42.degree. C.
[0046] f) 2.times.SSC at 65.degree. C. (low-stringency
condition).
[0047] Further, some applications have to be performed at low
stringency hybridization conditions, without any consequences for
the specificity of the hybridization. For example, a Southern blot
analysis of total DNA could be probed with a nucleic acid molecule
of the present invention and washed at low stringency (55.degree.
C. in 2.times.SSPE, 0.1% SDS). A further example of such
low-stringent hybridization conditions is 4.times.SSC at 50.degree.
C. or hybridization with 30 to 40% formamide at 42.degree. C. Such
molecules comprise those which are fragments, analogues or
derivatives of the polypeptide of the invention or used in the
methods of the invention and differ, for example, by way of amino
acid and/or nucleotide deletion(s), insertion(s), substitution (s),
addition(s) and/or recombination (s) or any other modification(s)
known in the art either alone or in combination from the
above-described amino acid sequences or their underlying nucleotide
sequence(s). However, it is preferred to use high stringency
hybridization conditions. Hybridization should advantageously be
carried out with fragments of at least 5, 10, 15, 20, 25, 30, 35 or
40 bp, advantageously at least 50, 60, 70 or 80 bp, preferably at
least 90, 100 or 110 bp. Most preferably are fragments of at least
15, 20, 25 or 30 bp. Preferably are also hybridizations with at
least 100 bp or 200, very especially preferably at least 400 bp in
length. In an especially preferred embodiment, the hybridization
should be carried out with the entire nucleic acid sequence with
conditions described above. The term "hybridizes under stringent
conditions" is defined above. In one embodiment, the term
"hybridizes under stringent conditions" is intended to describe
conditions for hybridization and washing under which nucleotide
sequences at least 30%, 40%, 50% or 65% identical to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 70%, more
preferably at least about 75% or 80%, and even more preferably at
least about 85%, 90% or 95% or more identical to each other
typically remain hybridized to each other. To determine the
percentage homology (=identity, herein used interchangeably) of two
amino acid sequences or of two nucleic acid molecules, the
sequences are written one underneath the other for an optimal
comparison (for example gaps may be inserted into the sequence of a
protein or of a nucleic acid in order to generate an optimal
alignment with the other protein or the other nucleic acid). The
amino acid residues or nucleic acid molecules at the corresponding
amino acid positions or nucleotide positions are then compared. If
a position in one sequence is occupied by the same amino acid
residue or the same nucleic acid molecule as the corresponding
position in the other sequence, the molecules are homologous at
this position (i.e. amino acid or nucleic acid "homology" as used
in the present context corresponds to amino acid or nucleic acid
"identity". The percentage homology between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e. % homology=number of identical positions/total
number of positions.times.100). The terms "homology" and "identity"
are thus to be considered as synonyms. For the determination of the
percentage homology (=identity) of two or more amino acids or of
two or more nucleotide sequences several computer software programs
have been developed. The homology of two or more sequences can be
calculated with for example the software fasta, which presently has
been used in the version fasta 3 (W. R. Pearson and D. J. Lipman
(1988), Improved Tools for Biological Sequence Comparison. PNAS
85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive Sequence
Comparison with FASTP and FASTA, Methods in Enzymology 183:63-98;
W. R. Pearson and D. J. Lipman (1988) Improved Tools for Biological
Sequence Comparison.PNAS 85:2444-2448; W. R. Pearson (1990); Rapid
and Sensitive Sequence Comparison with FASTP and FASTAMethods in
Enzymology 183:63-98). Another useful program for the calculation
of homologies of different sequences is the standard blast program,
which is included in the Biomax pedant software (Biomax, Munich,
Federal Republic of Germany). This leads unfortunately sometimes to
suboptimal results since blast does not always include complete
sequences of the subject and the query. Nevertheless as this
program is very efficient it can be used for the comparison of a
huge number of sequences. The following settings are typically used
for such a comparisons of sequences: -p Program Name [String]; -d
Database [String]; default=nr; -i Query File [File In];
default=stdin; -e Expectation value (E) [Real]; default=10.0; -m
alignment view options: 0=pairwise; 1=query-anchored showing
identities; 2=query-anchored no identities; 3=flat query-anchored,
show identities; 4=flat query-anchored, no identities;
5=query-anchored no identities and blunt ends; 6=flat
query-anchored, no identities and blunt ends; 7=XML Blast output;
8=tabular; 9 tabular with comment lines [Integer]; default=0; -o
BLAST report Output File [File Out] Optional; default=stdout; -F
Filter query sequence (DUST with blastn, SEG with others) [String];
default=T; -G Cost to open a gap (zero invokes default behavior)
[Integer]; default=0; -E Cost to extend a gap (zero invokes default
behavior) [Integer]; default=0; -X X dropoff value for gapped
alignment (in bits) (zero invokes default behavior); blastn 30,
megablast 20, tblastx 0, all others 15 [Integer]; default=0; -l
Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide
mismatch (blastn only) [Integer]; default=-3; -r Reward for a
nucleotide match (blastn only) [Integer]; default=1; -v Number of
database sequences to show one-line descriptions for (V) [Integer];
default=500; -b Number of database sequence to show alignments for
(B) [Integer]; default=250; -f Threshold for extending hits,
default if zero; blastp 11, blastn 0, blastx 12, tblastn 13;
tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped
alignment (not available with tblastx) [T/F]; default=T; -Q Query
Genetic code to use [Integer]; default=1; -D DB Genetic code (for
tblast[nx] only) [Integer]; default=1; -a Number of processors to
use [Integer]; default=1; -O SeqAlign file [File Out] Optional; -J
Believe the query define [T/F]; default=F; -M Matrix [String];
default=BLOSUM62; -W Word size, default if zero (blastn 11,
megablast 28, all others 3) [Integer]; default=0; -z Effective
length of the database (use zero for the real size) [Real];
default=0; -K Number of best hits from a region to keep (off by
default, if used a value of 100 is recommended) [Integer];
default=0; -P 0 for multiple hit, 1 for single hit [Integer];
default=0; -Y Effective length of the search space (use zero for
the real size) [Real]; default=0; -S Query strands to search
against database (for blast[nx], and tblastx); 3 is both, 1 is top,
2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];
default=F; -l Restrict search of database to list of GI's [String]
Optional; -U Use lower case filtering of FASTA sequence [T/F]
Optional; default=F; -y X dropoff value for ungapped extensions in
bits (0.0 invokes default behavior); blastn 20, megablast 10, all
others 7 [Real]; default=0.0; -Z X dropoff value for final gapped
alignment in bits (0.0 invokes default behavior); blastn/megablast
50, tblastx 0, all others 25 [Integer]; default=0; -R PSI-TBLASTN
checkpoint file [File In] Optional; -n MegaBlast search [T/F];
default=F; -L Location on query sequence [String] Optional; -A
Multiple Hits window size, default if zero (blastn/megablast 0, all
others 40 [Integer]; default=0; -w Frame shift penalty (OOF
algorithm for blastx) [Integer]; default=0; -t Length of the
largest intron allowed in tblastn for linking HSPs (0 disables
linking) [Integer]; default=0.
[0048] Results of high quality are reached by using the algorithm
of Needleman and Wunsch or Smith and Waterman. Therefore programs
based on said algorithms are preferred. Advantageously the
comparisons of sequences can be done with the program PileUp (J.
Mol. Evolution., 25, 351 (1987), Higgins et al., CABIOS 5, 151
(1989)) or preferably with the programs "Gap" and "Needle", which
are both based on the algorithms of Needleman and Wunsch (J. Mol,
Biol. 48; 443 (1970)), and "BestFit", which is based on the
algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)).
"Gap" and "BestFit" are part of the GCG software-package (Genetics
Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991);
Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), "Needle" is
part of the The European Molecular Biology Open Software Suite
(EMBOSS) (Trends in Genetics 16 (6), 276 (2000)). Therefore
preferably the calculations to determine the percentages of
sequence homology are done with the programs "Gap" or "Needle" over
the whole range of the sequences. The following standard
adjustments for the comparison of nucleic acid sequences were used
for "Needle": matrix: EDNAFULL, Gap_penalty: 10.0, Extend_penalty:
0.5. The following standard adjustments for the comparison of
nucleic acid sequences were used for "Gap": gap weight: 50, length
weight: 3, average match: 10.000, average mismatch: 0.000.
[0049] Identity of a sequence: In order to determine the percentage
to which two sequences are identical, e.g. nucleic acid sequences
or amino acid sequences as defined herein, preferably the amino
acid sequences encoded by a nucleic acid sequence of the polymeric
carrier as defined herein or the amino acid sequences themselves,
the sequences can be aligned in order to be subsequently compared
to one another. Therefore, e.g. a position of a first sequence may
be compared with the corresponding position of the second sequence.
If a position in the first sequence is occupied by the same
component (residue) as is the case at a position in the second
sequence, the two sequences are identical at this position. If this
is not the case, the sequences differ at this position. If
insertions occur in the second sequence in comparison to the first
sequence, gaps can be inserted into the first sequence to allow a
further alignment. If deletions occur in the second sequence in
comparison to the first sequence, gaps can be inserted into the
second sequence to allow a further alignment. The percentage to
which two sequences are identical is then a function of the number
of identical positions divided by the total number of positions
including those positions which are only occupied in one sequence.
The percentage to which two sequences are identical can be
determined using a mathematical algorithm. A preferred, but not
limiting, example of a mathematical algorithm which can be used is
the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or
Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an
algorithm is integrated in the BLAST program. Sequences which are
identical to the sequences of the present invention to a certain
extent can be identified by this program.
[0050] Immunogen: In the context of the present invention an
immunogen may be typically understood to be a compound that is able
to stimulate an immune response. Preferably, an immunogen is a
peptide, polypeptide, or protein. In a particularly preferred
embodiment, an immunogen in the sense of the present invention is
the product of translation of a provided nucleic acid molecule,
preferably an artificial nucleic acid molecule as defined herein.
Typically, an immunogen elicits at least an adaptive immune
response.
[0051] Immunostimulatory composition: In the context of the
invention, an immunostimulatory composition may be typically
understood to be a composition containing at least one component
which is able to induce an immune response or from which a
component which is able to induce an immune response is derivable.
Such immune response may be preferably an innate immune response or
a combination of an adaptive and an innate immune response.
Preferably, an immunostimulatory composition in the context of the
invention contains at least one artificial nucleic acid molecule,
more preferably an RNA, for example an mRNA molecule. The
immunostimulatory component, such as the mRNA may be complexed with
a suitable carrier. Thus, the immunostimulatory composition may
comprise an mRNA/carrier-complex. Furthermore, the
immunostimulatory composition may comprise an adjuvant and/or a
suitable vehicle for the immunostimulatory component, such as the
mRNA.
[0052] Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in
the context of the invention may typically be an RNA that is able
to induce an innate immune response itself. It usually does not
have an open reading frame and thus does not provide a
peptide-antigen or immunogen but elicits an innate immune response
e.g. by binding to a specific kind of Toll-like-receptor (TLR) or
other suitable receptors. However, of course also mRNAs having an
open reading frame and coding for a peptide/protein (e.g. an
antigenic function) may induce an innate immune response and, thus,
may be immunostimulatory RNAs.
[0053] Immune response: An immune response may typically be a
specific reaction of the adaptive immune system to a particular
antigen (so called specific or adaptive immune response) or an
unspecific reaction of the innate immune system (so called
unspecific or innate immune response), or a combination
thereof.
[0054] Immune system: The immune system may protect organisms from
infection. If a pathogen succeeds in passing a physical barrier of
an organism and enters this organism, the innate immune system
provides an immediate, but non-specific response. If pathogens
evade this innate response, vertebrates possess a second layer of
protection, the adaptive immune system. Here, the immune system
adapts its response during an infection to improve its recognition
of the pathogen. This improved response is then retained after the
pathogen has been eliminated, in the form of an immunological
memory, and allows the adaptive immune system to mount faster and
stronger attacks each time this pathogen is encountered. According
to this, the immune system comprises the innate and the adaptive
immune system. Each of these two parts typically contains so called
humoral and cellular components.
[0055] Innate immune system: The innate immune system, also known
as non-specific (or unspecific) immune system, typically comprises
the cells and mechanisms that defend the host from infection by
other organisms in a non-specific manner. This means that the cells
of the innate system may recognize and respond to pathogens in a
generic way, but unlike the adaptive immune system, it does not
confer long-lasting or protective immunity to the host. The innate
immune system may be, e.g., activated by ligands of Toll-like
receptors (TLRs) or other auxiliary substances such as
lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines,
monokines, lymphokines, interleukins or chemokines, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,
IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,
IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,
M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of
human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12
or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I like
receptor, an immunostimulatory nucleic acid, an immunostimulatory
RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral
agent. The pharmaceutical composition according to the present
invention may comprise one or more such substances. Typically, a
response of the innate immune system includes recruiting immune
cells to sites of infection, through the production of chemical
factors, including specialized chemical mediators, called
cytokines; activation of the complement cascade; identification and
removal of foreign substances present in organs, tissues, the blood
and lymph, by specialized white blood cells; activation of the
adaptive immune system; and/or acting as a physical and chemical
barrier to infectious agents.
[0056] Jet injection: The term "jet injection", as used herein,
refers to a needle-free injection method, wherein a fluid
containing at least one inventive nucleic acid sequence (e.g., RNA,
DNA, mRNA) and, optionally, further suitable excipients is forced
through an orifice, thus generating an ultra-fine liquid stream of
high pressure that is capable of penetrating mammalian skin and,
depending on the injection settings, subcutaneous tissue or muscle
tissue. In principle, the liquid stream forms a hole in the skin,
through which the liquid stream is pushed into the target tissue.
Preferably, jet injection is used for intradermal, subcutaneous or
intramuscular injection of the mRNA sequence according to the
invention. In a preferred embodiment, jet injection is used for
intramuscular injection of the mRNA sequence according to the
invention. In a further preferred embodiment, jet injection is used
for intradermal injection of the mRNA sequence according to the
invention.
[0057] Monocistronic nucleic acid: A monocistronic nucleic acid may
typically be a DNA or RNA, particularly an mRNA that comprises only
one coding sequences. A coding sequence in this context is a
sequence of several nucleotide triplets (codons) that can be
translated into a peptide or protein.
[0058] Monovalent/monovalent vaccine: A monovalent vaccine, also
called univalent vaccine, is designed against a single antigen for
a single organism. The term "monovalent vaccine" includes the
immunization against a single valence. In the context of the
invention, a monovalent Nipah virus vaccine would comprise a
vaccine comprising an artificial nucleic acid encoding one single
antigenic peptide or protein derived from one specific Nipah virus
strain and a monovalent Hendra virus vaccine would comprise a
vaccine comprising an artificial nucleic acid encoding one single
antigenic peptide or protein derived from one specific Hendra virus
strain
[0059] Nucleic acid molecule: A nucleic acid molecule is a molecule
comprising, preferably consisting of nucleic acid components. The
term nucleic acid molecule preferably refers to DNA or RNA
molecules. It is preferably used synonymous with the term
"polynucleotide". Preferably, a nucleic acid molecule is a polymer
comprising or consisting of nucleotide monomers, which are
covalently linked to each other by phosphodiester-bonds of a
sugar/phosphate-backbone. The term "nucleic acid molecule" also
encompasses modified nucleic acid molecules, such as base-modified,
sugar-modified or backbone-modified etc. DNA or RNA molecules.
[0060] Nucleic acid sequence/amino acid: The sequence of a nucleic
acid molecule is typically understood to be the particular and
individual order, i.e. the succession of its nucleotides. The
sequence of a protein or peptide is typically understood to be the
order, i.e. the succession of its amino acids.
[0061] Ortholoques and paraloques: Orthologues and paralogues (of a
sequence) encompass evolutionary concepts used to describe the
ancestral relationships of genes and their corresponding gene
products (proteins). Paralogues are genes (or proteins) within the
same species that have originated through duplication of an
ancestral gene; orthologues are genes (or proteins) from different
organisms that have originated through speciation, and are also
derived from a common ancestral gene. In the context of the
invention, an orthologue and/or a paralogue of a Nipah virus
nucleic acid sequence of the invention refers to a sequence having
in increasing order of preference at least 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the
sequence as represented by 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. In the context of the invention, an orthologue and/or a
paralogue of a Nipah virus amino acid sequence of the invention
refers to a sequence having in increasing order of preference at
least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or
more sequence identity to the sequence as represented by SEQ ID
NOs: 1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047,
1052-1058, 1513-1515. In the context of the invention, an
orthologue and/or a paralogue of a Hendra virus nucleic acid
sequence of the invention refers to a sequence having in increasing
order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% or more sequence identity to the sequence as
represented by 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. In the
context of the invention, an orthologue and/or a paralogue of a
Hendra virus amino acid sequence of the invention refers to a
sequence having in increasing order of preference at least 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more
sequence identity to the sequence as represented by SEQ ID NOs:
8-11, 19-26, 580-583, 591-598, 814-817, 825-832, 1048-1051,
1059-1066.
[0062] Peptide: A peptide or polypeptide is typically a polymer of
amino acid monomers, linked by peptide bonds. It typically contains
less than 50 monomer units. Nevertheless, the term peptide is not a
disclaimer for molecules having more than 50 monomer units. Long
peptides are also called polypeptides, typically having between 50
and 600 monomeric units. The term "polypeptide" as used herein,
however, is typically not limited by the length of the molecule it
refers to. In the context of the present invention, the term
"polypeptide" may also be used with respect to peptides comprising
less than 50 (e.g., 10) amino acids or peptides comprising even
more than 600 amino acids.
[0063] Pharmaceutically effective amount: A pharmaceutically
effective amount in the context of the invention is typically
understood to be an amount that is sufficient to induce a
pharmaceutical effect, such as an immune response, altering a
pathological level of an expressed peptide or protein, or
substituting a lacking gene product, e.g., in case of a
pathological situation.
[0064] Protein: A protein typically comprises one or more peptides
or polypeptides. A protein is typically folded into 3-dimensional
form, which may be required for the protein to exert its biological
function.
[0065] Poly(A) sequence: A poly(A) sequence, also called poly(A)
tail or 3'-poly(A) tail, is typically understood to be a sequence
of adenosine nucleotides, e.g., of up to about 400 adenosine
nucleotides, e.g. from about 20 to about 400, preferably from about
50 to about 400, more preferably from about 50 to about 300, even
more preferably from about 50 to about 250, most preferably from
about 60 to about 250 adenosine nucleotides. A poly(A) sequence is
typically located at the 3'-end of an mRNA. In the context of the
present invention, a poly(A) sequence may be located within an mRNA
or any other nucleic acid molecule, such as, e.g., in a vector, for
example, in a vector serving as template for the generation of an
RNA, preferably an mRNA, e.g., by transcription of the vector.
Moreover, poly(A) sequences, or poly(A) tails may be generated in
vitro by enzymatic polyadenylation of the RNA, e.g. using
Poly(A)polymerases (PAP) derived from Escherichia coli or yeast. In
addition, polyadenylation of RNA can be achieved by using
immobilized PAP enzymes e.g. in a polyadenylation reactor
(WO/2016/174271).
[0066] Poly(C) sequence: A poly(C) sequence is typically a long
sequence of cytosine nucleotides, typically about 10 to about 200
cytosine nucleotides, preferably about 10 to about 100 cytosine
nucleotides, more preferably about 10 to about 70 cytosine
nucleotides or even more, preferably about 20 to about 50, or even
about 20 to about 30 cytosine nucleotides. A poly(C) sequence may
preferably be located 3' of the coding sequence comprised by a
nucleic acid.
[0067] Polyadenylation: Polyadenylation is typically understood to
be the addition of a poly(A) sequence to a nucleic acid molecule,
such as an RNA molecule, e.g. to a premature mRNA. Polyadenylation
may be induced by a so called polyadenylation signal. This signal
is preferably located within a stretch of nucleotides at the 3'-end
of a nucleic acid molecule, such as an RNA molecule, to be
polyadenylated. A polyadenylation signal typically comprises a
hexamer consisting of adenine and uracil/thymine nucleotides,
preferably the hexamer sequence AAUAAA. Other sequences, preferably
hexamer sequences, are also conceivable. Polyadenylation typically
occurs during processing of a pre-mRNA (also called
premature-mRNA). Typically, RNA maturation (from pre-mRNA to mature
mRNA) comprises the step of polyadenylation.
[0068] Polyvalent/polyvalent vaccine: A polyvalent vaccine, called
also multivalent vaccine, containing antigens from more than one
strain of a virus, or different antigens of the same virus, or any
combination thereof. The term "polyvalent vaccine" describes that
this vaccine has more than one valence. In the context of the
invention, a polyvalent Nipah virus vaccine would comprise a
vaccine comprising an artificial nucleic acid encoding antigenic
peptides or proteins derived from several different Nipah virus
strains or comprising artificial nucleic acid encoding different
antigens from the same Nipah virus strain, or a combination
thereof. In preferred embodiment, a polyvalent Nipah virus vaccine
comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different
artificial nucleic acids each encoding at least one different
antigenic peptide or protein. A polyvalent Hendra virus vaccine
would comprise a vaccine comprising an artificial nucleic acid
encoding antigenic peptides or proteins derived from several
different Hendra virus strains or comprising artificial nucleic
acid encoding different antigens from the same Hendra virus strain,
or a combination thereof. In preferred embodiment, a polyvalent
Hendra virus vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or even
more different artificial nucleic acids each encoding at least one
different antigenic peptide or protein. A polyvalent Henipavirus
vaccine would comprise a vaccine comprising an artificial nucleic
acid encoding antigenic peptides or proteins derived from several
different Henipavirus strains or comprising artificial nucleic acid
encoding different antigens from the same Henipavirus strain, or a
combination thereof. In preferred embodiment, a polyvalent
Henipavirus vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or even
more different artificial nucleic acids each encoding at least one
different antigenic peptide or protein (e.g., at least one derived
from Nipah virus and at least one derived from Hendra virus).
[0069] Restriction site: A restriction site, also termed
restriction enzyme recognition site, is a nucleotide sequence
recognized by a restriction enzyme. A restriction site is typically
a short, preferably palindromic nucleotide sequence, e.g. a
sequence comprising 4 to 8 nucleotides. A restriction site is
preferably specifically recognized by a restriction enzyme. The
restriction enzyme typically cleaves a nucleotide sequence
comprising a restriction site at this site. In a double-stranded
nucleotide sequence, such as a double-stranded DNA sequence, the
restriction enzyme typically cuts both strands of the nucleotide
sequence.
[0070] RNA, mRNA: RNA is the usual abbreviation for
ribonucleic-acid. It is a nucleic acid molecule, i.e. a polymer
consisting of nucleotides. These nucleotides are usually
adenosine-monophosphate, uridine-monophosphate,
guanosine-monophosphate and cytidine-monophosphate monomers which
are connected to each other along a so-called backbone. The
backbone is formed by phosphodiester bonds between the sugar, i.e.
ribose, of a first and a phosphate moiety of a second, adjacent
monomer. The specific succession of the monomers is called the
RNA-sequence. Usually RNA may be obtainable by transcription of a
DNA-sequence, e.g., inside a cell. In eukaryotic cells,
transcription is typically performed inside the nucleus or the
mitochondria. Typically, transcription of DNA usually results in
the so-called premature RNA which has to be processed into
so-called messenger-RNA, usually abbreviated as mRNA. Processing of
the premature RNA, e.g. in eukaryotic organisms, comprises a
variety of different posttranscriptional-modifications such as
splicing, 5'-capping, polyadenylation, export from the nucleus or
the mitochondria and the like. The sum of these processes is also
called maturation of RNA. The mature messenger RNA usually provides
the nucleotide sequence that may be translated into an amino-acid
sequence of a particular peptide or protein. Typically, a mature
mRNA comprises a 5'-cap, a 5'-UTR, a coding sequence, a 3'-UTR and
a poly(A)sequence. Aside from messenger RNA, several non-coding
types of RNA exist which may be involved in regulation of
transcription and/or translation.
[0071] RNA in vitro transcription: The terms "RNA in vitro
transcription" or "in vitro transcription" relate to a process
wherein RNA is synthesized in a cell-free system (in vitro). DNA,
particularly plasmid DNA, is used as template for the generation of
RNA transcripts. RNA may be obtained by DNA-dependent in vitro
transcription of an appropriate DNA template, which according to
the present invention is preferably a linearized plasmid DNA
template. The promoter for controlling in vitro transcription can
be any promoter for any DNA-dependent RNA polymerase. Particular
examples of DNA-dependent RNA polymerases are the T7, T3, and SP6
RNA polymerases. A DNA template for in vitro RNA transcription may
be obtained by cloning of a nucleic acid, in particular cDNA
corresponding to the respective RNA to be in vitro transcribed, and
introducing it into an appropriate vector for in vitro
transcription, for example into plasmid DNA. In a preferred
embodiment of the present invention the DNA template is linearized
with a suitable restriction enzyme, before it is transcribed in
vitro. The cDNA may be obtained by reverse transcription of mRNA or
chemical synthesis. Moreover, the DNA template for in vitro RNA
synthesis may also be obtained by gene synthesis. Methods for in
vitro transcription are known in the art (see, e.g., Geall et al.
(2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013)
Methods Enzymol. 530:101-14). Reagents used in said method
typically include:
1) a linearized DNA template with a promoter sequence that has a
high binding affinity for its respective RNA polymerase such as
bacteriophage-encoded RNA polymerases; 2) ribonucleoside
triphosphates (NTPs) for the four bases (adenine, cytosine, guanine
and uracil); 3) optionally, a cap analogue as defined above (e.g.
m7G(5'')ppp(5')G (m7G)); 4) a DNA-dependent RNA polymerase capable
of binding to the promoter sequence within the linearized DNA
template (e.g. T7, T3 or SP6 RNA polymerase); 5) optionally, a
ribonuclease (RNase) inhibitor to inactivate any contaminating
RNase; 6) optionally, a pyrophosphatase to degrade pyrophosphate,
which may inhibit transcription; 7) MgCl.sub.2, which supplies
Mg.sup.2+ ions as a co-factor for the polymerase; 8) a buffer to
maintain a suitable pH value, which can also contain antioxidants
(e.g. DTT), and/or polyamines such as spermidine at optimal
concentrations.
[0072] Sequence identity: Two or more sequences are identical if
they exhibit the same length and order of nucleotides or amino
acids. The percentage of identity typically describes the extent,
to which two sequences are identical, i.e. it typically describes
the percentage of nucleotides that correspond in their sequence
position with identical nucleotides of a reference sequence. In
order to determine the degree of identity, the sequences to be
compared are considered to exhibit the same length, i.e. the length
of the longest sequence of the sequences to be compared. This means
that a first sequence consisting of 8 nucleotides is 80% identical
to a second sequence consisting of 10 nucleotides comprising the
first sequence. Hence, in the context of the present invention,
identity of sequences preferably relates to the percentage of
nucleotides of a sequence which have the same position in two or
more sequences having the same length. Therefore, e.g. a position
of a first sequence may be compared with the corresponding position
of the second sequence. If a position in the first sequence is
occupied by the same component (residue) as is the case at a
position in the second sequence, the two sequences are identical at
this position. If this is not the case, the sequences differ at
this position. If insertions occur in the second sequence in
comparison to the first sequence, gaps can be inserted into the
first sequence to allow a further alignment. If deletions occur in
the second sequence in comparison to the first sequence, gaps can
be inserted into the second sequence to allow a further alignment.
The percentage to which two sequences are identical is then a
function of the number of identical positions divided by the total
number of positions including those positions which are only
occupied in one sequence. The percentage to which two sequences are
identical can be determined using a mathematical algorithm. A
preferred, but not limiting, example of a mathematical algorithm
which can be used is the algorithm of Karlin et al. (1993), PNAS
USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res.,
25:3389-3402. Such an algorithm is integrated in the BLAST program.
Sequences which are identical to the sequences of the present
invention to a certain extent can be identified by this
program.
[0073] Serotype, serotype of a virus: A serotype or a serotype of a
virus is a group of viruses classified together based on their
antigens on the surface of the virus, allowing the epidemiologic
classification of organisms to the sub-species level.
[0074] Strain, strain of a virus: A strain or a strain of a virus
is a group of viruses that are genetically distinct from other
groups of the same species. The strain that is defined by a genetic
variant is also defined as a "subtype".
[0075] Stabilized nucleic acid molecule: A stabilized nucleic acid
molecule is a nucleic acid molecule, preferably a DNA or RNA
molecule that is modified such, that it is more stable to
disintegration or degradation, e.g., by environmental factors or
enzymatic digest, such as by an exo- or endonuclease degradation,
than the nucleic acid molecule without the modification.
Preferably, a stabilized nucleic acid molecule in the context of
the present invention is stabilized in a cell, such as a
prokaryotic or eukaryotic cell, preferably in a mammalian cell,
such as a human cell. The stabilization effect may also be exerted
outside of cells, e.g. in a buffer solution etc., for example, in a
manufacturing process for a pharmaceutical composition comprising
the stabilized nucleic acid molecule.
[0076] Transfection: The term "transfection" refers to the
introduction of nucleic acid molecules, such as DNA or RNA (e.g.
mRNA) molecules, into cells, preferably into eukaryotic cells. In
the context of the present invention, the term "transfection"
encompasses any method known to the skilled person for introducing
nucleic acid molecules into cells, preferably into eukaryotic
cells, such as into mammalian cells. Such methods encompass, for
example, electroporation, lipofection, e.g. based on cationic
lipids and/or liposomes, calcium phosphate precipitation,
nanoparticle based transfection, virus based transfection, or
transfection based on cationic polymers, such as DEAE-dextran or
polyethylenimine etc. Preferably, the introduction is
non-viral.
[0077] Vaccine: A vaccine is typically understood to be a
prophylactic or therapeutic material providing at least one
antigen, preferably an immunogen. The antigen or immunogen may be
derived from any material that is suitable for vaccination. For
example, the antigen or immunogen may be derived from a pathogen,
such as from bacteria or virus particles etc., or from a tumor or
cancerous tissue. The antigen or immunogen stimulates the adaptive
immune system of a mammalian subject to provide an adaptive immune
response. In the context of the present invention, the antigen is
preferably provided via an artificial nucleic acid.
[0078] Variant of a nucleic acid sequence: A variant of a nucleic
acid sequence refers to a variant of nucleic acid sequences which
forms the basis of a nucleic acid sequence. For example, a variant
nucleic acid sequence may exhibit one or more nucleotide deletions,
insertions, additions and/or substitutions compared to the nucleic
acid sequence from which the variant is derived. Preferably, a
variant of a nucleic acid sequence is at least 40%, preferably at
least 50%, more preferably at least 60%, more preferably at least
70%, even more preferably at least 80%, even more preferably at
least 90%, most preferably at least 95% identical to the nucleic
acid sequence the variant is derived from. Preferably, the variant
is a functional variant. A "variant" of a nucleic acid sequence may
have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide
identity over a stretch of 10, 20, 30, 50, 75 or 100 nucleotide of
such nucleic acid sequence.
[0079] Variants of proteins: "Variants" of proteins or peptides as
defined in the context of the present invention may be generated,
having an amino acid sequence which differs from the original
sequence in one or more mutation(s), such as one or more
substituted, inserted and/or deleted amino acid(s). Preferably,
these fragments and/or variants have the same biological function
or specific activity compared to the full-length native protein,
e.g. its specific antigenic property. "Variants" of proteins or
peptides as defined in the context of the present invention may
comprise conservative amino acid substitution(s) compared to their
native, i.e. non-mutated physiological, sequence. Those amino acid
sequences as well as their encoding nucleotide sequences in
particular fall under the term variants as defined herein.
Substitutions in which amino acids, which originate from the same
class, are exchanged for one another are called conservative
substitutions. In particular, these are amino acids having
aliphatic side chains, positively or negatively charged side
chains, aromatic groups in the side chains or amino acids, the side
chains of which can enter into hydrogen bridges, e.g. side chains
which have a hydroxyl function. This means that e.g. an amino acid
having a polar side chain is replaced by another amino acid having
a likewise polar side chain, or, for example, an amino acid
characterized by a hydrophobic side chain is substituted by another
amino acid having a likewise hydrophobic side chain (e.g. serine
(threonine) by threonine (serine) or leucine (isoleucine) by
isoleucine (leucine)). Insertions and substitutions are possible,
in particular, at those sequence positions which cause no
modification to the three-dimensional structure or do not affect
the binding region. Modifications to a three-dimensional structure
by insertion(s) or deletion(s) can easily be determined e.g. using
CD spectra (circular dichroism spectra) (Urry, 1985, Absorption,
Circular Dichroism and ORD of Polypeptides, in: Modern Physical
Methods in Biochemistry, Neuberger et al. (ed.), Elsevier,
Amsterdam). A "variant" of a protein or peptide may have at least
70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a
stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or
peptide. Furthermore, variants of proteins or peptides as defined
herein, which may be encoded by a nucleic acid molecule, may also
comprise those sequences, wherein nucleotides of the encoding
nucleic acid sequence are exchanged according to the degeneration
of the genetic code, without leading to an alteration of the
respective amino acid sequence of the protein or peptide, i.e. the
amino acid sequence or at least part thereof may not differ from
the original sequence within the above meaning. Preferably, a
variant of a protein comprises a functional variant of the protein,
which means that the variant exerts the same effect or
functionality as the protein it is derived from.
[0080] Vector: The term "vector" refers to a nucleic acid molecule,
preferably to an artificial nucleic acid molecule. A vector in the
context of the present invention is suitable for incorporating or
harboring a desired nucleic acid sequence, such as a nucleic acid
sequence comprising a coding sequence. Such vectors may be storage
vectors, expression vectors, cloning vectors, transfer vectors etc.
A storage vector is a vector which allows the convenient storage of
a nucleic acid molecule, for example, of an mRNA molecule. Thus,
the vector may comprise a sequence corresponding, e.g., to a
desired mRNA sequence or a part thereof, such as a sequence
corresponding to the coding sequence and the 3'-UTR and/or the
5''-UTR of an mRNA. An expression vector may be used for production
of expression products such as RNA, e.g. mRNA, or peptides,
polypeptides or proteins. For example, an expression vector may
comprise sequences needed for transcription of a sequence stretch
of the vector, such as a promoter sequence, e.g. an RNA polymerase
promoter sequence. A cloning vector is typically a vector that
contains a cloning site, which may be used to incorporate nucleic
acid sequences into the vector. A cloning vector may be, e.g., a
plasmid vector or a bacteriophage vector. A transfer vector may be
a vector which is suitable for transferring nucleic acid molecules
into cells or organisms, for example, viral vectors. A vector in
the context of the present invention may be, e.g., an RNA vector or
a DNA vector. Preferably, a vector is a DNA molecule. Preferably, a
vector in the sense of the present application comprises a cloning
site, a selection marker, such as an antibiotic resistance factor,
and a sequence suitable for multiplication of the vector, such as
an origin of replication. Preferably, a vector in the context of
the present application is a plasmid vector.
[0081] Vehicle: A vehicle is typically understood to be a material
that is suitable for storing, transporting, and/or administering a
compound, such as a pharmaceutically active compound. For example,
it may be a physiologically acceptable liquid which is suitable for
storing, transporting, and/or administering a pharmaceutically
active compound.
[0082] 3'-untranslated region (3'-UTR): Generally, the term
"3"-UTR'' refers to a part of a nucleic acid molecule, which is
located 3' (i.e. "downstream") of a coding sequence and which is
not translated into protein. Typically, a 3'-UTR is the part of an
mRNA which is located between the protein coding sequence (coding
region or coding sequence (CDS)) and the poly(A) sequence of the
mRNA. In the context of the invention, the term 3'-UTR may also
comprise elements, which are not encoded in the template, from
which an RNA is transcribed, but which are added after
transcription during maturation, e.g. a poly(A) sequence. A 3'-UTR
of the mRNA is not translated into an amino acid sequence. The
3'-UTR sequence is generally encoded by the gene which is
transcribed into the respective mRNA during the gene expression
process. The genomic sequence is first transcribed into pre-mature
mRNA, which comprises optional introns. The pre-mature mRNA is then
further processed into mature mRNA in a maturation process. This
maturation process comprises the steps of 5''-capping, splicing the
pre-mature mRNA to excize optional introns and modifications of the
3'-end, such as polyadenylation of the 3'-end of the pre-mature
mRNA and optional endo-/or exonuclease cleavages etc. In the
context of the present invention, a 3'-UTR corresponds to the
sequence of a mature mRNA which is located between the stop codon
of the protein coding sequence, preferably immediately 3' to the
stop codon of the protein coding sequence, and the poly(A) sequence
of the mRNA. The term "corresponds to" means that the 3'-UTR
sequence may be an RNA sequence, such as in the mRNA sequence used
for defining the 3'-UTR sequence, or a DNA sequence which
corresponds to such RNA sequence. In the context of the present
invention, the term "a 3'-UTR of a gene", is the sequence which
corresponds to the 3'-UTR of the mature mRNA derived from this
gene, i.e. the mRNA obtained by transcription of the gene and
maturation of the pre-mature mRNA. The term "3'-UTR of a gene"
encompasses the DNA sequence and the RNA sequence (both sense and
antisense strand and both mature and immature) of the 3'-UTR.
Preferably, the 3'-UTRs have a length of more than 20, 30, 40 or 50
nucleotides.
[0083] 5'-untranslated region (5'-UTR): Generally, the term
"5"-UTR'' refers to a part of a nucleic acid molecule, which is
located 5' (i.e. "upstream") of a coding sequence and which is not
translated into protein. A 5'-UTR is typically understood to be a
particular section of messenger RNA (mRNA), which is located 5' of
the coding sequence of the mRNA. Typically, the 5'-UTR starts with
the transcriptional start site and ends one nucleotide before the
start codon of the coding sequence. Preferably, the 5'-UTRs have a
length of more than 20, 30, 40 or 50 nucleotides. The 5'-UTR may
comprise elements for controlling gene expression, also called
regulatory elements. Such regulatory elements may be, for example,
ribosomal binding sites. The 5'-UTR may be post-transcriptionally
modified, for example by addition of a 5'-cap. A 5'-UTR of the mRNA
is not translated into an amino acid sequence. The 5'-UTR sequence
is generally encoded by the gene which is transcribed into the
respective mRNA during the gene expression process. The genomic
sequence is first transcribed into pre-mature mRNA, which comprises
optional introns. The pre-mature mRNA is then further processed
into mature mRNA in a maturation process. This maturation process
comprises the steps of 5'-capping, splicing the pre-mature mRNA to
excise optional introns and modifications of the 3'-end, such as
polyadenylation of the 3'-end of the pre-mature mRNA and optional
endo-/or exonuclease cleavages etc. In the context of the present
invention, a 5'-UTR corresponds to the sequence of a mature mRNA
which is located between the start codon and, for example, the
5'-cap. Preferably, the 5'-UTR corresponds to the sequence which
extends from a nucleotide located 3' to the 5'-cap, more preferably
from the nucleotide located immediately 3' to the 5'-cap, to a
nucleotide located 5' to the start codon of the protein coding
sequence, preferably to the nucleotide located immediately 5' to
the start codon of the protein coding sequence. The nucleotide
located immediately 3' to the 5'-cap of a mature mRNA typically
corresponds to the transcriptional start site. The term
"corresponds to" means that the 5'-UTR sequence may be an RNA
sequence, such as in the mRNA sequence used for defining the 5'-UTR
sequence, or a DNA sequence which corresponds to such RNA sequence.
In the context of the present invention, the term "a 5'-UTR of a
gene" is the sequence which corresponds to the 5'-UTR of the mature
mRNA derived from this gene, i.e. the mRNA obtained by
transcription of the gene and maturation of the pre-mature mRNA.
The term "5'-UTR of a gene" encompasses the DNA sequence and the
RNA sequence (both sense and antisense strand and both mature and
immature) of the 5'-UTR.
[0084] 5'-terminal oligopyrimidine tract (TOP): The 5'-terminal
oligopyrimidine tract (TOP) is typically a stretch of pyrimidine
nucleotides located in the 5' terminal region of a nucleic acid
molecule, such as the 5' terminal region of certain mRNA molecules
or the 5' terminal region of a functional entity, e.g. the
transcribed region, of certain genes. The sequence starts with a
cytidine, which usually corresponds to the transcriptional start
site, and is followed by a stretch of usually about 3 to 30
pyrimidine nucleotides. For example, the TOP may comprise 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidine
stretch and thus the 5' TOP ends one nucleotide 5' to the first
purine nucleotide located downstream of the TOP. Messenger RNA that
contains a 5' terminal oligopyrimidine tract is often referred to
as TOP mRNA. Accordingly, genes that provide such messenger RNAs
are referred to as TOP genes. TOP sequences have, for example, been
found in genes and mRNAs encoding peptide elongation factors and
ribosomal proteins.
[0085] TOP motif: In the context of the present invention, a TOP
motif is a nucleic acid sequence which corresponds to a 5' TOP as
defined above. Thus, a TOP motif in the context of the present
invention is preferably a stretch of pyrimidine nucleotides having
a length of 3-30 nucleotides. Preferably, the TOP-motif consists of
at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine
nucleotides, preferably at least 5 pyrimidine nucleotides, more
preferably at least 6 nucleotides, more preferably at least 7
nucleotides, most preferably at least 8 pyrimidine nucleotides,
wherein the stretch of pyrimidine nucleotides preferably starts at
its 5'-end with a cytosine nucleotide. In TOP genes and TOP mRNAs,
the TOP-motif preferably starts at its 5'-end with the
transcriptional start site and ends one nucleotide 5' to the first
purin residue in said gene or mRNA. A TOP motif in the sense of the
present invention is preferably located at the 5'-end of a sequence
which represents a 5'-UTR or at the 5'-end of a sequence which
codes for a 5'-UTR. Thus, preferably, a stretch of 3 or more
pyrimidine nucleotides is called "TOP motif" in the sense of the
present invention if this stretch is located at the 5'-end of a
respective sequence, such as the artificial nucleic acid molecule,
the 5'-UTR element of the artificial nucleic acid molecule, or the
nucleic acid sequence which is derived from the 5'-UTR of a TOP
gene as described herein. In other words, a stretch of 3 or more
pyrimidine nucleotides, which is not located at the 5''-end of a
5'-UTR or a 5'-UTR element but anywhere within a 5'-UTR or a 5'-UTR
element, is preferably not referred to as "TOP motif".
[0086] TOP gene: TOP genes are typically characterised by the
presence of a 5' terminal oligopyrimidine tract. Furthermore, most
TOP genes are characterized by a growth-associated translational
regulation. However, also TOP genes with a tissue specific
translational regulation are known. As defined above, the 5'-UTR of
a TOP gene corresponds to the sequence of a 5'-UTR of a mature mRNA
derived from a TOP gene, which preferably extends from the
nucleotide located 3' to the 5'-cap to the nucleotide located 5' to
the start codon. A 5'-UTR of a TOP gene typically does not comprise
any start codons, preferably no upstream AUGs (uAUGs) or upstream
coding sequences (uORFs). Therein, upstream AUGs and upstream
coding sequences are typically understood to be AUGs and coding
sequences that occur 5' of the start codon (AUG) of the coding
sequence that should be translated. The 5'-UTRs of TOP genes are
generally rather short. The lengths of 5'-UTRs of TOP genes may
vary between 20 nucleotides up to 500 nucleotides, and are
typically less than about 200 nucleotides, preferably less than
about 150 nucleotides, more preferably less than about 100
nucleotides. Exemplary 5'-UTRs of TOP genes in the sense of the
present invention are the nucleic acid sequences extending from the
nucleotide at position 5 to the nucleotide located immediately 5'
to the start codon (e.g. the ATG) in the sequences according to SEQ
ID NOs: 1-1363 of the patent application WO2013/143700, whose
disclosure is incorporated herewith by reference. In this context a
particularly preferred fragment of a 5'-UTR of a TOP gene is a
5'-UTR of a TOP gene lacking the 5'TOP motif. The terms "5'-UTR of
a TOP gene" or "5"-TOP UTR'' preferably refer to the 5'-UTR of a
naturally occurring TOP gene.
Short Description of the Invention
[0087] The present invention is based on the surprising finding
that at least one Henipavirus peptide or protein, particularly at
least one Hendra virus and/or Nipah virus peptide or protein
encoded by an artificial nucleic acid can efficiently be expressed
in a mammalian cell. Further unexpectedly, the artificial nucleic
acid, e.g. an mRNA sequence of the invention, is suitable for
eliciting an immune response against Hendra virus and/or Nipah
virus in a mammalian subject, in particular, in a human subject.
The artificial nucleic acid invention, the composition comprising
said artificial nucleic acid, and the vaccine induces very
efficiently antigen-specific immune responses against the encoded
antigenic peptide or protein. Moreover, the the artificial nucleic
acid invention, the composition comprising said artificial nucleic
acid, and the vaccine can be stored without cold chain
(lyophilizable) enabling rapid and scalable vaccine production
which is of major importance in the context of pandemic Hendra
virus and/or Nipah virus outbreaks.
[0088] In a first aspect, the present invention relates to
artificial nucleic acids comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from a
Henipavirus or a fragment or variant thereof, wherein the at least
one antigenic peptide or protein comprises or consists of a
Henipavirus RNA-directed RNA polymerase (L), Henipavirus fusion
protein (F), Henipavirus non-structural protein (V), Henipavirus
glycoprotein (G), Henipavirus nucleoprotein (N), Henipavirus matrix
protein (M), Henipavirus phosphoprotein (P), Henipavirus protein C,
and Henipavirus protein W, or a fragment or variant of any of
these.
[0089] In a preferred embodiment, the invention relates to an
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.
[0090] In a preferred embodiment, the Henipavirus is Nipah virus or
Hendra virus.
[0091] In another preferred embodiment, the at least one antigenic
peptide or protein comprises or consists of a Hendra virus fusion
protein, and/or Hendra virus glycoprotein, and/or Nipah virus
fusion protein and/or Nipah virus glycoprotein, a fragment or
variant of any of these.
[0092] The at least one antigenic peptide or protein, provided by
the artificial nucleic acid, may additionally comprise an
N-terminal heterologous signal peptide, preferably selected from an
IgE-leader or an HA-A signal peptide, to improve secretion of the
antigenic peptide or protein.
[0093] The artificial nucleic acid may be monocistronic,
bicistronic or multicistronic.
[0094] The artificial nucleic acid sequence according to the
invention may be a modified nucleic acid sequence.
[0095] The artificial nucleic acid may comprises an untranslated
region (UTR), e.g. a 3''-UTR and/or 5''-UTR, preferably a
heterologous 3''-UTR and/or 5'-UTR, preferably derived from a gene
encoding a stable mRNA.
[0096] In a preferred embodiment, the artificial nucleic acid is an
RNA, preferably an mRNA, wherein the RNA is a stabilized RNA.
[0097] The artificial nucleic acid may further comprise a 5''-cap
structure, and/or a 5''-UTR, and/or a Poly(A)sequence and/or a
Poly(C) sequence and/or a histone stem-loop, and/or a 3''-UTR.
[0098] In another aspect, the invention relates to a composition
comprising at least one artificial nucleic acid as described herein
and at least one pharmaceutically acceptable carrier.
[0099] The composition may comprise a plurality or at least more
than one of the artificial nucleic acids encoding a different
antigenic peptide or protein derived from a Henipavirus or from a
homolog, fragment or variant thereof, wherein the Henipavirus may
be selected from Hendra virus and/or Nipah virus.
[0100] The artificial nucleic acid comprised in the composition may
additionally be 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 lipids.
[0101] In an embodiment, the at least one artificial nucleic acid
is complexed with protamine.
[0102] In an embodiment, the at least one artificial nucleic acid
of the invention is complexed with a polymeric, preferably a
polymer (e.g. peptide polymer) in conjunction with a lipidoid (e.g.
3-C12 OH).
[0103] The composition may comprise at least one protamine
complexed artificial nucleic acid and at least one free artificial
nucleic acid, wherein the molar ratio of the complexed nucleic acid
to the free nucleic acid about 1:1.
[0104] In another embodiment, the composition may comprise the
artificial nucleic acid of the invention complexed with one or more
lipids, thereby forming liposomes, lipid nanoparticles and/or
lipoplexes.
[0105] The composition may further comprise at least one adjuvant
component.
[0106] The present invention is also directed to the use of the
artificial nucleic acid in treatment or prophylaxis of an infection
with Henipavirus.
[0107] In particular, the present invention is directed to the use
of the artificial nucleic acid in treatment or prophylaxis of an
infection with Hendra virus or a disorder related to such an
infection.
[0108] Moreover, the present invention is directed to the use of
the artificial nucleic acid in treatment or prophylaxis of an
infection with Nipah virus or a disorder related to such an
infection.
[0109] The present invention also concerns a Henipavirus vaccine,
in particular a Nipah virus vaccine and a Hendra virus vaccine.
[0110] The invention further concerns a method of treating or
preventing a disorder or a disease in a mammalian subject or an
avian subject, first and second medical uses of the artificial
nucleic acid, compositions and vaccines. Further, the invention is
directed to a kit, particularly to a kit of parts, comprising the
artificial nucleic acid, compositions and vaccines.
DETAILED DESCRIPTION OF THE INVENTION
[0111] The present application is filed together with a sequence
listing in electronic format, which is part of the description of
the present application. The information contained in the
electronic format of the sequence listing filed together with this
application is incorporated herein by reference in its entirety.
Where reference is made herein to a "SEQ ID NO:" the corresponding
nucleic acid sequence or amino acid (aa) sequence in the sequence
listing having the respective identifier is referred to. For many
sequences, the sequence listing also provides detailed information,
e.g. regarding certain structural features, sequence optimizations,
GenBank identifiers, or regarding its coding capacity. In
particular, such information may be provided under the identifier
<223> in the sequence listing. Accordingly, information
provided under identifier <223> is explicitly included herein
in its entirety and has to be understood as part of the description
of the invention.
[0112] In a first aspect, the invention relates to an artificial
nucleic acid comprising at least one coding sequence encoding at
least one antigenic peptide or protein derived from a Henipavirus
or a fragment or variant thereof.
[0113] Henipavirus:
[0114] In the context of the present invention, the term
"Henipavirus" comprises any Henipavirus irrespective of genotype,
species, strain, isolate, or serotype (NCBI taxonomy ID: 260964).
Preferably, the term Henipavirus relates to a virus genus
comprising virus strains selected from Cedar henipavirus or Cedar
virus (NCBI taxonomy ID: 1221391), Ghanaian bat henipavirus or Bat
paramyxovirus (NCBI taxonomy ID: 665603), Mojiang henipavirus or
Mojiang virus (NCBI taxonomy ID: 1474807), Hendra virus (NCBI
taxonomy ID: 928303), Nipah virus (NCBI taxonomy ID: 121791).
[0115] Henipavirus Peptides or Proteins:
[0116] Henipavirus is a genus of negative sense single stranded RNA
viruses belonging to the Paramyxovirinae virus superfamily (NCBI
Taxonomy ID: 11158). The Henipavirus genome is about 18 kb in size,
encoding for nine proteins, comprising RNA-directed RNA polymerase
(L), fusion protein (F), non-structural protein (V), glycoprotein
(G), nucleoprotein (N), matrix protein (M), phosphoprotein (P),
protein C, and protein W.
[0117] In particular, the term "Henipavirus protein" as used herein
comprises or consists of an individual structural or non-structural
Henipavirus protein. A Henipavirus peptide or protein in the
meaning of the present invention may be any full length protein or
fragment derived from Henipavirus RNA-directed RNA polymerase (L),
Henipavirus fusion protein (F), Henipavirus non-structural protein
(V), Henipavirus glycoprotein (G), Henipavirus nucleoprotein (N),
Henipavirus matrix protein (M), Henipavirus phosphoprotein (P),
Henipavirus protein C, and Henipavirus protein W.
[0118] Accordingly, the term "Henipavirus protein" as used in the
present invention may relate to an amino acid sequence
corresponding to any Henipavirus RNA-directed RNA polymerase (L),
Henipavirus fusion protein (F), Henipavirus non-structural protein
(V), Henipavirus glycoprotein (G), Henipavirus nucleoprotein (N),
Henipavirus matrix protein (M), Henipavirus phosphoprotein (P),
Henipavirus protein C, and Henipavirus protein W.
[0119] The term "Henipavirus antigenic peptide or protein" as used
in the present invention may relate to an amino acid sequence
corresponding to any Henipavirus RNA-directed RNA polymerase (L),
Henipavirus fusion protein (F), Henipavirus non-structural protein
(V), Henipavirus glycoprotein (G), Henipavirus nucleoprotein (N),
Henipavirus matrix protein (M), Henipavirus phosphoprotein (P),
Henipavirus protein C, and Henipavirus protein W capable. Any
Henipavirus peptide or protein provided herein, or any a fragment
or variant thereof, can cause an immune response when administered
to a subject. Therefore, all Henipavirus proteins or peptides
provided herein can be considered as antigens in the context of the
present invention.
[0120] Any Henipavirus peptide or protein provided herein, or any a
fragment or variant thereof, can cause an immune response when
administered to a subject. Therefore, all Henipavirus proteins or
peptides provided herein can be considered as antigens in the
context of the present invention.
[0121] In an embodiment, the Henipavirus peptide or protein as
defined above is selected from Cedar henipavirus or Cedar virus
(NCBI taxonomy ID: 1221391), Ghanaian bat henipavirus or Bat
paramyxovirus (NCBI taxonomy ID: 665603), Mojiang henipavirus or
Mojiang virus (NCBI taxonomy ID: 1474807), Hendra virus (NCBI
taxonomy ID: 928303), Nipah virus (NCBI taxonomy ID: 121791).
[0122] Accordingly, in preferred embodiments, the artificial
nucleic acid as defined herein, comprising at least one coding
sequence encoding at least one antigenic peptide or protein,
wherein the at least one antigenic peptide or protein comprises or
consists of a RNA-directed RNA polymerase (L), fusion protein,
non-structural protein, glycoprotein, nucleoprotein, matrix
protein, phosphoprotein, protein C, and protein W, or a fragment or
variant of any of these.
[0123] In embodiments, the Henipavirus is selected from Hendra
virus (NCBI taxonomy ID: 928303) and Nipah virus (NCBI taxonomy ID:
121791).
[0124] Accordingly, in a preferred embodiment, the Henipavirus
peptide or protein is selected from a Hendra virus peptide or
protein or Nipah virus peptide or protein.
[0125] More preferably, the at least one antigenic peptide or
protein comprises or consists of a Hendra virus fusion protein,
and/or Hendra virus glycoprotein, and/or Nipah virus fusion protein
and/or Nipah virus glycoprotein, a fragment or variant of any of
these.
[0126] In preferred embodiments, the at least one encoded antigenic
peptide or protein comprises at least one of the amino acid
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 any one of SEQ ID NOs: 1-26, 573-598, 807-832,
1041-1066, 1513-1515 or provided in Table 1, Table 1B, Table 2, and
Table 2B, or a fragment or variant or orthologue or paralogue of
any of these.
[0127] Hendra Virus Peptides or Proteins:
[0128] The term "Hendra virus protein" or "Hendra virus protein" as
used in the present invention may relate to an amino acid sequence
corresponding to any Hendra virus RNA-directed RNA polymerase (L),
Hendra virus fusion protein (F), Hendra virus non-structural
protein (V), Hendra virus glycoprotein (G), Hendra virus
nucleoprotein (N), Hendra virus matrix protein (M), Hendra virus
phosphoprotein (P), Hendra virus protein C, and Hendra virus
protein W.
[0129] Any Hendra virus peptide or protein provided herein, or any
a fragment or variant thereof, can cause an immune response when
administered to a subject. Therefore, all Hendra virus proteins or
peptides provided herein can be considered as antigens in the
context of the present invention.
[0130] In some embodiments described herein, the at least one
Hendra virus antigenic peptide or protein encoded by the at least
one coding sequence of the artificial nucleic acid may consist of
an individual Hendra virus protein, the amino acid sequence of
which does typically not comprise an N-terminal Methionine residue.
It is thus understood that the phrase "artificial nucleic acid
comprising at least one coding sequence encoding at least antigenic
peptide or protein derived from a Hendra virus . . . " relates to a
protein or peptide comprising the amino acid sequence of said
Hendra virus protein and--if the amino acid sequence of the
respective Hendra virus protein does not comprise such an
N-terminal Methionine residue--an introduced N-terminal Methionine
residue.
[0131] In the context of the present invention a fragment of a
protein or a variant thereof encoded by the at least one coding
sequence of the artificial nucleic acid according to the invention
may typically comprise an amino acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with an amino acid sequence of the respective naturally occurring
full-length Hendra virus protein or a variant thereof, preferably
as disclosed in Table 1 or Table 1B.
[0132] In a preferred embodiment, the at least one coding sequence
of the artificial nucleic acid sequence according to the invention
preferably encodes Hendra virus proteins selected from the proteins
provided in Table 1 or Table 1B, or a fragment or variant thereof.
Any Hendra virus protein provided in Table 1 or Table 1B, or any a
fragment or variant thereof, can cause an immune response when
administered to an individual. Therefore, all Hendra virus proteins
provided in Table 1 or Table 1B can be considered as preferred
Hendra virus antigens in the context of the present invention.
[0133] It is further preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes a Hendra virus protein or peptide, or a fragment
or variant thereof, wherein the Hendra virus protein or peptide is
an antigen selected from the antigens listed in Table 1. Therein,
each row corresponds to a Hendra virus antigenic peptide or protein
as identified by the respective gene name (first column "Name") and
the NCBI database accession number of the corresponding protein
(second column "Accession No."). The third column ("A", "Protein")
in Table 1 indicates the SEQ ID NOs corresponding to the respective
amino acid sequence as provided herein. The SEQ ID NOs
corresponding to the nucleic acid sequence of the wild type nucleic
acid sequence encoding the Hendra virus antigenic protein or
peptide is indicated in the fourth column ("B", "CDS wt"). The
following columns ("C"-"J") provides the SEQ ID NOs corresponding
to modified nucleic acid sequences (opt1, opt2, opt3, opt4, opt5,
opt6, opt7) of the nucleic acid sequences as described herein that
encode the Hendra virus protein or peptide preferably having the
amino acid sequence as defined by the SEQ ID NOs indicated in the
third column ("A") or by the database entry indicated in the second
column ("Accession No."). Additional information regarding each of
the sequences provided in Table 1 may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223>.
TABLE-US-00001 TABLE 1 List of Hendra virus antigens: A B C D E F G
H J Name Accession No. Protein CDS wt opt1 opt2 opt3 opt4 opt5 opt6
opt7 F NP_047111.2 8 34 60 86 112 138 164 190 216 F AEB21233.1 9 35
61 87 113 139 165 191 217 F AEQ38114.1 10 36 62 88 114 140 166 192
218 F AAB39505.1 11 37 63 89 115 141 167 193 219 G NP_047112.2 19
45 71 97 123 149 175 201 227 G AEB21225.1 20 46 72 98 124 150 176
202 228 G AEB21216.1 21 47 73 99 125 151 177 203 229 G AEB21206.1
22 48 74 100 126 152 178 204 230 G AEQ38052.1 23 49 75 101 127 153
179 205 231 G AEQ38115.1 24 50 76 102 128 154 180 206 232 G
AEQ38108.1 25 51 77 103 129 155 181 207 233 G AAV80426.1 26 52 78
104 130 156 182 208 234
[0134] According to preferred embodiments, the inventive artificial
nucleic acid comprises or consists of at least one coding sequence
encoding at least one Hendra virus antigenic peptide or peptide as
described herein, wherein the at least one Hendra virus antigenic
peptide or protein comprises at least one 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: 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.
[0135] According to a preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one Hendra virus antigenic
peptide or peptide as described herein, wherein the at least one
Hendra virus antigenic peptide or protein comprises an amino acid
sequence according to any one of SEQ ID NOs: 8-11 and 19-26, or a
homolog, fragment or variant of any of these sequences (see Table
1, column "A").
[0136] In an embodiment the Hendra virus antigenic peptide or
protein is derived from a Hendra virus Fusion protein (F) according
to SEQ ID NOs: 8-11.
[0137] In this context it is further preferred that the at least
one coding sequence of the artificial nucleic acid sequence of the
present invention encodes at least one Hendra virus peptide or
protein which is derived from Hendra virus fusion protein (F), or a
fragment or variant thereof, wherein the Hendra virus fusion
protein (F) is selected from the Hendra virus fusion protein amino
acid sequences listed in Table 1.
[0138] Therein, rows corresponding to a Hendra virus fusion protein
(F) (SEQ ID NOs: 8-11) can be identified by the respective gene
name (first column "Name": "F") and the database accession number
of the corresponding protein (second column "Accession No."). The
SEQ ID NOs corresponding to the nucleic acid sequence of the wild
type nucleic acid encoding the Hendra virus fusion protein (F) or
peptide is indicated in the fourth column ("B"). The further
columns ("C"-"J") provide the SEQ ID NOs corresponding to modified
nucleic acid sequences of the nucleic acids as described herein
that encode the Hendra virus fusion protein (F) or peptide
preferably having the amino acid sequence as defined by the SEQ ID
NOs: 8-11 or by the database entry indicated in the second column
("Accession No.").
[0139] In an embodiment the Hendra virus antigenic peptide or
protein is derived from a Hendra virus glycoprotein (G) according
to SEQ ID NOs: 19-26.
[0140] In this context it is further preferred that the at least
one coding sequence of the artificial nucleic acid sequence of the
present invention encodes at least one Hendra virus peptide or
protein which is derived from Hendra virus glycoprotein (G), or a
fragment or variant thereof, wherein the Hendra virus glycoprotein
(G) is selected from the Hendra virus fusion protein amino acid
sequences listed in Table 1.
[0141] Therein, rows corresponding to a Hendra virus glycoprotein
(G) (SEQ ID NOs: 19-26) can be identified by the respective gene
name (first column "Name": "F") and the database accession number
of the corresponding protein (second column "Accession No."). The
SEQ ID NOs corresponding to the nucleic acid sequence of the wild
type nucleic acid encoding the Hendra virus glycoprotein (G) or
peptide is indicated in the fourth column ("B"). The further
columns ("C"-"J") provide the SEQ ID NOs corresponding to modified
nucleic acid sequences of the nucleic acids as described herein
that encode the Hendra virus glycoprotein (G) or peptide preferably
having the amino acid sequence as defined by the SEQ ID NOs: 19-26
or by the database entry indicated in the second column ("Accession
No.").
[0142] In a specific embodiment, Hendra virus glycoprotein (G) (SEQ
ID NOs: 19-26) is N-terminally truncated to generate a soluble form
of the protein (solG). The N-terminal truncation has to be adapted
in a way that membrane-bound domains of the protein are removed.
The membrane topology of a protein can be determined using
prediction algorithms as commonly known in the art (e.g. HMMTop
(Tusnady and Simon (1998) Principles Governing Amino Acid
Composition of Integral Membrane Proteins: Applications to Topology
Prediction." J. Mol. Biol. 283, 489-506) or TMHMM (Krogh et al.
Predicting transmembrane protein topology with a hidden Markov
model: Application to complete genomes. Journal of Molecular
Biology, 305(3):567-580, January 2001).
[0143] Hendra virus full length glycoprotein (G) polypeptides
consist of 604 amino acids (see Table 1).
[0144] In embodiments, soluble forms of the Hendra virus G protein
(solG) are generated by truncating the first 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, or 80 amino acids of the protein
according to SEQ ID NOs: 19-26. In other words, amino acids 69-604,
70-604, 71-604, 72-604, 73-604, 74-604, 75-604, 76-604, 77-604,
78-604, 79-604, 80-604, 81-604 of the proteins according to SEQ ID
NOs: 19-26 are soluble forms of glycoprotein (solG).
[0145] In preferred embodiments, soluble forms of the protein
(solG) are generated by truncating the first 70 amino acids of the
protein according to SEQ ID NOs: 19-26. In other words, amino acids
71-604 of the proteins according to SEQ ID NOs: 19-26 are soluble
forms of glycoprotein (solG).
[0146] In another specific embodiment, soluble forms of the protein
(solG) are generated by truncating the first 73 amino acids of the
protein according to SEQ ID NOs: 19-26. In other words, amino acids
74-604 of the proteins according to SEQ ID NOs: 19-26 are soluble
forms of glycoprotein (solG).
[0147] In this context it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one Hendra virus antigenic peptide or
protein which is derived from Hendra virus soluble glycoprotein
(solG) as defined above, or a fragment or variant thereof. Suitable
Hendra virus soluble glycoprotein (solG) proteins (and respective
nucleic acid coding sequences) are provided in Table 1B.
[0148] To facilitate secretion of the truncated solG protein,
elements that promote secretion may be N-terminally fused to the
(N-terminally truncated) Hendra virus solG proteins as specified
above. In specific embodiments, heterologous secretory signal
peptides may be used, preferably selected from the list comprising
SEQ ID NOs: 258-282, 310-316 wherein IgE leader (SEQ ID NO: 264)
and HA signal peptide (SEQ ID NO: 282) are particularly preferred.
Further details about secretory signal peptides are provided in the
paragraph "Secretory signal peptides" of the present application
and in Table 3.
[0149] Accordingly, in this context it is preferred that the at
least one coding sequence of the artificial nucleic acid sequence
of the present invention encodes at least one Hendra virus
antigenic peptide or protein which is derived from a truncated
Hendra virus solG as defined above, or a fragment or variant
thereof, additionally comprising an N-terminal heterologous
secretory signal peptide as defined above, or a fragment or variant
thereof. Suitable Hendra virus soluble glycoprotein (solG) proteins
comprising an N-terminal heterologous secretory signal peptide (and
respective nucleic acid coding sequences) are provided in Table
1B.
[0150] In a specific embodiment, Hendra virus fusion protein (F)
(SEQ ID NOs: 8-11) is N-terminally truncated to remove the
endogenous secretory signal peptide to generate truncated forms of
Hendra virus fusion protein (F). The N-terminal truncation has to
be adapted in a way that secretory signal peptide of the protein
are removed. Secretory signal peptides of a protein can be
determined using prediction algorithms as commonly known in the art
(e.g. SignalP (SignalP 4.0: discriminating signal peptides from
transmembrane regions Thomas Nordahl Petersen, Soren Brunak, Gunnar
von Heijne & Henrik Nielsen, Nature Methods, 8:785-786,
2011).
[0151] Hendra virus full length Fusion (F) proteins commonly
consist of 546 amino acids (see Table 1).
[0152] In embodiments, Hendra F proteins lacking the endogenous
signal peptide (SS) are generated by truncating the first 25 amino
acids of the protein according to SEQ ID NOs: 8-11. In other words,
amino acids 26-546 of the proteins according to SEQ ID NOs: 8-11
are truncated forms of glycoprotein (FdeISS).
[0153] In embodiments, Hendra F proteins lacking the endogenous
signal peptide (SS) are generated by truncating the first 26 amino
acids of the protein according to SEQ ID NOs: 8-11. In other words,
amino acids 27-546 of the proteins according to SEQ ID NOs: 8-11
are truncated forms of F protein (FdeISS).
[0154] In this context it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one Hendra virus antigenic peptide or
protein which is derived from Hendra virus FdeISS as defined above,
or a fragment or variant thereof. Suitable Hendra virus truncated
forms of Hendra F protein (FdeISS) (and respective nucleic acid
coding sequences) are provided in Table 1B.
[0155] To facilitate secretion or improve secretion of the
truncated F proteins (FdeISS), elements that promote secretion may
be N-terminally fused to the (N-terminally truncated) Hendra virus
FdeISS proteins as specified above. In specific embodiments,
heterologous secretory signal peptides may be used, preferably
selected from the list comprising SEQ ID NOs: 258-282, 310-316,
wherein IgE leader (SEQ ID NO: 264) and HA signal peptide (SEQ ID
NO: 282) are particularly preferred. Further details about
secretory signal peptides are provided in the paragraph "Secretory
signal peptides" of the present application and in Table 3.
[0156] Accordingly it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one Hendra virus antigenic peptide or
protein which is derived from a truncated Hendra virus FdeISS as
defined above, or a fragment or variant thereof, additionally
comprising an N-terminal heterologous secretory signal peptide as
defined above, or a fragment or variant thereof. Suitable Hendra
virus FdeISS proteins comprising an N-terminal heterologous
secretory signal peptide (and respective nucleic acid coding
sequences) are provided in Table 1B.
[0157] In this context, it is particularly preferred that the at
least one coding sequence of the artificial nucleic acid sequence
of the present invention encodes a Hendra virus FdeISS proteins as
provided in Table 1B. Particularly preferred are Hendra virus
FdeISS proteins additionally comprising an N-terminal heterologous
secretory signal peptide (ICE-leader or HA signal peptide),
preferably selected from the antigens listed in Table 1B. In
addition, Table 1B provides suitable nucleic acid sequences
encoding FdeISS and FdeISS proteins additionally comprising an
N-terminal heterologous secretory signal peptides.
[0158] In Table 1B provided herein, each row corresponds to a
Hendra virus antigenic peptide or protein as identified by the
respective construct name (first column--Name). The second column
("A", "Protein") in Table 1B indicates the SEQ ID NOs corresponding
to the respective amino acid sequence as provided herein. The SEQ
ID NOs corresponding to the nucleic acid sequence of the wild type
nucleic acid sequence encoding the indicated Hendra virus antigenic
protein or peptide is indicated in the fourth column ("B", "CDS
wt"). The following columns ("C"-"J") provides the SEQ ID NOs
corresponding to modified nucleic acid sequences (opt1, opt2, opt3,
opt4, opt5, opt6, opt7) of the nucleic acid sequences as described
herein that encode the Hendra virus protein or peptide preferably
having the amino acid sequence as defined by the SEQ ID NOs
indicated in the second column ("A"). Additional information
regarding each of the sequences provided in Table 1B may also be
derived from the sequence listing, in particular from the details
provided therein under identifier <223>.
TABLE-US-00002 TABLE 1B List of truncated Hendra virus antigens and
Signal-peptide fusion proteins: A B C D E F G H J Name Protein CDS
wt CDS opt1 CDS opt2 CDS opt3 CDS opt4 CDS opt5 CDS opt6 CDS opt7
F(27-546) (FdelSS) 580 606 632 658 684 710 736 762 788 F(27-546)
(FdelSS) 581 607 633 659 685 711 737 763 789 F(26-546) (FdelSS) 582
608 634 660 686 712 738 764 790 F(27-546) (FdelSS) 583 609 635 661
687 713 739 765 791 HsIgE(1-18)_F(27-546) 814 840 866 892 918 944
970 996 1022 HsIgE(1-18)_F(27-546) 815 841 867 893 919 945 971 997
1023 HsIgE(1-18)_F(26-546) 816 842 868 894 920 946 972 998 1024
HsIgE(1-18)_F(27-546) 817 843 869 895 921 947 973 999 1025
H1N1-HA(1-17)_F(27-546) 1048 1074 1100 1126 1152 1178 1204 1230
1256 H1N1-HA(1-17)_F(27-546) 1049 1075 1101 1127 1153 1179 1205
1231 1257 H1N1-HA(1-17)_F(26-546) 1050 1076 1102 1128 1154 1180
1206 1232 1258 H1N1-HA(1-17)_F(27-546) 1051 1077 1103 1129 1155
1181 1207 1233 1259 G(70-604) (solG) 591 617 643 669 695 721 747
773 799 G(70-604) (solG) 592 618 644 670 696 722 748 774 800
G(70-604) (solG) 593 619 645 671 697 723 749 775 801 G(70-604)
(solG) 594 620 646 672 698 724 750 776 802 G(70-604) (solG) 595 621
647 673 699 725 751 777 803 G(70-604) (solG) 596 622 648 674 700
726 752 778 804 G(70-604) (solG) 597 623 649 675 701 727 753 779
805 G(70-604) (solG) 598 624 650 676 702 728 754 780 806
HsIgE(1-18)_G(70-604) 825 851 877 903 929 955 981 1007 1033
HsIgE(1-18)_G(70-604) 826 852 878 904 930 956 982 1008 1034
HsIgE(1-18)_G(70-604) 827 853 879 905 931 957 983 1009 1035
HsIgE(1-18)_G(70-604) 828 854 880 906 932 958 984 1010 1036
HsIgE(1-18)_G(70-604) 829 855 881 907 933 959 985 1011 1037
HsIgE(1-18)_G(70-604) 830 856 882 908 934 960 986 1012 1038
HsIgE(1-18)_G(70-604) 831 857 883 909 935 961 987 1013 1039
HsIgE(1-18)_G(70-604) 832 858 884 910 936 962 988 1014 1040
H1N1-HA(1-17)_G(70-604) 1059 1085 1111 1137 1163 1189 1215 1241
1267 H1N1-HA(1-17)_G(70-604) 1060 1086 1112 1138 1164 1190 1216
1242 1268 H1N1-HA(1-17)_G(70-604) 1061 1087 1113 1139 1165 1191
1217 1243 1269 H1N1-HA(1-17)_G(70-604) 1062 1088 1114 1140 1166
1192 1218 1244 1270 H1N1-HA(1-17)_G(70-604) 1063 1089 1115 1141
1167 1193 1219 1245 1271 H1N1-HA(1-17)_G(70-604) 1064 1090 1116
1142 1168 1194 1220 1246 1272 H1N1-HA(1-17)_G(70-604) 1065 1091
1117 1143 1169 1195 1221 1247 1273 H1N1-HA(1-17)_G(70-604) 1066
1092 1118 1144 1170 1196 1222 1248 1274
[0159] According to preferred embodiments, the inventive artificial
nucleic acid comprises or consists of at least one coding sequence
encoding at least one Hendra virus antigenic peptide or peptide as
provided herein, wherein the at least one Hendra virus antigenic
peptide or protein comprises at least one 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: 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.
[0160] In other embodiments, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from a Hendra virus
RNA-directed RNA polymerase (L), Hendra virus fusion protein (F),
Hendra virus non-structural protein (V), Hendra virus glycoprotein
(G), Hendra virus nucleoprotein (N), Hendra virus matrix protein
(M), Hendra virus phosphoprotein (P), Hendra virus protein C, and
Hendra virus protein W or a fragment or variant of any of
these.
[0161] In another embodiment, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from Hendra virus
RNA-directed RNA polymerase (L), or a fragment or variant thereof.
In another embodiment, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from Hendra virus
non-structural protein (V), or a fragment or variant thereof. In
another embodiment, the inventive artificial nucleic acid comprises
or consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Hendra virus
nucleoprotein (N), or a fragment or variant thereof. In another
embodiment, the inventive artificial nucleic acid comprises or
consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Hendra virus matrix
protein (M), or a fragment or variant thereof. In another
embodiment, the inventive artificial nucleic acid comprises or
consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Hendra virus
phosphoprotein (P), or a fragment or variant thereof. In another
embodiment, the inventive artificial nucleic acid comprises or
consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Hendra virus protein C,
or a fragment or variant thereof. In another embodiment, the
inventive artificial nucleic acid comprises or consists of at least
one coding sequence encoding at least one antigenic peptide or
protein derived from Hendra virus protein W, or a fragment or
variant thereof.
Nipah virus peptides or proteins:
[0162] The term "Nipah virus protein" or "Nipah virus peptide" as
used in the present invention may relate to an amino acid sequence
corresponding to any Nipah virus RNA-directed RNA polymerase (L),
Nipah virus fusion protein (F), Nipah virus non-structural protein
(V), Nipah virus glycoprotein (G), Nipah virus nucleoprotein (N),
Nipah virus matrix protein (M), Nipah virus phosphoprotein (P),
Nipah virus protein C, and Nipah virus protein W.
[0163] Any Nipah virus peptide or protein provided herein, or any a
fragment or variant thereof, can cause an immune response when
administered to a subject. Therefore, all Nipah virus proteins or
peptides provided herein can be considered as antigens in the
context of the present invention.
[0164] In some embodiments described herein, the at least one Nipah
virus antigenic peptide or protein encoded by the at least one
coding sequence of the artificial nucleic acid may consist of an
individual Nipah virus protein, the amino acid sequence of which
does typically not comprise an N-terminal Methionine residue. It is
thus understood that the phrase "artificial nucleic acid comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from a Nipah virus . . . " relates to a
protein or peptide comprising the amino acid sequence of said Nipah
virus protein and--if the amino acid sequence of the respective
Nipah virus protein does not comprise such an N-terminal Methionine
residue--an introduced N-terminal Methionine residue.
[0165] In the context of the present invention a fragment of a
protein or a variant thereof encoded by the at least one coding
sequence of the artificial nucleic acid according to the invention
may typically comprise an amino acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with an amino acid sequence of the respective naturally occurring
full-length Nipah virus protein or a variant thereof, preferably as
disclosed in Table 2 or Table 2B.
[0166] In a preferred embodiment, the at least one coding sequence
of the artificial nucleic acid sequence according to the invention
preferably encodes Nipah virus proteins selected from the proteins
provided in Table 2 or Table 2B, or a fragment or variant thereof.
Any Nipah virus protein provided in Table 2 or Table 2B, or any a
fragment or variant thereof, can cause an immune response when
administered to an individual. Therefore, all Nipah virus proteins
provided in Table 2 or Table 2B can be considered as preferred
Nipah virus antigens in the context of the present invention.
[0167] It is further preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes a Nipah virus protein or peptide, or a fragment
or variant thereof, wherein the Nipah virus protein or peptide is
an antigen selected from the antigens listed in Table 2. Therein,
each row corresponds to a Nipah virus antigenic peptide or protein
as identified by the respective gene name (first column "Name") and
the NCBI database accession number of the corresponding protein
(second column "Accession No."). The third column ("A", "protein")
in Table 2 indicates the SEQ ID NOs corresponding to the respective
amino acid sequence as provided herein. The SEQ ID NOs
corresponding to the nucleic acid sequence of the wild type nucleic
acid sequence encoding the Nipah virus antigenic protein or peptide
is indicated in the fourth column ("B", "CDS wt"). The following
columns ("C"-"J") provides the SEQ ID NOs corresponding to modified
nucleic acid sequences (opt1, opt2, opt3, opt4, opt5, opt6, opt7)
of the nucleic acid sequences as described herein that encode the
Nipah virus protein or peptide preferably having the amino acid
sequence as defined by the SEQ ID NOs indicated in the third column
("A") or by the database entry indicated in the second column
("Accession No."). Additional information regarding each of the
sequences provided in Table 2 may also be derived from the sequence
listing, in particular from the details provided therein under
identifier <223>.
TABLE-US-00003 TABLE 2 List of Nipah virus antigens: A B C D E F G
H J Name Accession No. Protein CDS wt opt1 opt2 opt3 opt4 opt5 opt6
opt7 F AAK50553.1 1 27 53 79 105 131 157 183 209 F AEZ01388.1 2 28
54 80 106 132 158 184 210 F AAY43915.1 3 29 55 81 107 133 159 185
211 F CAF25496.1 4 30 56 82 108 134 160 186 212 F AAM13405.1 5 31
57 83 109 135 161 187 213 F CBM41033.1 6 32 58 84 110 136 162 188
214 F AEZ01396.1 7 33 59 85 111 137 163 189 215 G AAK50554.1 12 38
64 90 116 142 168 194 220 G AEZ01389.1 13 39 65 91 117 143 169 195
221 G ACT32615.1 14 40 66 92 118 144 170 196 222 G CAF25497.1 15 41
67 93 119 145 171 197 223 G CBM41034.1 16 42 68 94 120 146 172 198
224 G AAX51853.1 17 43 69 95 121 147 173 199 225 G AEZ01397.1 18 44
70 96 122 148 174 200 226
[0168] According to preferred embodiments, the inventive artificial
nucleic acid comprises or consists of at least one coding sequence
encoding at least one Nipah virus antigenic peptide or peptide as
described herein, wherein the at least one Nipah virus antigenic
peptide or protein comprises at least one 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: 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.
[0169] According to a preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one Nipah virus antigenic peptide
or peptide as described herein, wherein the at least one Nipah
virus antigenic peptide or protein comprises an amino acid sequence
according to any one of SEQ ID NOs: 1-7 and 12-18, or a homolog,
fragment or variant of any of these sequences (see Table 2, column
"A").
[0170] In an embodiment the Nipah virus antigenic peptide or
protein is derived from a Nipah virus Fusion protein (F) according
to SEQ ID NOs: 1-7.
[0171] In this context it is further preferred that the at least
one coding sequence of the artificial nucleic acid sequence of the
present invention encodes at least one Nipah virus peptide or
protein which is derived from Nipah virus fusion protein (F), or a
fragment or variant thereof, wherein the Nipah virus fusion protein
(F) is selected from the Nipah virus fusion protein amino acid
sequences listed in Table 2.
[0172] Therein, rows corresponding to a Nipah virus fusion protein
(F) (SEQ ID NOs: 1-7) can be identified by the respective gene name
(first column "Name": "F") and the database accession number of the
corresponding protein (second column "Accession No."). The SEQ ID
NOs: corresponding to the nucleic acid sequence of the wild type
nucleic acid encoding the Nipah virus fusion protein (F) or peptide
is indicated in the fourth column ("B"). The further columns
("C"-"J") provide the SEQ ID NOs corresponding to modified nucleic
acid sequences of the nucleic acids as described herein that encode
the Nipah virus fusion protein (F) or peptide preferably having the
amino acid sequence as defined by the SEQ ID NOs: 1-7 or by the
database entry indicated in the second column ("Accession
No.").
[0173] In an embodiment the Nipah virus antigenic peptide or
protein is derived from a Nipah virus glycoprotein (G) according to
SEQ ID NOs: 12-18.
[0174] In this context it is further preferred that the at least
one coding sequence of the artificial nucleic acid sequence of the
present invention encodes at least one Nipah virus peptide or
protein which is derived from Nipah virus glycoprotein (G), or a
fragment or variant thereof, wherein the Nipah virus glycoprotein
(G) is selected from the Nipah virus fusion protein amino acid
sequences listed in Table 2.
[0175] Therein, rows corresponding to a Nipah virus glycoprotein
(G) (SEQ ID NOs: 12-18) can be identified by the respective gene
name (first column "Name": "F") and the database accession number
of the corresponding protein (second column "Accession No."). The
SEQ ID NOs: corresponding to the nucleic acid sequence of the wild
type nucleic acid encoding the Nipah virus glycoprotein (G) or
peptide is indicated in the fourth column ("B"). The further
columns ("C"-"J") provide the SEQ ID NOs corresponding to modified
nucleic acid sequences of the nucleic acids as described herein
that encode the Nipah virus glycoprotein (G) or peptide preferably
having the amino acid sequence as defined by the SEQ ID NOs: 12-18
or by the database entry indicated in the second column ("Accession
No.").
[0176] In a specific embodiment, Nipah virus glycoprotein (G) (SEQ
ID NOs: 12-18) is N-terminally truncated to generate a soluble form
of the protein (solG). The N-terminal truncation has to be adapted
in a way that membrane-bound domains of the protein are removed.
The membrane topology of a protein can be determined using
prediction algorithms as commonly known in the art.
[0177] Nipah virus glycoprotein (G) polypeptides commonly consist
of 602 amino acids (see Table 2).
[0178] In embodiments, soluble forms of the Nipah virus G protein
(solG) are generated by truncating the first 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, or 80 amino acids of the protein
according to SEQ ID NOs: 12-18. In other words, amino acids 69-602,
70-602, 71-602, 72-602, 73-602, 74-602, 75-602, 76-602, 77-602,
78-602, 79-602, 80-602, 81-602 of the proteins according to SEQ ID
NOs: 12-18 are soluble forms of glycoprotein (solG).
[0179] In a preferred embodiments, soluble forms of the Nipah virus
protein (solG) are generated by truncating the first 70 amino acids
of the protein according to SEQ ID NOs: 12-18. In other words,
amino acids 71-602 of the proteins according to SEQ ID NOs: 12-18
are soluble forms of glycoprotein (solG).
[0180] In another specific embodiment, soluble forms of the Nipah
virus protein (solG) are generated by truncating the first 72 amino
acids of the protein according to SEQ ID NOs 12-18. In other words,
amino acids 73 602 of the proteins according to SEQ ID NOs 12-18
are soluble forms of glycoprotein (solG).
[0181] In this context it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one Nipah virus antigenic peptide or
protein which is derived from Nipah virus soluble glycoprotein
(solG) as defined above, or a fragment or variant thereof. Suitable
Nipah virus soluble glycoprotein (solG) proteins (and respective
nucleic acid coding sequences) are provided in Table 2B.
[0182] To facilitate secretion of the truncated protein, elements
that promote secretion may be N-terminally fused to the
(N-terminally truncated) Nipah virus solG proteins. In specific
embodiments, secretory signal peptides may be used, preferably
selected from the list comprising SEQ ID NOs: 258-282, 310-316,
wherein IgE leader (SEQ ID NO: 264) and HA signal peptide (SEQ ID
NO: 282) are particularly preferred. Further details about
secretory signal peptides are provided in the paragraph "Secretory
signal peptides" of the present application and in Table 3.
[0183] Accordingly, in this context it is preferred that the at
least one coding sequence of the artificial nucleic acid sequence
of the present invention encodes at least one Hendra virus
antigenic peptide or protein which is derived from a truncated
Nipah virus solG as defined above, or a fragment or variant
thereof, additionally comprising an N-terminal heterologous
secretory signal peptide as defined above, or a fragment or variant
thereof. Suitable Nipah virus soluble glycoprotein (solG) proteins
comprising an N-terminal heterologous secretory signal peptide (and
respective nucleic acid coding sequences) are provided in Table
2B.
[0184] In a specific embodiment, Nipah virus fusion protein (F)
(SEQ ID NOs: 1-7) is N-terminally truncated to remove the
endogenous secretory signal peptide to generate truncated forms of
Nipah virus fusion protein (F). The N-terminal truncation has to be
adapted in a way that secretory signal peptide of the protein are
removed. Secretory signal peptides of a protein can be determined
using prediction algorithms as commonly known in the art (e.g.
SignalP (SignalP 4.0: discriminating signal peptides from
transmembrane regions Thomas Nordahl Petersen, Soren Brunak, Gunnar
von Heijne & Henrik Nielsen, Nature Methods, 8:785-786,
2011).
[0185] Nipah virus full length Fusion (F) proteins commonly consist
of 546 amino acids (see Table 2).
[0186] In embodiments, Nipah F proteins lacking the endogenous
signal peptide (SS) are generated by truncating the first 25 amino
acids of the protein according to SEQ ID NOs: 1-7. In other words,
amino acids 26-546 of the proteins according to SEQ ID NOs: 1-7 are
truncated forms of glycoprotein (FdeISS).
[0187] In embodiments, Nipah F proteins lacking the endogenous
signal peptide (SS) are generated by truncating the first 26 amino
acids of the protein according to SEQ ID NOs: 1-7. In other words,
amino acids 27-546 of the proteins according to SEQ ID NOs: 1-7 are
truncated forms of F protein (FdeISS).
[0188] In this context it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one Nipah virus antigenic peptide or
protein which is derived from Nipah virus FdeISS as defined above,
or a fragment or variant thereof. Suitable truncated forms of Nipah
F protein (FdeISS) (and respective nucleic acid coding sequences)
are provided in Table 2B.
[0189] To facilitate secretion or improve secretion of the
truncated Nipah F proteins (FdeISS), elements that promote
secretion may be N-terminally fused to the (N-terminally truncated)
Nipah virus FdeISS proteins as specified above. In specific
embodiments, heterologous secretory signal peptides may be used,
preferably selected from the list comprising SEQ ID NOs: 258-282,
310-316, wherein IgE leader (SEQ ID NO: 264) and HA signal peptide
(SEQ ID NO: 282) are particularly preferred. Further details about
secretory signal peptides are provided in the paragraph "Secretory
signal peptides" of the present application and in Table 3.
[0190] Accordingly it is preferred that the at least one coding
sequence of the artificial nucleic acid sequence of the present
invention encodes at least one antigenic peptide or protein which
is derived from a truncated Nipah virus FdeISS as defined above, or
a fragment or variant thereof, additionally comprising an
N-terminal heterologous secretory signal peptide as defined above,
or a fragment or variant thereof. Suitable Nipah virus FdeISS
proteins comprising an N-terminal heterologous secretory signal
peptide (and respective nucleic acid coding sequences) are provided
in Table 2B.
[0191] In this context, it is particularly preferred that the at
least one coding sequence of the artificial nucleic acid sequence
of the present invention encodes a Nipah virus FdeISS proteins as
provided in Table 2B. Particularly preferred are Nipah virus FdeISS
proteins additionally comprising an N-terminal heterologous
secretory signal peptide (IGE-leader or HA signal peptide),
preferably selected from the antigens listed in Table 2B. In
addition, Table 2B provides suitable nucleic acid sequences
encoding FdeISS and FdeISS proteins additionally comprising an
N-terminal heterologous secretory signal peptides.
[0192] In Table 2B provided herein, each row corresponds to a Nipah
virus antigenic peptide or protein as identified by the respective
construct name (first column "Name"). The second column ("A",
"Protein") in Table 2B indicates the SEQ ID NOs corresponding to
the respective amino acid sequence as provided herein. The SEQ ID
NOs corresponding to the nucleic acid sequence of the wild type
nucleic acid sequence encoding the indicated Nipah virus antigenic
protein or peptide is indicated in the fourth column ("B", "CDS
wt"). The following columns ("C"-"J") provides the SEQ ID NOs
corresponding to modified nucleic acid sequences (opt1, opt2, opt3,
opt4, opt5, opt6, opt7) of the nucleic acid sequences as described
herein that encode the Nipah virus protein or peptide preferably
having the amino acid sequence as defined by the SEQ ID NOs
indicated in the second column ("A"). Additional information
regarding each of the sequences provided in Table 2B may also be
derived from the sequence listing, in particular from the details
provided therein under identifier <223>.
TABLE-US-00004 TABLE 2B List of truncated Nipah virus antigens and
Signal-peptide fusion proteins: A B C D E F G H J Name Protein CDS
wt CDS opt1 CDS opt2 CDS opt3 CDS opt4 CDS opt5 CDS opt6 CDS opt7
F(27-546) (FdelSS) 573 599 625 651 677 703 729 755 781 F(27-546)
(FdelSS) 574 600 626 652 678 704 730 756 782 F(27-546) (FdelSS) 575
601 627 653 679 705 731 757 783 F(27-546) (FdelSS) 576 602 628 654
680 706 732 758 784 F(27-546) (FdelSS) 577 603 629 655 681 707 733
759 785 F(27-546) (FdelSS) 578 604 630 656 682 708 734 760 786
F(27-546) (FdelSS) 579 605 631 657 683 709 735 761 787
HsIgE(1-18)_F(27-546) 807 833 859 885 911 937 963 989 1015
HsIgE(1-18)_F(27-546) 808 834 860 886 912 938 964 990 1016
HsIgE(1-18)_F(27-546) 809 835 861 887 913 939 965 991 1017
HsIgE(1-18)_F(27-546) 810 836 862 888 914 940 966 992 1018
HsIgE(1-18)_F(27-546) 811 837 863 889 915 941 967 993 1019
HsIgE(1-18)_F(27-546) 812 838 864 890 916 942 968 994 1020
HsIgE(1-18)_F(27-546) 813 839 865 891 917 943 969 995 1021
H1N1-HA(1-17)_F(27-546) 1041 1067 1093 1119 1145 1171 1197 1223
1249 H1N1-HA(1-17)_F(27-546) 1042 1068 1094 1120 1146 1172 1198
1224 1250 H1N1-HA(1-17)_F(27-546) 1043 1069 1095 1121 1147 1173
1199 1225 1251 H1N1-HA(1-17)_F(27-546) 1044 1070 1096 1122 1148
1174 1200 1226 1252 H1N1-HA(1-17)_F(27-546) 1045 1071 1097 1123
1149 1175 1201 1227 1253 H1N1-HA(1-17)_F(27-546) 1046 1072 1098
1124 1150 1176 1202 1228 1254 H1N1-HA(1-17)_F(27-546) 1047 1073
1099 1125 1151 1177 1203 1229 1255 G(70-602) (solG) 584 610 636 662
688 714 740 766 792 G(70-602) (solG) 585 611 637 663 689 715 741
767 793 G(70-602) (solG) 586 612 638 664 690 716 742 768 794
G(70-602) (solG) 587 613 639 665 691 717 743 769 795 G(70-602)
(solG) 588 614 640 666 692 718 744 770 796 G(70-602) (solG) 589 615
641 667 693 719 745 771 797 G(70-602) (solG) 590 616 642 668 694
720 746 772 798 HsIgE(1-18)_G(70-602) 818 844 870 896 922 948 974
1000 1026 HsIgE(1-18)_G(70-602) 819 845 871 897 923 949 975 1001
1027 HsIgE(1-18)_G(70-602) 820 846 872 898 924 950 976 1002 1028
HsIgE(1-18)_G(70-602) 821 847 873 899 925 951 977 1003 1029
HsIgE(1-18)_G(70-602) 822 848 874 900 926 952 978 1004 1030
HsIgE(1-18)_G(70-602) 823 849 875 901 927 953 979 1005 1031
HsIgE(1-18)_G(70-602) 824 850 876 902 928 954 980 1006 1032
H1N1-HA(1-17)_G(70-602) 1052 1078 1104 1130 1156 1182 1208 1234
1260 H1N1-HA(1-17)_G(70-602) 1053 1079 1105 1131 1157 1183 1209
1235 1261 H1N1-HA(1-17)_G(70-602) 1054 1080 1106 1132 1158 1184
1210 1236 1262 H1N1-HA(1-17)_G(70-602) 1055 1081 1107 1133 1159
1185 1211 1237 1263 H1N1-HA(1-17)_G(70-602) 1056 1082 1108 1134
1160 1186 1212 1238 1264 H1N1-HA(1-17)_G(70-602) 1057 1083 1109
1135 1161 1187 1213 1239 1265 H1N1-HA(1-17)_G(70-602) 1058 1084
1110 1136 1162 1188 1214 1240 1266 HsSPARC(1-17)_F(27-546) 1513
1516 1519 1522 1525 1528 1531 1534 1537 HsCTRB2(1-18)_F(27-546)
1514 1517 1520 1523 1526 1529 1532 1535 1538 Nipah 1515 1518 1521
1524 1527 1530 1533 1536 1539
henipavirus_AAK50553_F(1-26)_F(27-546)
[0193] According to preferred embodiments, the inventive artificial
nucleic acid comprises or consists of at least one coding sequence
encoding at least one Nipah virus antigenic peptide or peptide as
provided herein, wherein the at least one Nipah virus antigenic
peptide or protein comprises at least one 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: 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.
[0194] In other embodiments, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from a Nipah virus
RNA-directed RNA polymerase (L), Nipah virus fusion protein (F),
Nipah virus non-structural protein (V), Nipah virus glycoprotein
(G), Nipah virus nucleoprotein (N), Nipah virus matrix protein (M),
Nipah virus phosphoprotein (P), Nipah virus protein C, and Nipah
virus protein W or a fragment or variant of any of these.
[0195] In another embodiment, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from Nipah virus
RNA-directed RNA polymerase (L), or a fragment or variant thereof.
In another embodiment, the inventive artificial nucleic acid
comprises or consists of at least one coding sequence encoding at
least one antigenic peptide or protein derived from Nipah virus
non-structural protein (V), or a fragment or variant thereof. In
another embodiment, the inventive artificial nucleic acid comprises
or consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Nipah virus nucleoprotein
(N), or a fragment or variant thereof. In another embodiment, the
inventive artificial nucleic acid comprises or consists of at least
one coding sequence encoding at least one antigenic peptide or
protein derived from Nipah virus matrix protein (M), or a fragment
or variant thereof. In another preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one antigenic peptide or protein
derived from Nipah virus phosphoprotein (P), or a fragment or
variant thereof. In another embodiment, the inventive artificial
nucleic acid comprises or consists of at least one coding sequence
encoding at least one antigenic peptide or protein derived from
Nipah virus protein C, or a fragment or variant thereof. In another
embodiment, the inventive artificial nucleic acid comprises or
consists of at least one coding sequence encoding at least one
antigenic peptide or protein derived from Nipah virus protein W, or
a fragment or variant thereof.
[0196] Additional Peptide or Protein Elements:
[0197] According to another particularly preferred embodiment, the
artificial nucleic acid sequence, particularly the RNA sequence
according to the invention may additionally encode a further,
preferably heterologous peptide or protein elements, that e.g.,
promote secretion of the protein (secretory signal peptides),
promote anchoring of the encoded antigen in the plasma membrane
(transmembrane domains), promote virus-like particle formation (VLP
forming domains). In addition, the artificial nucleic acid sequence
according to the present invention may additionally encode peptide
linker elements, self-cleaving peptides or helper peptides.
Further, the artificial nucleic acid sequence according to the
present invention may additionally encode an immunologic adjuvant
sequence, and/or a dendritic cell targeting sequence.
[0198] Secretory Signal Peptides:
[0199] According another preferred embodiment, the artificial
nucleic acid sequence, particularly the RNA sequence according to
the invention may additionally or alternatively encode at least one
secretory signal peptide. Such signal peptides are sequences, which
typically exhibit a length of about 15 to 30 amino acids and are
preferably located at the N-terminus of the encoded peptide,
without being limited thereto. Signal peptides as defined herein
preferably allow the transport of the Henipavirus and/or Hendra
virus and/or Nipah virus antigenic peptide or proteins as encoded
by the at least one artificial nucleic acid sequence into a defined
cellular compartment, preferably the cell surface, the endoplasmic
reticulum (ER) or the endosomal-lysosomal compartment. Examples of
secretory signal peptide sequences as defined herein include,
without being limited thereto, signal sequences of classical or
non-classical MHC-molecules (e.g. signal sequences of MHC I and II
molecules, e.g. of the MHC class I molecule HLA-A*0201), signal
sequences of cytokines or immunoglobulins as defined herein, signal
sequences of the invariant chain of immunoglobulins or antibodies
as defined herein, signal sequences of Lamp1, Tapasin, Erp57,
Calretikulin, Calnexin, and further membrane associated proteins or
of proteins associated with the endoplasmic reticulum (ER) or the
endosomal-lysosomal compartment. Most preferably, signal sequences
of MHC class I molecule HLA-A*0201 may be used according to the
present invention. For example, a signal peptide derived from HLA-A
is preferably used in order to promote secretion of the encoded
Henipavirus and/or Hendra virus and/or Nipah virus antigen as
defined herein or a fragment or variant thereof. More preferably,
an HLA-A signal peptide is fused to an encoded Henipavirus and/or
Hendra virus and/or Nipah virus antigen as defined herein or to a
fragment or variant thereof. Particularly preferred secretory
signal peptides according to the present invention are provided in
the sequence listing (SEQ ID NOs: 258-282, 310-316). Further
suitable secretory signal peptides may be selected from the list of
amino acid sequences according to SEQ ID NOs: 1-1115 and SEQ ID NO:
1728 of the patent application WO2017/081082, or fragments or
variants of these sequences, herewith incorporated by reference.
Particularly preferred secretory signal peptides in the context of
the invention are IgE leader (HsIgE(1-18)) (SEQ ID NO: 264) HA
signal peptide (H1N1-HA(1-17)) (SEQ ID NO: 282), HsSPARC (SEQ ID
NO: 281), and HsCTRB2 (SEQ ID NO: 267).
[0200] On nucleic acid level, particularly RNA level, any
nucleotide sequence moiety can be employed that encodes any of
secretory signal peptides used in the context of the present
invention. Owing to the degenerated genetic code, e.g. in the case
of most peptide sequences according to SEQ ID NOs: 258-282, 310-316
more than one particular nucleic acid sequence is conceivable as
encoding the respective polypeptide. While each and every such
nucleic acid may generally be used in the context of the present
invention, it is preferable that the nucleic acid sequence that
encodes the polypeptide sequence is selected such that its sequence
is optimized according to the general guidance provided in this
specification.
[0201] In the context of the invention, it is particularly
preferred that the secretory signal peptide as defined herein is
located at the N-terminus of the at least one antigenic peptide or
protein, followed by a F-protein lacking its endogenous secretory
signal peptide (FdeISS) as defined above or followed by a G-protein
lacking its endogenous transmembrane domain (solG) as defined above
(see also Table 1B and Table 2B).
[0202] In preferred embodiments, secretory signal peptides may
suitably be selected from those provided in Table 3. In Table 3,
each row corresponds to a secretory signal peptide as identified by
the respective name (first column "Name") and the Accession number
(second column "NCBI Accession No.") The third column ("A",
"Protein") in Table 3 indicates the SEQ ID NOs corresponding to the
respective amino acid sequence as provided herein. The SEQ ID NOs
corresponding to the nucleic acid sequence of the wild type nucleic
acid sequence encoding the indicated secretory signal peptide is
indicated in the fourth column ("B", "CDS wt"). The following
columns ("C-J") provides the SEQ ID NOs corresponding to modified
nucleic acid sequences (opt1, opt2, opt3, opt4, opt5, opt6, opt7)
of the nucleic acid sequences as described herein that encode the
secretory signal peptide preferably having the amino acid sequence
as defined by the SEQ ID NOs indicated in the third column ("A") or
by the database entry indicated in the second column ("Accession
No."). Additional information regarding each of the sequences
provided in Table 3 may also be derived from the sequence listing,
in particular from the details provided therein under identifier
<223>.
TABLE-US-00005 TABLE 3 List of suitable secretory signal peptides:
NCBI Accession A B C D E F G H J Name No. Protein CDS wt CDS opt1
CDS opt2 CDS opt3 CDS opt4 CDS opt5 CDS opt6 CDS opt7
HsHLA-A2(1-24) AAA59606 258 317 349 381 413 445 477 509 541
HsPLAT(1-23) AAA61213 259 318 350 382 414 446 478 510 542
HsPLAT(1-21) AAA61213 260 319 351 383 415 447 479 511 543
HsPLAT(1-22) AAA61213 261 320 352 384 416 448 480 512 544
HsEPO(1-27) NP_000790 262 321 353 385 417 449 481 513 545
HsALB(1-18) NP_000468 263 322 354 386 418 450 482 514 546
HsIgE(1-18) AAB59424 264 323 355 387 419 451 483 515 547
HsCD5(1-24) NP_055022 265 324 356 388 420 452 484 516 548
HsIL2(1-20) NP_000577 266 325 357 389 421 453 485 517 549
HsCTRB2(1-18) NP_001020371 267 326 358 390 422 454 486 518 550
HsIgG-HC(1-19) BAC87457 268 327 359 391 423 455 487 519 551
HsIg-HC(1-19) AAA52897 269 328 360 392 424 456 488 520 552
HsIg-LC(1-19) AAA59018 270 329 361 393 425 457 489 521 553
GpLuc(1-17) AAG54095 271 330 362 394 426 458 490 522 554
MmIgkappa(1-21) BAR42292 272 331 363 395 427 459 491 523 555
NrChit1(1-26) ABF74624 273 332 364 396 428 460 492 524 556
CILp1.1(1-21) AAS93426 274 333 365 397 429 461 493 525 557
NgNep1(1-24) AB114914 275 334 366 398 430 462 494 526 558
HsAzu1(1-19) NP_001691 276 335 367 399 431 463 495 527 559
HsCD33(1-16) AAA51948 277 336 368 400 432 464 496 528 560
VcCtxB(1-19) BAA06291 278 337 369 401 433 465 497 529 561
HsCST4(1-20) NP_001890 279 338 370 402 434 466 498 530 562
HsIns-iso1(1-24) AAA59172 280 339 371 403 435 467 499 531 563
HsSPARC(1-17) CAA68724 281 340 372 404 436 468 500 532 564
H1N1-HA(1-17) ACQ45338 282 341 373 405 437 469 501 533 565
HsMHCII(1-25) CAA23783 310 342 374 406 438 470 502 534 566 F(1-26)
AAK50553 311 343 375 407 439 471 503 535 567 F(1-26) AEZ01388 312
344 376 408 440 472 504 536 568 F(1-26) AAY43915 313 345 377 409
441 473 505 537 569 F(1-26) CBM41033 314 346 378 410 442 474 506
538 570 F(1-26) NP_047111 315 347 379 411 443 475 507 539 571
F(1-25) AEQ38114 316 348 380 412 444 476 508 540 572
[0203] Accordingly, in preferred embodiments, the at least one
coding sequence of the artificial nucleic acid of the invention
additionally encodes at least one further peptide or protein
element selected from a secretory signal peptide, 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 (as provided in Table 3),
or a fragment or variant of any of these sequences.
[0204] According to preferred embodiments, secretory signal
peptides as provided in Table 3 may be N-terminally fused to Hendra
virus F_deISS or SoIG proteins as provided in Table 1B or Hendra
virus F_deISS or SoIG proteins as provided in Table 2B to generate
antigenic Hendra and Nipah virus proteins optimized for secretion.
Preferred embodiments of Hendra and Nipah virus proteins comprising
heterologous N-terminal signal peptides are provided in Table 18
and Table 2B.
[0205] According to preferred embodiments, the nucleic acid
sequence according to the invention comprises at least one coding
sequence encoding a heterologous secretory signal sequence as
defined above and, in addition, 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 or a fragment or variant of any of these
sequences.
[0206] Transmembrane domains VLP forming domains Peptide linker
Self-cleaving peptides Helper peptides: According to another
embodiment, the artificial nucleic acid sequence, particularly the
RNA sequence according to the invention may additionally encode at
least one transmembrane domain element. Transmembrane elements or
membrane spanning polypeptide elements are present in proteins that
are integrated or anchored in plasma membranes of cells. Typical
transmembrane elements are alpha-helical transmembrane elements.
Such transmembrane elements are composed essentially of amino acids
with hydrophobic side chains, because the interior of a cell
membrane (lipid bilayer) is also hydrophobic. From the structural
perspective, transmembrane elements are commonly single hydrophobic
alpha helices or beta barrel structures; whereas hydrophobic alpha
helices are usually present in proteins that are present in
membrane anchored proteins (e.g., seven transmembrane domain
receptors), beta-barrel structures are often present in proteins
that generate pores or channels. For target proteins, such as
antigenic peptides or proteins according to the present invention
(derived from Henipavirus, Hendra virus, Nipah virus) it may be
beneficial to introduce a transmembrane element into the respective
constructs. By addition of a transmembrane element to the target
peptide/protein it may be possible to further enhance the immune
response, wherein the translated target peptide/protein, e.g. a
viral antigen, anchors to a target membrane, e.g. the plasma
membrane of a cell, thereby increasing immune responses. This
effect is also referred to as antigen clustering. When used in
combination with a polypeptide or protein of interest in the
context of the present invention, such transmembrane element can be
placed N-terminal or C-terminal to the Henipavirus and/or Hendra
virus and/or Nipah virus antigenic peptide or protein of interest.
On nucleic acid level, the coding sequence for such transmembrane
element is typically placed in frame (i.e. in the same reading
frame), 5' or 3' to the coding sequence of the polypeptide as
defined herein. The transmembrane domain may be selected from the
transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env
of HIV-1. EIAV (equine infectious anaemia virus), MLV (murine
leukaemia virus), mouse mammary tumor virus, G protein of VSV
(vesicular stomatitis virus), Rabies virus, or a transmembrane
element of a seven transmembrane domain receptor. Specific elements
suitable in the context of the present invention are provided in
the sequence listing (SEQ ID NOs: 283-294). On nucleic acid level,
particularly RNA level, any nucleotide sequence moiety can be
employed that encodes any transmembrane domain used in the present
invention. Owing to the degenerated genetic code, in the case of
most polypeptides SEQ ID NOs: 283-294, more than one particular
nucleic acid sequence is conceivable as encoding the respective
polypeptide. While each and every such nucleic acid may generally
be used in the context of the present invention, it is preferable
that the nucleic acid sequence that encodes the polypeptide
sequence is selected such that its sequence is optimized according
to the general guidance provided in this specification.
Alternatively, any polypeptide element may be selected which is
characterized by at least 80% identity, at least 85% identity,
preferably at least 90% identity, and more preferably at least 95%
identity to any of the sequences SEQ ID NOs: 283-294. On nucleic
acid level, any polynucleotide (e.g. RNA) moiety may be selected
which encodes such polypeptide element.
[0207] According to another embodiment, the artificial nucleic acid
sequence, particularly the RNA sequence according to the invention
may additionally encode at least one VLP forming domain. VLPs are
self-assembled viral structural proteins (envelope proteins or
capsid proteins) that structurally resemble viruses (without
containing viral genetic material). VLPs contain repetitive high
density displays of antigens which present conformational epitopes
that can elicit strong T cell and B cell immune responses. When
used in combination with a Henipavirus and/or Hendra virus and/or
Nipah virus antigenic peptide or protein in the context of the
present invention, such VLP forming element can be placed
N-terminal or C-terminal to the polypeptide of interest. On nucleic
acid level, the coding sequence for such VLP forming element is
typically placed in frame (i.e. in the same reading frame), 5' or
3' to the coding sequence of the polypeptide as defined herein. For
nucleic acid (e.g. RNA) encoding a polypeptide or protein of
interest, particularly antigenic polypeptides or proteins
(Henipavirus, Hendra virus, Nipah virus), it may be beneficial to
introduce a VLP forming element into the respective constructs. In
addition to the "clustering" of epitopes, an improved secretion of
the VLP particle may also increase the immunogenicity of the
respective antigen. VLP forming elements fused to an antigen may
generate virus like particles containing repetitive high density
displays of antigens. VLP forming elements may be selected e.g.
from any one of SEQ ID NOs: 295-296. Essentially, such VLP forming
elements can be chosen from any viral or phage capsid or envelope
protein. VLP forming elements may be used as additional elements to
promote or improve the particle formation of the target protein.
Suitably, the polypeptide sequence of the VLP forming element used
in the present invention is selected from the following list of
polypeptide sequences (SEQ ID NOs: 295-296). On nucleic acid level,
particularly RNA level, any nucleotide sequence moiety can be
employed that encodes any of VLP forming element used in the
present invention. Owing to the degenerated genetic code, in the
case of most polypeptides SEQ ID NOs: 295-296, more than one
particular nucleic acid sequence is conceivable as encoding the
respective polypeptide of the below list. While each and every such
nucleic acid may generally be used in the context of the present
invention, it is preferable that the nucleic acid sequence that
encodes the polypeptide sequence is selected such that its sequence
is codon-optimized according to the general guidance provided in
this specification. Alternatively, any polypeptide element may be
selected which is characterized by at least 80% identity, at least
85% identity, preferably at least 90% identity, and more preferably
at least 95% identity to any of the sequences SEQ ID NOs: 295-296.
On nucleic acid level, any polynucleotide (e.g. RNA) moiety may be
selected which encodes such polypeptide element.
[0208] According to another embodiment, the artificial nucleic acid
sequence, particularly the RNA sequence according to the invention
may additionally encode at least one peptide linker element. In
protein constructs composed of several elements (e.g., Henipavirus
antigenic peptide or protein fused to a transmembrane domain), the
protein elements may be separated by peptide linker elements. Such
elements may be beneficial because they allow for a proper folding
of the individual elements and thereby the proper functionality of
each element. Alternatively, the term "spacer" or "peptide spacer"
is used herein. When used in the context of the present invention,
such linkers or spacers are particularly useful when encoded by a
nucleic acid encoding at least two functional protein elements,
such as at least one polypeptide or protein of interest (Nipah
virus and/or Hendra virus antigens) and at least one further
protein or polypeptide element (e.g., VLP forming domain,
transmembrane domain). In that case, the linker is typically
located on the polypeptide chain in between the polypeptide of
interest and the at least one further protein element. On nucleic
acid level, the coding sequence for such linker is typically placed
in the reading frame, 5' or 3' to the coding sequence for the
polypeptide or protein of interest, or placed between coding
regions for individual polypeptide domains of a given protein of
interest. Peptide linkers are preferably composed of small,
non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The
small size of these amino acids provides flexibility, and allows
for mobility of the connecting functional domains. The
incorporation of Ser or Thr can maintain the stability of the
linker in aqueous solutions by forming hydrogen bonds with the
water molecules, and therefore reduces an interaction between the
linker and the protein moieties. Rigid linkers generally maintain
the distance between the protein domains and they may be based on
helical structures and/or they have a sequence that is rich in
proline. Cleavable linkers (also termed "cleavage linkers") allow
for in vivo separation of the protein domains. The mechanism of
cleavage may be based e.g. on reduction of disulfide bonds within
the linker sequence or proteolytic cleavage. The cleavage may be
mediated by an enzyme (enzymatic cleavage), e.g. the cleavage
linker may provide a protease sensitive sequence (e.g., furin
cleavage). A typical sequence of a flexible linker is composed of
repeats of the amino acids Glycine (G) and Serine (S). For
instance, the linker may have the following sequence: GS, GSG, SGG,
SG, GGS, SGS, GSS, SSG. In some embodiments, the same sequence is
repeated multiple times (e.g. two, three, four, five or six times)
to create a longer linker. In other embodiments, a single amino
acid residue such as S or G can be used as a linker. Linkers or
spacers may be used as additional elements to promote or improve
the secretion of the target protein (Henipavirus and/or Hendra
virus and/or Nipah virus antigenic peptides or proteins). Suitably,
the polypeptide sequence of the linker or spacer used in the
present invention is selected from the following list of
polypeptide sequences (SEQ ID NOs: 297-299). On nucleic acid level,
particularly RNA level, any nucleotide sequence moiety can be
employed that encodes any of linker or spacer used in the present
invention. Owing to the degenerated genetic code, in the case of
most polypeptides of SEQ ID NOs: 297-299, more than one particular
nucleic acid sequence is conceivable as encoding the respective
polypeptide list. While each and every such nucleic acid may
generally be used in the context of the present invention, it is
preferable that the nucleic acid sequence that encodes the
polypeptide sequence is selected such that its sequence is
optimized according to the general guidance provided in this
specification. Alternatively, any polypeptide element may be
selected which is characterized by at least 80% identity, at least
85% identity, preferably at least 90% identity, and more preferably
at least 95% identity to any of the sequences SEQ ID NOs: 297-299.
On nucleic acid level, any polynucleotide (e.g. RNA) moiety may be
selected which encodes such polypeptide element.
[0209] According to another embodiment, the artificial nucleic acid
sequence, particularly the RNA sequence according to the invention
may additionally encode at least one self-cleaving peptide. Viral
self-cleaving peptides (2A peptides) allow the expression of
multiple proteins from a single open reading frame. The terms 2A
peptide and 2A element are used interchangeably herein. The
mechanism by the 2A sequence for generating two proteins from one
transcript is by ribosome skipping--a normal peptide bond is
impaired at 2A, resulting in two discontinuous protein fragments
from one translation event. When used in the context of the present
invention, such 2A peptides are particularly useful when encoded by
a nucleic acid encoding at least two functional protein elements
(e.g. Henipavirus and/or Hendra virus and/or Nipah virus antigenic
peptides or proteins). In general, a 2A element is useful when the
nucleic acid molecule encodes at least one polypeptide or protein
of interest and at least one further protein element. In a
preferred embodiment, a 2A element is present when the
polynucleotide of the invention encodes two proteins or
polypeptides of interest, e.g. two antigens. The coding sequence
for such 2A peptide is typically located in between the coding
sequence of the polypeptide of interest and the coding sequence of
the least one further protein element (which may also be a
polypeptide of interest), so that cleavage of the 2A peptide leads
to two separate polypeptide molecules, at least one of them being a
polypeptide or protein of interest. For example, for expressing
target proteins (Henipavirus and/or Hendra virus and/or Nipah virus
antigenic peptides or proteins) that are composed of several
polypeptide chains, such as antibodies, it may be beneficial to
provide coding information for both polypeptide chains on a single
nucleic acid molecule, separated by a nucleic acid sequence
encoding a 2A peptide. 2A peptides may also be beneficial when
cleavage of the protein of interest from another encoded
polypeptide element is desired. 2A peptides may be derived from
foot-and-mouth diseases virus, from equine rhinitis A virus, Thosea
asigna virus, Porcine teschovirus-1. Suitably, the polypeptide
sequence of the 2A peptide used in the present invention may be
selected from the following list of polypeptide sequences (SEQ ID
NOs: 300-303). On nucleic acid level, particularly RNA level, any
nucleotide sequence moiety can be employed that encodes any of 2A
peptide used in the present invention. Owing to the degenerated
genetic code, in the case of most polypeptides (SEQ ID NOs:
300-303), more than one particular nucleic acid sequence is
conceivable as encoding the respective polypeptide. While each and
every such nucleic acid may generally be used in the context of the
present invention, it is preferable that the nucleic acid sequence
that encodes the polypeptide sequence is selected such that its
sequence is optimized according to the general guidance provided in
this specification. Alternatively, any polypeptide element may be
selected which is characterized by at least 80% identity, at least
85% identity, preferably at least 90% identity, and more preferably
at least 95% identity to any of the sequences SEQ ID NOs: 300-303.
On nucleic acid level, any polynucleotide (e.g. RNA) moiety may be
selected which encodes such polypeptide element.
[0210] According to another embodiment, the artificial nucleic acid
sequence, particularly the RNA sequence according to the invention
may additionally encode at least one helper peptide. In essence,
helper peptides binds to class II MHC molecules as a nonspecific
vaccine helper epitope (adjuvant) and induces an increased (and
long term) immune response by increasing the helper T-cell
response. In an embodiment, such a helper peptide may be
N-terminally and/or C-terminally fused to the antigenic peptide or
protein derived from Henipavirus, Nipah virus or Hendra virus. In
an embodiment, the helper peptide is derived from tetanus toxin,
according to SEQ ID NO: 257. On nucleic acid level, particularly
RNA level, any nucleotide sequence moiety can be employed that
encodes any helper peptide used in the present invention. Owing to
the degenerated genetic code, in the case of most polypeptides (SEQ
ID NO: 257), more than one particular nucleic acid sequence is
conceivable as encoding the respective polypeptide. While each and
every such nucleic acid may generally be used in the context of the
present invention, it is preferable that the nucleic acid sequence
that encodes the polypeptide sequence is selected such that its
sequence is optimized according to the general guidance provided in
this specification. Alternatively, any polypeptide element may be
selected which is characterized by at least 80% identity, at least
85% identity, preferably at least 90% identity, and more preferably
at least 95% identity to any of the sequences SEQ ID NO: 257. On
nucleic acid level, any polynucleotide (e.g. RNA) moiety may be
selected which encodes such polypeptide element.
[0211] Henipavirus Nucleic Acids:
[0212] In the context of the invention, the coding sequence
encoding the at least one Henipavirus antigenic peptide or protein
or fragment, variant or derivative thereof, may be selected from
any nucleic acid sequence comprising a coding sequence encoding
Henipavirus RNA-directed RNA polymerase (L), Henipavirus fusion
protein (F), Henipavirus non-structural protein (V), Henipavirus
glycoprotein (G), Henipavirus nucleoprotein (N), Henipavirus matrix
protein (M), Henipavirus phosphoprotein (P), Henipavirus protein C,
and Henipavirus protein W. In the context of the invention, said
artificial nucleic acid sequences may be derived from any
Henipavirus strain, species, serotype, subtype fragment or variant
thereof (e.g. as provided above in the section "Henipavirus").
[0213] The artificial nucleic acid of the invention may comprise or
consist of at least one coding sequence encoding at least one
Henipavirus antigenic peptide or protein as defined herein,
preferably encoding any one of SEQ ID NOs: 1-26, 573-598, 807-832,
1041-1066, 1513-1515 or fragments of variants thereof. It has to be
understood that, on nucleic acid level, any nucleic acid sequence
(e.g. DNA sequence, RNA sequence) which encodes an amino acid
sequences being identical to SEQ ID NOs: 1-26, 573-598, 807-832,
1041-1066, 1513-1515 or fragments or variants thereof, or any
nucleic acid sequence (e.g. DNA sequence, RNA sequence) which
encodes amino acid sequences being 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 fragments or variants thereof, may
be selected and may accordingly be understood as suitable coding
sequence and may therefore be comprised in the artificial nucleic
acid of the invention.
[0214] According to a preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one Henipavirus antigenic peptide
or protein as described herein. Preferably, the inventive
artificial nucleic acid comprises or consists of a coding sequence
according to any one of SEQ ID NOs: 27-234, 599-806, 833-1040,
1067-1274, 1275-1508, 1516-1539, 1540-1548 or a homolog, fragment
or variant of any of these sequences.
[0215] 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.
[0216] It is further preferred that the nucleic acid sequence
according to the invention comprises at least one coding sequence
encoding a heterologous secretory signal sequence comprising 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 nucleic acid
sequences according to SEQ ID NOs 317-572 or a fragment or variant
thereof and, in addition, at least one coding sequence encoding a
Henipavirus antigenic peptide or protein comprising 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 nucleic acid sequences
according to SEQ ID NOs: 599-806 or a fragments or variants any of
these sequences.
[0217] In this context, it is preferred that the nucleic acid
sequence according to the invention comprises at least one coding
sequence encoding a heterologous secretory signal sequence and, in
addition, at least one coding sequence encoding a Henipavirus
antigenic peptide or protein comprising 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 nucleic acid sequences according
to SEQ ID NOs: 833-1040, 1067-1274, 1516-1539 or a fragments or
variants any of these sequences.
[0218] Hendra Virus Nucleic Acids:
[0219] In the context of the invention, the coding sequence
encoding the at least one Hendra virus antigenic peptide or protein
or fragment, variant or derivative thereof, may be selected from
any nucleic acid sequence comprising a coding sequence encoding
Hendra virus RNA-directed RNA polymerase (L), Hendra virus fusion
protein (F), Hendra virus non-structural protein (V), Hendra virus
glycoprotein (G), Hendra virus nucleoprotein (N), Hendra virus
matrix protein (M), Hendra virus phosphoprotein (P), Hendra virus
protein C, and Hendra virus protein W. In the context of the
invention, said artificial nucleic acid sequences may be derived
from any Hendra virus strain, species, serotype, subtype fragment
or variant thereof.
[0220] According to a preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one Hendra virus antigenic
peptide or protein as described herein. Preferably, the inventive
artificial nucleic acid comprises or consists of a coding sequence
according to any one of 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 homolog, fragment or variant of any of these sequences.
[0221] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Hendra
virus antigenic peptide or protein comprising 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 nucleic acid sequences
according 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 a homolog, fragment
or variant of any of these sequences, or at least one of the
nucleic acid 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.
[0222] In a preferred embodiment, the present invention thus
provides artificial nucleic acid sequences comprising at least one
coding sequence, wherein the coding sequence encoding Hendra virus
fusion protein (F) comprises or consists of any one of the nucleic
acid sequences defined in Table 1 and Table 1B, a homolog, fragment
or variant of any one of these sequences.
[0223] In particularly preferred embodiments the nucleic acid
sequence comprises or consists of at least one coding sequence
encoding Hendra virus fusion protein (F) according to SEQ ID NOs:
34-37, 60-63, 86-89, 112-115, 138-141, 164-167, 190-193, 216-219,
606-609, 632-635, 658-661, 684-687, 710-713, 736-739, 762-765,
788-791, 840-843, 866-869, 892-895, 918-921, 944-947, 970-973,
996-999, 1022-1025, 1074-1077, 1100-1103, 1126-1129, 1152-1155,
1178-1181, 1204-1207, 1230-1233, 1256-1259, or a homolog, fragment
or variant of any of these sequences.
[0224] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Hendra
virus fusion protein (F) comprising 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 nucleic acid sequences according
to SEQ ID NOs: 34-37, 60-63, 86-89, 112-115, 138-141, 164-167,
190-193, 216-219, 606-609, 632-635, 658-661, 684-687, 710-713,
736-739, 762-765, 788-791, 840-843, 866-869, 892-895, 918-921,
944-947, 970-973, 996-999, 1022-1025, 1074-1077, 1100-1103,
1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259
and as disclosed in Table 1 and Table 1B.
[0225] In a preferred embodiment, the present invention thus
provides artificial nucleic acid sequences comprising at least one
coding sequence, wherein the coding sequence encoding Hendra virus
glycoprotein (G) comprises or consists of any one of the nucleic
acid sequences defined in Table 1 and Table 1B, a homolog, fragment
or variant of any one of these sequences.
[0226] In particularly preferred embodiments the nucleic acid
sequence comprises or consists of at least one coding sequence
encoding Hendra virus glycoprotein (G) according to SEQ ID NOs:
45-52, 71-78, 97-104, 123-130, 149-156, 175-182, 201-208, 227-234,
617-624, 643-650, 669-676, 695-702, 721-728, 747-754, 773-780,
799-806, 851-858, 877-884, 903-910, 929-936, 955-962, 981-988,
1007-1014, 1033-1040, 1085-1092, 1111-1118, 1137-1144, 1163-1170,
1189-1196, 1215-1222, 1241-1248, 1267-1274, or a homolog, fragment
or variant of any of these sequences.
[0227] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Hendra
virus glycoprotein (G) comprising 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 nucleic acid sequences according to SEQ ID
NOs: 45-52, 71-78, 97-104, 123-130, 149-156, 175-182, 201-208,
227-234, 617-624, 643-650, 669-676, 695-702, 721-728, 747-754,
773-780, 799-806, 851-858, 877-884, 903-910, 929-936, 955-962,
981-988, 1007-1014, 1033-1040, 1085-1092, 1111-1118, 1137-1144,
1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274, and as
disclosed in Table 1 and Table 1B.
[0228] It is further preferred that the nucleic acid sequence
according to the invention comprises at least one coding sequence
encoding a heterologous secretory signal sequence comprising 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 nucleic acid
sequences according to SEQ ID NOs: 317-572 or a fragment or variant
thereof and, in addition, at least one coding sequence encoding a
Hendra virus antigenic peptide or protein comprising 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 nucleic acid sequences
according to SEQ ID NOs: 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 or a homolog, fragment
or variant of any of these sequences.
[0229] In this context, it is preferred that the nucleic acid
sequence according to the invention comprises at least one coding
sequence encoding a heterologous secretory signal sequence and, in
addition, at least one coding sequence encoding a Hendra virus
antigenic peptide or protein comprising 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 nucleic acid sequences according
to SEQ ID NOs: 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 or a homolog, fragment or variant of any of
these sequences.
[0230] In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Hendra virus
RNA-directed RNA polymerase (L), or a fragment or variant thereof.
In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Hendra virus
non-structural protein (V), or a fragment or variant thereof. In
another embodiment the nucleic acid sequence comprises or consists
of at least one coding sequence encoding Hendra virus nucleoprotein
(N), or a fragment or variant thereof. In another embodiment the
nucleic acid sequence comprises or consists of at least one coding
sequence encoding Hendra virus matrix protein (M), or a fragment or
variant thereof. In another embodiment the nucleic acid sequence
comprises or consists of at least one coding sequence encoding
Hendra virus phosphoprotein (P), or a fragment or variant thereof.
In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Hendra virus
protein C, or a fragment or variant thereof. In another embodiment
the nucleic acid sequence comprises or consists of at least one
coding sequence encoding Hendra virus protein W, or a fragment or
variant thereof.
[0231] The inventive artificial nucleic acid encoding Hendra virus
antigenic peptide or protein, preferably the at least one coding
sequence of the artificial nucleic acid according to the invention,
may comprise or consist of a variant of a nucleic acid sequence as
defined herein, preferably of a nucleic acid sequence encoding a
protein or a fragment thereof as defined herein. The expression
"variant of a nucleic acid sequence" as used herein in the context
of a nucleic acid sequence encoding a protein or a fragment
thereof, typically refers to a nucleic acid sequence, which differs
by at least one nucleic acid residue from the respective naturally
occurring nucleic acid sequence encoding a protein or a fragment
thereof. More preferably, the expression "variant of a nucleic acid
sequence" refers to a nucleic acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with a nucleic acid sequence, from which it is derived.
[0232] Nipah Virus Nucleic Acids:
[0233] In the context of the invention, the coding sequence
encoding the at least one Nipah virus antigenic peptide or protein
or fragment, variant or derivative thereof, may be selected from
any nucleic acid sequence comprising a coding sequence encoding
Nipah virus RNA-directed RNA polymerase (L), Nipah virus fusion
protein (F), Nipah virus non-structural protein (V), Nipah virus
glycoprotein (G), Nipah virus nucleoprotein (N), Nipah virus matrix
protein (M), Nipah virus phosphoprotein (P), Nipah virus protein C,
and Nipah virus protein W. In the context of the invention, said
artificial nucleic acid sequences may be derived from any Nipah
virus strain, species, serotype, subtype fragment or variant
thereof.
[0234] According to a preferred embodiment, the inventive
artificial nucleic acid comprises or consists of at least one
coding sequence encoding at least one Nipah virus antigenic peptide
or protein as described herein. Preferably, the inventive
artificial nucleic acid comprises or consists of a coding sequence
according to any one of 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 homolog, fragment or variant of any of these
sequences.
[0235] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Nipah
virus antigenic peptide or protein comprising 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 nucleic acid sequences
according 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
a homolog, fragment or variant of any of these sequences, or at
least one of the nucleic acid 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.
[0236] In a preferred embodiment, the present invention provides
artificial nucleic acid sequences comprising at least one coding
sequence, wherein the coding sequence encoding Nipah virus fusion
protein (F) comprises or consists of any one of the nucleic acid
sequences defined in Table 2 and Table 2B, a homolog, fragment or
variant of any one of these sequences.
[0237] In particularly preferred embodiments the nucleic acid
sequence comprises or consists of at least one coding sequence
encoding Nipah virus fusion protein (F) according to SEQ ID NOs:
27-33, 53-59, 79-85, 105-111, 131-137, 157-163, 183-189, 209-215,
599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761,
781-787, 833-839, 859-865, 885-891, 911-917, 937-943, 963-969,
989-995, 1015-1021, 1067-1073, 1093-1099, 1119-1125, 1145-1151,
1171-1177, 1197-1203, 1223-1229, 1249-1255, 1516-1539 or a homolog,
fragment or variant of any of these sequences.
[0238] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Nipah
virus fusion protein (F) comprising 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 nucleic acid sequences according
to SEQ ID NOs: 27-33, 53-59, 79-85, 105-111, 131-137, 157-163,
183-189, 209-215, 599-605, 625-631, 651-657, 677-683, 703-709,
729-735, 755-761, 781-787, 833-839, 859-865, 885-891, 911-917,
937-943, 963-969, 989-995, 1015-1021, 1067-1073, 1093-1099,
1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,
1516-1539 and as disclosed in Table 2 and Table 2B.
[0239] In a preferred embodiment, the present invention provides
artificial nucleic acid sequences comprising at least one coding
sequence, wherein the coding sequence encoding Nipah virus
glycoprotein (G) comprises or consists of any one of the nucleic
acid sequences defined in Table 2 and Table 2B, a homolog, fragment
or variant of any one of these sequences.
[0240] In particularly preferred embodiments the nucleic acid
sequence comprises or consists of at least one coding sequence
encoding Nipah virus glycoprotein (G) according to SEQ ID NOs:
38-44, 64-70, 90-96, 116-122, 142-148, 168-174, 194-200, 220-226,
610-616, 636-642, 662-668, 688-694, 714-720, 740-746, 766-772,
792-798, 844-850, 870-876, 896-902, 922-928, 948-954, 974-980,
1000-1006, 1026-1032, 1078-1084, 1104-1110, 1130-1136, 1156-1162,
1182-1188, 1208-1214, 1234-1240, 1260-1266, or a homolog, fragment
or variant of any of these sequences.
[0241] Preferably, the nucleic acid sequence according to the
invention comprises at least one coding sequence encoding Nipah
virus glycoprotein (G) comprising 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 nucleic acid sequences according to SEQ ID
NOs: 38-44, 64-70, 90-96, 116-122, 142-148, 168-174, 194-200,
220-226, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,
766-772, 792-798, 844-850, 870-876, 896-902, 922-928, 948-954,
974-980, 1000-1006, 1026-1032, 1078-1084, 1104-1110, 1130-1136,
1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266, and as
disclosed in Table 2 and Table 2B.
[0242] It is further preferred that the nucleic acid sequence
according to the invention comprises at least one coding sequence
encoding a heterologous secretory signal sequence comprising 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 nucleic acid
sequences according to SEQ ID NOs: 317-572 or a fragment or variant
thereof and, in addition, at least one coding sequence encoding a
Nipah virus antigenic peptide or protein comprising 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 nucleic acid sequences
according to SEQ ID NOs: 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 or a homolog, fragment
or variant of any of these sequences.
[0243] In this context, it is preferred that the nucleic acid
sequence according to the invention comprises at least one coding
sequence encoding a heterologous secretory signal sequence and, in
addition, at least one coding sequence encoding a Nipah virus
antigenic peptide or protein comprising 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 nucleic acid sequences according
to SEQ ID NOs: 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, 1516-1539 or a homolog, fragment or variant
of any of these sequences.
[0244] In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Nipah virus
RNA-directed RNA polymerase (L), or a fragment or variant thereof.
In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Nipah virus
non-structural protein (V), or a fragment or variant thereof. In
another embodiment the nucleic acid sequence comprises or consists
of at least one coding sequence encoding Nipah virus nucleoprotein
(N), or a fragment or variant thereof. In another embodiment the
nucleic acid sequence comprises or consists of at least one coding
sequence encoding Nipah virus matrix protein (M), or a fragment or
variant thereof. In another embodiment the nucleic acid sequence
comprises or consists of at least one coding sequence encoding
Nipah virus phosphoprotein (P), or a fragment or variant thereof.
In another embodiment the nucleic acid sequence comprises or
consists of at least one coding sequence encoding Nipah virus
protein C, or a fragment or variant thereof. In another embodiment
the nucleic acid sequence comprises or consists of at least one
coding sequence encoding Nipah virus protein W, or a fragment or
variant thereof.
[0245] The inventive artificial nucleic acid encoding Nipah virus
antigenic peptide or protein, preferably the at least one coding
sequence of the artificial nucleic acid according to the invention,
may comprise or consist of a variant of a nucleic acid sequence as
defined herein, preferably of a nucleic acid sequence encoding a
protein or a fragment thereof as defined herein. The expression
"variant of a nucleic acid sequence" as used herein in the context
of a nucleic acid sequence encoding a protein or a fragment
thereof, typically refers to a nucleic acid sequence, which differs
by at least one nucleic acid residue from the respective naturally
occurring nucleic acid sequence encoding a protein or a fragment
thereof. More preferably, the expression "variant of a nucleic acid
sequence" refers to a nucleic acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with a nucleic acid sequence, from which it is derived.
[0246] In specific embodiments, the at least one coding sequence
comprises at least one of the DNA 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 any one of the
sequences SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274,
1275-1508, 1516-1539, 1540-1548 wherein the indicated uridine
nucleotides are substituted with thymidine nucleotides, or a
fragment or variant or orthologue or paralogue of any of these.
[0247] Mono-, Bi- and Multicistronic and Multi-Antigen Nucleic
Acids:
[0248] According to certain embodiments of the present invention,
the artificial nucleic acid is mono-, bi-, or multicistronic,
preferably as defined herein.
[0249] In specific embodiments the artificial nucleic acid of the
invention is monocistronic, wherein the (one) coding sequence
encodes at least two different Hendra virus and/or Nipah virus
antigenic peptides or proteins, or a fragment or variant
thereof.
[0250] The coding sequences in a bi- or multicistronic nucleic acid
molecule preferably encode distinct Henipavirus antigenic proteins
or peptides, Hendra virus antigenic proteins or peptides, Nipah
virus antigenic proteins or peptides as defined herein or a
fragment or variant thereof. Preferably, the coding sequences
encoding two or more antigenic proteins or peptides may be
separated in the bi- or multicistronic nucleic acid by at least one
IRES (internal ribosomal entry site) sequence, as defined
below.
[0251] In specific embodiments the artificial nucleic acid of the
invention 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.
[0252] Thus, the term "encoding two or more antigenic peptides or
proteins" or "encode at least two different Hendra virus and/or
Nipah virus antigenic peptides or proteins" may mean, without being
limited thereto, that the bi- or even multicistronic nucleic acid,
may encode e.g. at least two, three, four, five, six or more
(preferably different) Henipavirus antigenic peptides or proteins
and/or Hendra virus antigenic proteins or peptides and/or Nipah
virus antigenic proteins or peptides derived from different viruses
or their fragments or variants within the definitions provided
herein. More preferably, without being limited thereto, the bi- or
even multicistronic nucleic acid, may encode, for example, at least
two, three, four, five, six or more (preferably different)
Henipavirus antigenic peptides or proteins and/or Hendra virus
antigenic proteins or peptides and/or Nipah virus antigenic
proteins or peptides as defined herein or their fragments or
variants as defined herein. In this context, a so-called IRES
(internal ribosomal entry site) sequence as defined above can
function as a sole ribosome binding site, but it can also serve to
provide a bi- or even multicistronic nucleic acid as defined above,
which encodes several Henipavirus antigenic peptides or proteins
and/or Hendra virus antigenic proteins or peptides and/or Nipah
virus antigenic proteins or peptides which are to be translated by
the ribosomes independently of one another. Examples of IRES
sequences, which can be used according to the invention, are those
from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses
(PV), encephalomyocarditis viruses (ECMV), foot and mouth disease
viruses (FMDV), hepatitis C viruses (HCV), classical swine fever
viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency
viruses (Sly) or cricket paralysis viruses (CrPV).
[0253] Particular suitable IRES sequences that may be used in the
context of the invention may be IRES sequences derived from EMCV
(SEQ ID NO: 304) and IRES sequences derived from FMDV (SEQ ID NO:
305).
[0254] According to a further embodiment the at least one coding
sequence of the nucleic acid sequence according to the invention
may encode at least two, three, four, five, six, seven, eight and
more Henipavirus antigenic peptides or proteins and/or Hendra virus
antigenic proteins or peptides and/or Nipah virus antigenic
proteins or peptides (or fragments and derivatives thereof) as
defined herein linked with or without an amino acid linker
sequence, wherein said linker sequence can comprise rigid linkers,
flexible linkers, cleavable linkers (e.g., self-cleaving peptides)
or a combination thereof (see paragraph "Peptide linker elements"
of the present invention). Therein, the Henipavirus antigenic
peptides or proteins and/or Hendra virus antigenic proteins or
peptides and/or Nipah virus antigenic proteins or peptides may be
identical or different or a combination thereof. Particular
Henipavirus antigenic peptides or proteins and/or Hendra virus
antigenic proteins or peptides and/or Nipah virus antigenic
proteins or peptides combinations can be encoded by said nucleic
acid encoding at least two Henipavirus antigenic peptides or
proteins and/or Hendra virus antigenic proteins or peptides and/or
Nipah virus antigenic proteins or peptides as explained herein
(also herein referred to as "multi-antigen-constructs/nucleic
acid").
[0255] It has to be noted, that in the context of the invention,
certain combinations of coding sequences (e.g., comprising at least
two different Henipavirus antigenic peptides or proteins and/or
Hendra virus antigenic proteins or peptides and/or Nipah virus
antigenic proteins or peptides and/or comprising at least two
antigenic peptides or proteins derived from a genetically different
Henipavirus, Hendra virus, Nipah virus) may be generated by any
combination of moni- bi- and multicistronic nucleic acids and/or
multi-antigen-constructs/nucleic acid to obtain a poly- or even
multivalent nucleic acid mixture.
[0256] Preferably, the at least one coding sequence of the nucleic
acid sequence according to the invention comprises at least two,
three, four, five, six, seven, eight or more nucleic acid sequences
identical to or having a sequence identity of at least 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at
least 70%, more preferably of at least 80%, even more preferably at
least 85%, even more preferably of at least 90% and most preferably
of at least 95% or even 97%, with any one of the nucleic acid
sequences disclosed according to SEQ ID NOs: 27-234, 599-806,
833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548 or a fragment
or variant of any one of said nucleic acid sequences.
[0257] Preferably, the nucleic acid sequence comprising at least
one coding sequence as defined herein typically comprises a length
of about 50 to about 20000, or 100 to about 20000 nucleotides,
preferably of about 250 to about 20000 nucleotides, more preferably
of about 500 to about 10000, even more preferably of about 500 to
about 5000.
[0258] According to a further embodiment, the nucleic acid sequence
according to the invention is an artificial nucleic sequence as
defined herein.
[0259] RNA:
[0260] In embodiments, the artificial nucleic acid is an RNA, in
particular a circular RNA. As used herein, "circular RNA" has to be
understood as a circular polynucleotide that can encode at least
one antigenic peptide or protein as defined herein. Accordingly, in
preferred embodiments, said circular RNA comprises at least one
coding sequence encoding at least one antigenic peptide or protein
derived from a Henipavirus or a fragment or variant thereof as
defined herein.
[0261] The production of circRNAs can be performed using various
methods provided in the art. For example, U.S. Pat. No. 6,210,931
teaches a method of synthesizing circRNAs by inserting DNA
fragments into a plasmid containing sequences having the capability
of spontaneous cleavage and self-circularization. U.S. Pat. No.
5,773,244 teaches producing circRNAs by making a DNA construct
encoding an RNA cyclase ribozyme, expressing the DNA construct as
an RNA, and then allowing the RNA to self-splice, which produces a
circRNA free from intron in vitro. WO1992001813 teaches a process
of making single strand circular nucleic acids by synthesizing a
linear polynucleotide, combining the linear nucleotide with a
complementary linking oligonucleotide under hybridization
conditions, and ligating the linear polynucleotide. The person
skilled in the art may also use methods provided in WO2015034925 or
WO2016011222 to produce circular RNA. Accordingly, methods for
producing circular RNA as provided in U.S. Pat. Nos. 6,210,931,
5,773,244, WO1992001813, WO2015034925 and WO2016011222 are
incorporated herewith by reference.
[0262] In a preferred embodiment, the artificial nucleic acid is an
RNA, preferably an mRNA.
[0263] The artificial RNA according to the present invention may be
prepared using any method known in the art, including chemical
synthesis such as e.g. solid phase RNA synthesis, as well as in
vitro methods, such as RNA in vitro transcription reactions.
[0264] In a preferred embodiment, the artificial nucleic acid as
defined herein, preferably the RNA as defined herein, is obtained
by RNA in vitro transcription. Accordingly, the RNA of the
invention is preferably an in vitro transcribed RNA.
[0265] The terms "RNA in vitro transcription" or "in vitro
transcription" relate to a process wherein RNA is synthesized in a
cell-free system (in vitro) as defined above. DNA, particularly
plasmid DNA (or PCR product), is typically used as template for the
generation of RNA transcripts.
[0266] In embodiments, the nucleotide mixture used in RNA in vitro
transcription may additionally contain modified nucleotides as
defined herein. In embodiments, the nucleotide mixture (i.e. the
fraction of each nucleotide in the mixture) may be optimized for
the given RNA sequence, preferably as described WO2015/188933.
[0267] In embodiment where more than one different artificial
nucleic acid as defined herein has to be produced, e.g. where 2, 3,
4, 5, 6, 7, 8, 9, 10 or even more different artificial nucleic
acids have to be produced (e.g. encoding different antigenic
peptides, proteins of Hendra and/or Nipah virus), procedures as
described in WO2017/109134 may be suitably used.
[0268] In the context of nucleic acid vaccine production, it may be
required to provide GMP-grade RNA. GMP-grade RNA may be suitably
produced using a manufacturing process approved by regulatory
authorities. Accordingly, in a particularly preferred embodiment,
RNA production is performed under current good manufacturing
practice (GMP), implementing various quality control steps on DNA
and RNA level, according to WO2016/180430. Accordingly, the RNA of
the invention is a GMP-grade RNA, particularly a GMP-grade
mRNA.
[0269] The obtained RNA products are preferably purified using
PureMessenger.RTM. (CureVac, Tubingen, Germany; RP-HPLC according
to WO2008/077592) and/or tangential flow filtration (as described
in WO2016/193206).
[0270] In a preferred embodiment, the RNA, particularly the
purified RNA, is lyophilized according to WO2016/165831 or
WO2011/069586 to yield a temperature stable dried artificial
nucleic acid (powder) as defined herein. The RNA of the invention,
particularly the purified RNA may also be dried using spray-drying
or spray-freeze drying according to WO2016/184575 or WO2016184576
to yield a temperature stable artificial nucleic acid (powder) as
defined herein. Accordingly, in the context of manufacturing and
purifying nucleic acids, particularly RNA, the disclosures of
WO2017/109161, WO2015/188933, WO2016/180430, WO2008/077592,
WO2016/193206, WO2016/165831, WO2011/069586, WO2016/184575, and
WO2016/184576 are incorporated herewith by reference.
[0271] Accordingly, in preferred embodiments the RNA is a dried
RNA, particularly a dried mRNA.
[0272] The term "dried RNA" as used herein has to be understood as
RNA that has been lyophilized, or spray-dried, or spray-freeze
dried as defined above to obtain a temperature stable dried RNA
(powder).
[0273] Accordingly, in preferred embodiments the RNA is a purified
RNA, particularly purified mRNA.
[0274] The term "purified RNA" as used herein has to be understood
as RNA which has a higher purity after certain purification steps
(e.g. HPLC, TFF, precipitation steps) than the starting material
(e.g. in vitro transcribed RNA). Typical impurities that are
essentially not present in purified RNA comprise peptides or
proteins (e.g. enzymes derived from DNA dependent RNA in vitro
transcription, e.g. RNA polymerases, RNases, BSA, pyrophosphatase,
restriction endonuclease, DNase), spermidine, abortive RNA
sequences, RNA fragments, free nucleotides (modified nucleotides,
conventional NTPs, cap analogue), plasmid DNA fragments, buffer
components (HEPES, TRIS, MgCl.sub.2) etc. Other impurities that may
be derived from e.g. fermentation procedures comprise bacterial
impurities (bioburden, bacterial DNA) or impurities derived from
purification procedures (organic solvents etc.). Accordingly, it is
desirable in this regard for the "degree of RNA purity" to be as
close as possible to 100%. It is also desirable for the degree of
RNA purity that the amount of full length RNA transcripts is as
close as possible to 100%. Accordingly "purified RNA" as used
herein has a degree of purity of more than 70%, 75%, 80%, 85%, very
particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most
favorably 99% or more. The degree of purity may for example be
determined by an analytical HPLC, wherein the percentages provided
above correspond to the ratio between the area of the peak for the
target RNA and the total area of all peaks representing the
by-products. Alternatively, the degree of purity may for example be
determined by an analytical agarose gel electrophoresis or
capillary gel electrophoresis.
[0275] It has to be understood that "dried RNA" as defined herein
and "purified RNA" as defined herein or "GMP-grade mRNA" as defined
herein may have superior stability characteristics and improved
efficiency (e.g. better translatability of the mRNA in vivo).
[0276] Nucleic Acid Modifications:
[0277] According to a further embodiment, the nucleic acid sequence
according to the invention is a modified nucleic acid sequence,
preferably a modified RNA sequence as described herein. In this
context, a modification as defined herein preferably leads to a
stabilization of the nucleic acid sequence according to the
invention. More preferably, the invention thus provides a nucleic
acid sequence, more preferably a stabilized RNA sequence comprising
at least one coding sequence as defined herein.
[0278] According to one embodiment, the nucleic acid sequence of
the present invention may thus be provided as a "stabilized nucleic
acid sequence", preferably as a "stabilized RNA sequence", that is
to say as an nucleic acid sequence or the RNA that is essentially
resistant to in vivo degradation (e.g. by an exo- or
endo-nuclease).
[0279] Such stabilization may be effected by providing a "dried
RNA" and/or a "purified RNA" as specified herein. Alternatively or
in addition to that, such stabilization can be effected, for
example, by a modified phosphate backbone of the nucleic acid,
particularly of the RNA of the present invention. A backbone
modification in connection with the present invention is a
modification in which phosphates of the backbone of the nucleotides
contained in the nucleic acid or the RNA are chemically modified.
Nucleotides that may be preferably used in this connection contain
e.g. a phosphorothioate-modified phosphate backbone, preferably at
least one of the phosphate oxygens contained in the phosphate
backbone being replaced by a sulfur atom. Stabilized nucleic acids
or RNAs may further include, for example: non-ionic phosphate
analogues, such as, for example, alkyl and aryl phosphonates, in
which the charged phosphonate oxygen is replaced by an alkyl or
aryl group, or phosphodiesters and alkylphosphotriesters, in which
the charged oxygen residue is present in alkylated form. Such
backbone modifications typically include, without implying any
limitation, modifications from the group consisting of
methylphosphonates, phosphoramidates and phosphorothioates (e.g.
cytidine-5''-O-(1-thiophosphate)).
[0280] In the following, specific modifications are described,
which are preferably capable of "stabilizing" the RNA as defined
herein.
Chemical Modifications of Nucleic Acids:
[0281] The term "RNA modification" as used herein may refer to
chemical modifications comprising backbone modifications as well as
sugar modifications or base modifications. In this context, a
modified RNA (sequence) as defined herein may contain nucleotide
analogues/modifications, e.g. backbone modifications, sugar
modifications or base modifications. A backbone modification in
connection with the present invention is a modification, in which
phosphates of the backbone of the nucleotides contained in an RNA
as defined herein are chemically modified. A sugar modification in
connection with the present invention is a chemical modification of
the sugar of the nucleotides of the RNA as defined herein.
Furthermore, a base modification in connection with the present
invention is a chemical modification of the base moiety of the
nucleotides of the RNA. In this context, nucleotide analogues or
modifications are preferably selected from nucleotide analogues,
which are applicable for transcription and/or translation. The
modified nucleosides and nucleotides, which may be incorporated
into a modified nucleic acid or RNA as described herein, can be
modified in the sugar moiety. For example, the 2' hydroxyl group
(OH) can be modified or replaced with a number of different "oxy"
or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group
modifications include, but are not limited to, alkoxy or aryloxy
(--OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl
or sugar); polyethyleneglycols (PEG), --O(CH2CH2O)nCH2CH2OR;
"locked" nucleic acids (SNA) in which the 2' hydroxyl is connected,
e.g., by a methylene bridge, to the 4' carbon of the same ribose
sugar; and amino groups (--O-amino, wherein the amino group, e.g.,
NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino,
diarylamino, heteroarylamino, or diheteroaryl amino, ethylene
diamine, polyamino) or aminoalkoxy. "Deoxy" modifications include
hydrogen, amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diary) amino, heteroaryl amino, diheteroaryl amino, or
amino acid); or the amino group can be attached to the sugar
through a linker, wherein the linker comprises one or more of the
atoms C, N, and 0. The sugar group can also contain one or more
carbons that possess the opposite stereochemical configuration than
that of the corresponding carbon in ribose. Thus, a modified RNA
can include nucleotides containing, for instance, arabinose as the
sugar. The phosphate backbone may further be modified in the
modified nucleosides and nucleotides, which may be incorporated
into a modified nucleic acid or a modified RNA as described herein.
The phosphate groups of the backbone can be modified by replacing
one or more of the oxygen atoms with a different substituent.
Further, the modified nucleosides and nucleotides can include the
full replacement of an unmodified phosphate moiety with a modified
phosphate as described herein. Examples of modified phosphate
groups include, but are not limited to, phosphorothioate,
phosphoroselenates, borano phosphates, borano phosphate esters,
hydrogen phosphonates, phosphoroamidates, alkyl or aryl
phosphonates and phosphotriesters. Phosphorodithioates have both
non-linking oxygens replaced by sulfur. The phosphate linker can
also be modified by the replacement of a linking oxygen with
nitrogen (bridged phosphoroamidates), sulfur (bridged
phosphorothioates) and carbon (bridged methylene-phosphonates). The
modified nucleosides and nucleotides, which may be incorporated
into a modified nucleic acid or particularly into a modified RNA as
described herein, can further be modified in the nucleobase moiety.
Examples of nucleobases particularly found in RNA include, but are
not limited to, adenine, guanine, cytosine and uracil. For example,
the nucleosides and nucleotides described herein can be chemically
modified on the major groove face. In some embodiments, the major
groove chemical modifications can include an amino group, a thiol
group, an alkyl group, or a halo group. In particularly preferred
embodiments of the present invention, the nucleotide
analogues/modifications are selected from base modifications, which
are preferably selected from
2-amino-6-chloropurineriboside-5''-triphosphate,
2-Aminopurine-riboside-5'-triphosphate;
2-aminoadenosine-5'-triphosphate,
2''-Amino-2'-deoxycytidine-triphosphate,
2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate,
2''-Fluorothymidine-5'-triphosphate,
2'-O-Methyl-inosine-5'-triphosphate 4-thiouridine-5'-triphosphate,
5-aminoallylcytidine-5'-triphosphate,
5-aminoallyluridine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, 5-bromouridine-5''-triphosphate,
5-Bromo-2'-deoxycytidine-5''-triphosphate,
5-Bromo-2'-deoxyuridine-5''-triphosphate,
5-iodocytidine-5'-triphosphate, 5-lodo-2%
deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate,
5-lodo-2'-deoxyuridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 5-methyluridine-5''-triphosphate,
5-Propynyl-2'-deoxycytidine-5'-triphosphate,
5-Propynyl-2'-deoxyuridine-5''-triphosphate,
6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate,
6-chloropurineriboside-5''-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N1-methyladenosine-5'-triphosphate,
N1-methylguanosine-5'-triphosphate,
N6-methyladenosine-5''-triphosphate,
06-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate,
or puromycin-5''-triphosphate, xanthosine-5'-triphosphate.
Particular preference is given to nucleotides for base
modifications selected from the group of base-modified nucleotides
consisting of 5-methylcytidine-5'-triphosphate,
7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate,
and pseudouridine-5'-triphosphate. In some embodiments, modified
nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine,
2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,
5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyluridine, 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine,
5-methyl-uridine, 1-methyl-pseudouridine,
4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine. In some embodiments, modified
nucleosides include 5-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,
N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine. In other embodiments,
modified nucleosides include 2-aminopurine, 2, 6-diaminopurine,
7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine. In other embodiments, modified nucleosides
include inosine, 1-methyl-inosine, wyosine, wybutosine,
7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0282] In some embodiments, the nucleotide can be modified on the
major groove face and can include replacing hydrogen on C-5 of
uracil with a methyl group or a halo group. In specific
embodiments, a modified nucleoside is
5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine,
5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or
5'-O-(1-thiophosphate)-pseudouridine. In further specific
embodiments, nucleoside modifications selected from 6-aza-cytidine,
2-thio-cytidine, .alpha.-thio-cytidine, Pseudo-iso-cytidine,
5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine,
5,6-dihydrouridine, .alpha.-thio-uridine, 4-thio-uridine,
6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine,
5-methyl-uridine, Pyrrolo-cytidine, inosine,
.alpha.-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,
8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,
2-amino-6-Chloro-purine, N6-methyl-2-amino-purine,
Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine,
.alpha.-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.
[0283] Particularly preferred and suitable in the context of the
invention are pseudouridine (.psi.), N1-methylpseudouridine
(m1.psi.), 5-methylcytosine, and 5-methoxyuridine. Accordingly, the
artificial nucleic acid as defined herein may comprise at least one
modified nucleotide selected from pseudouridine (.psi.),
N1-methylpseudouridine (m1.psi.), 5-methylcytosine, and
5-methoxyuridine.
[0284] According to a further embodiment, a modified nucleic acid,
particularly a modified RNA as defined herein can contain a lipid
modification. Such a lipid-modified nucleic acid typically
comprises a nucleic acid as defined herein. Such a lipid-modified
nucleic acid or RNA as defined herein typically further comprises
at least one linker covalently linked with that nucleic acid or
RNA, and at least one lipid covalently linked with the respective
linker. Alternatively, the lipid-modified nucleic acid comprises at
least one nucleic acid as defined herein and at least one
(bifunctional) lipid covalently linked (without a linker) with that
nucleic acid. According to a third alternative, the lipid-modified
nucleic acid comprises an nucleic acid molecule as defined herein,
at least one linker covalently linked with that RNA, and at least
one lipid covalently linked with the respective linker, and also at
least one (bifunctional) lipid covalently linked (without a linker)
with that nucleic acid. In this context, it is particularly
preferred that the lipid modification is present at the terminal
ends of a linear nucleic acid sequence.
Sequence Modified Henipavirus Sequences:
[0285] According to preferred embodiments, the artificial nucleic
acid of the invention may be sequence-modified. Accordingly, in
embodiments, 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 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 the codons in the at
least one coding sequence are adapted to human codon usage, and/or
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.
[0286] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Henipavirus, wherein the at least
one coding sequence comprises a (sequence modified) 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.
[0287] Sequence Modified Hendra Virus Sequences:
[0288] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Hendra virus, comprising at least
one coding sequence, wherein the coding sequence comprises or
consists of any one of the (modified) RNA sequences as defined in
the columns "C-J" of Table 1 and Table 1B, or of a fragment or
variant of any one of these sequences.
[0289] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus, comprises
or consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (modified) RNA sequences as defined
in columns "C-J" of Table 1 and Table 1B, or of a fragment or
variant of any one of these sequences.
[0290] According to a particularly preferred embodiment, the at
least one coding sequence of the nucleic acid sequence, encoding at
least one antigenic peptide or protein derived from Hendra virus,
comprises or consists of an RNA sequence having a sequence identity
of at least 80% with any one of the (modified) RNA sequences as
defined in the columns "C-J" of Table 1 and Table 1B, or of a
fragment or variant of any one of these sequences.
[0291] Sequence Modified Nipah Virus Sequences:
[0292] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Nipah virus, comprising at least
one coding sequence, wherein the coding sequence comprises or
consists of any one of the (modified) RNA sequences as defined in
the columns "C-J" of Table 2 and columns "C-J" of Table 2B, or of a
fragment or variant of any one of these sequences.
[0293] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Nipah virus, comprises or
consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (modified) RNA sequences as defined
in columns "C-J" of Table 2 and in columns "C-J" of Table 2B, or of
a fragment or variant of any one of these sequences.
[0294] According to a particularly preferred embodiment, the at
least one coding sequence of the nucleic acid sequence, encoding at
least one antigenic peptide or protein derived from Nipah virus,
comprises or consists of an RNA sequence having a sequence identity
of at least 80% with any one of the (modified) RNA sequences
according as defined in the columns "C-J" of Table 2 and in columns
"C-J" of Table 2B, or of a fragment or variant of any one of these
sequences.
[0295] G/C Content Modification:
[0296] According to an embodiment, the nucleic acid sequence of the
present invention, may be modified, and thus stabilized, by
modifying the guanosine/cytosine (G/C) content of the nucleic acid
sequence, preferably of the at least one coding sequence of the
nucleic acid sequence of the present invention.
[0297] In a particularly preferred embodiment of the present
invention, the G/C content of the coding sequence of the nucleic
acid sequence of the present invention is modified, particularly
increased, compared to the G/C content of the coding sequence of
the respective wild type nucleic acid sequence, i.e. the unmodified
nucleic acid. The amino acid sequence encoded by the nucleic acid
is preferably not modified as compared to the amino acid sequence
encoded by the respective wild type nucleic acid. This modification
of the nucleic acid sequence of the present invention is based on
the fact that the sequence of any nucleic acid region, particularly
the sequence of any RNA region to be translated is important for
efficient translation of that nucleic acid, particularly of that
RNA. Thus, the composition of the nucleic acid and the sequence of
various nucleotides are important. In particular, in case of RNA,
sequences having an increased G (guanosine)/C (cytosine) content
are more stable than sequences having an increased A (adenosine)/U
(uracil) content. According to the invention, the codons of the
nucleic acid are therefore varied compared to the respective wild
type nucleic acid, while retaining the translated amino acid
sequence, such that they include an increased amount of G/C
nucleotides. In respect to the fact that several codons code for
one and the same amino acid (so-called degeneration of the genetic
code), the most favourable codons for the stability can be
determined (so-called alternative codon usage). Depending on the
amino acid to be encoded by the nucleic acid, there are various
possibilities for modification of the nucleic acid sequence,
compared to its wild type sequence.
[0298] The following modifications may apply for RNA molecules, but
may also be transferrable to DNA molecules: In the case of amino
acids, which are encoded by codons, containing exclusively G or C
nucleotides, no modification of the codon is necessary. Thus, the
codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and
Gly (GGC or GGG) require no modification, since no A or U is
present. In contrast, codons which contain A and/or U nucleotides
can be modified by substitution of other codons, which code for the
same amino acids but contain no A and/or U. Examples of these are:
the codons for Pro can be modified from CCU or CCA to CCC or CCG;
the codons for Arg can be modified from CGU or CGA or AGA or AGG to
CGC or CGG; the codons for Ala can be modified from GCU or GCA to
GCC or GCG; the codons for Gly can be modified from GGU or GGA to
GGC or GGG. In other cases, although A or U nucleotides cannot be
eliminated from the codons, it is however possible to decrease the
A and U content by using codons which contain a lower content of A
and/or U nucleotides. Examples of these are: the codons for Phe can
be modified from UUU to UUC; the codons for Leu can be modified
from UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can be
modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for
Tyr can be modified from UAU to UAC; the codon for Cys can be
modified from UGU to UGC; the codon for His can be modified from
CAU to CAC; the codon for Gln can be modified from CAA to CAG; the
codons for Ile can be modified from AUU or AUA to AUC; the codons
for Thr can be modified from ACU or ACA to ACC or ACG; the codon
for Asn can be modified from AAU to AAC; the codon for Lys can be
modified from AAA to AAG; the codons for Val can be modified from
GUU or GUA to GUC or GUG; the codon for Asp can be modified from
GAU to GAC; the codon for Glu can be modified from GAA to GAG; the
stop codon UAA can be modified to UAG or UGA. In the case of the
codons for Met (AUG) and Trp (UGG), on the other hand, there is no
possibility of sequence modification. The substitutions listed
above can be used either individually or in all possible
combinations to increase the G/C content of the RNA sequence of the
present invention compared to its particular wild type RNA (i.e.
the original sequence). Thus, for example, all codons for Thr
occurring in the wild type sequence can be modified to ACC (or
ACG). Preferably, however, for example, combinations of the above
substitution possibilities are used: substitution of all codons
coding for Thr in the original sequence (wild type RNA) to ACC (or
ACG) and substitution of all codons originally coding for Ser to
UCC (or UCG or AGC); substitution of all codons coding for Ile in
the original sequence to AUC and substitution of all codons
originally coding for Lys to AAG and substitution of all codons
originally coding for Tyr to UAC; substitution of all codons coding
for Val in the original sequence to GUC (or GUG) and substitution
of all codons originally coding for Glu to GAG and substitution of
all codons originally coding for Ala to GCC (or GCG) and
substitution of all codons originally coding for Arg to CGC (or
CGG); substitution of all codons coding for Val in the original
sequence to GUC (or GUG) and substitution of all codons originally
coding for Glu to GAG and substitution of all codons originally
coding for Ala to GCC (or GCG) and substitution of all codons
originally coding for Gly to GGC (or GGG) and substitution of all
codons originally coding for Asn to MC; substitution of all codons
coding for Val in the original sequence to GUC (or GUG) and
substitution of all codons originally coding for Phe to UUC and
substitution of all codons originally coding for Cys to UGC and
substitution of all codons originally coding for Leu to CUG (or
CUC) and substitution of all codons originally coding for Gln to
CAG and substitution of all codons originally coding for Pro to CCC
(or CCG); etc.
[0299] Preferably, the G/C content of the coding sequence of the
RNA sequence of the present invention is increased by at least 7%,
more preferably by at least 15%, particularly preferably by at
least 20%, compared to the GIC content of the coding sequence of
the wild type RNA, which codes for an NIPAH virus antigen as
defined herein or a fragment or variant thereof. According to a
specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more
preferably at least 70%, even more preferably at least 80% and most
preferably at least 90%, 95% or even 100% of the substitutable
codons in the region coding for a peptide or protein as defined
herein or a fragment or variant thereof or the whole sequence of
the wild type RNA sequence are substituted, thereby increasing the
GC/content of said sequence. In this context, it is particularly
preferable to increase the GIC content of the RNA sequence of the
present invention, preferably of the at least one coding sequence
of the RNA sequence according to the invention, to the maximum
(i.e. 100% of the substitutable codons) as compared to the wild
type sequence. According to the invention, a further preferred
modification of the RNA sequence of the present invention is based
on the finding that the translation efficiency is also determined
by a different frequency in the occurrence of tRNAs in cells. Thus,
if so-called "rare codons" are present in the RNA sequence of the
present invention to an increased extent, the corresponding
modified RNA sequence is translated to a significantly poorer
degree than in the case where codons coding for relatively
"frequent" tRNAs are present. According to the invention, in the
modified RNA sequence of the present invention, the region which
codes for a peptide or protein as defined herein or a fragment or
variant thereof is modified compared to the corresponding region of
the wild type RNA sequence such that at least one codon of the wild
type sequence, which codes for a tRNA which is relatively rare in
the cell, is exchanged for a codon, which codes for a tRNA which is
relatively frequent in the cell and carries the same amino acid as
the relatively rare tRNA. By this modification, the sequence of the
RNA of the present invention is modified such that codons for which
frequently occurring tRNAs are available are inserted. In other
words, according to the invention, by this modification all codons
of the wild type sequence, which code for a tRNA which is
relatively rare in the cell, can in each case be exchanged for a
codon, which codes for a tRNA which is relatively frequent in the
cell and which, in each case, carries the same amino acid as the
relatively rare tRNA. Which tRNAs occur relatively frequently in
the cell and which, in contrast, occur relatively rarely is known
to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet.
Dev. 2001, 11(6): 660-666. The codons, which use for the particular
amino acid the tRNA which occurs the most frequently, e.g. the Gly
codon, which uses the tRNA, which occurs the most frequently in the
(human) cell, are particularly preferred. According to the
invention, it is particularly preferable to link the sequential GIC
content which is increased, in particular maximized, in the
modified RNA sequence of the present invention, with the "frequent"
codons without modifying the amino acid sequence of the protein
encoded by the coding sequence of the RNA sequence. This preferred
embodiment allows provision of a particularly efficiently
translated and stabilized (modified) RNA sequence of the present
invention. The determination of a modified RNA sequence of the
present invention as described above (increased G/C content;
exchange of tRNAs) can be carried out using the computer program
explained in WO 02/098443--the disclosure content of which is
included in its full scope in the present invention. Using this
computer program, the nucleotide sequence of any desired RNA
sequence can be modified with the aid of the genetic code or the
degenerative nature thereof such that a maximum G/C content
results, in combination with the use of codons which code for tRNAs
occurring as frequently as possible in the cell, the amino acid
sequence coded by the modified RNA sequence preferably not being
modified compared to the non-modified sequence. Alternatively, it
is also possible to modify only the G/C content or only the codon
usage compared to the original sequence. The source code in Visual
Basic 6.0 (development environment used: Microsoft Visual Studio
Enterprise 6.0 with Servicepack 3) is also described in WO
02/098443. In a further preferred embodiment of the present
invention, the A/U content in the environment of the ribosome
binding site of the RNA sequence of the present invention is
increased compared to the A/U content in the environment of the
ribosome binding site of its respective wild type RNA. This
modification (an increased A/U content around the ribosome binding
site) increases the efficiency of ribosome binding to the RNA. An
effective binding of the ribosomes to the ribosome binding site
(Kozak sequence: SEQ ID NO: 255, 256; the AUG forms the start
codon) in turn has the effect of an efficient translation of the
RNA. According to a further embodiment of the present invention,
the RNA sequence of the present invention may be modified with
respect to potentially destabilizing sequence elements.
Particularly, the coding sequence and/or the 5' and/or 3'
untranslated region of this RNA sequence may be modified compared
to the respective wild type RNA such that it contains no
destabilizing sequence elements, the encoded amino acid sequence of
the modified RNA sequence preferably not being modified compared to
its respective wild type RNA. It is known that, for example in
sequences of eukaryotic RNAs, destabilizing sequence elements (DSE)
occur, to which signal proteins bind and regulate enzymatic
degradation of RNA in vivo. For further stabilization of the
modified RNA sequence, optionally in the region which encodes at
least one peptide or protein as defined herein or a fragment or
variant thereof, one or more such modifications compared to the
corresponding region of the wild type RNA can therefore be carried
out, so that no or substantially no destabilizing sequence elements
are contained there. According to the invention, DSE present in the
untranslated regions (3'- and/or 5'-UTR) can also be eliminated
from the RNA sequence of the present invention by such
modifications. Such destabilizing sequences are e.g. AU-rich
sequences (AURES), which occur in 3'-UTR sections of numerous
unstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:
1670 to 1674). The RNA sequence of the present invention is
therefore preferably modified compared to the respective wild type
RNA such that the RNA sequence of the present invention contains no
such destabilizing sequences. This also applies to those sequence
motifs which are recognized by possible endonucleases, e.g. the
sequence GAACAAG, which is contained in the 3'-UTR segment of the
gene encoding the transferrin receptor (Binder et al., EMBO J.
1994, 13: 1969 to 1980). These sequence motifs are also preferably
removed in the RNA sequence of the present invention.
[0300] G/C Content Modified Henipavirus Sequences:
[0301] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Henipavirus, wherein the at least
one coding sequence comprises a (G/C modified) 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 any one of the (modified) RNA sequences as defined in
the columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 1, Table 1B,
Table 2, and Table 2B.
[0302] According to a particularily preferred embodiment, the
artificial nucleic acid according to the invention comprises at
least one coding sequence, 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 53-78, 625-635,
859-869, 1093-1103, 636-650, 870-884, 1104-1118, 1275-1508,
1519-1521 and as defined in columns "C" (opt1) of Table 1, Table
1B, Table 2, and Table 2B, or a fragment or variant of any of these
sequences.
[0303] G/C Content Modified Hendra Virus Sequences:
[0304] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Hendra virus, comprising at least
one coding sequence, wherein the coding sequence comprises or
consists of any one of the (GIC modified) RNA sequences as defined
in columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 1 and columns
"C, G-J" (opt1, opt5, opt6, opt7) of Table 1B, or of a fragment or
variant of any one of these sequences.
[0305] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (G/C modified) RNA sequences according to SEQ ID
NOs: 60-63, 164-167, 190-193, 216-219, 632-635, 866-869, 1100-1103,
736-739, 970-973, 1204-1207, 762-765, 996-999, 1230-1233, 788-791,
1022-1025, 1256-1259 and as defined in columns "C, G-J" (opt1,
opt5, opt6, opt7) of Table 1 and columns "C, G-J" (opt1, opt5,
opt6, opt7) of Table 1B, or of a fragment or variant of any one of
these sequences.
[0306] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (G/C modified) RNA sequences according to SEQ ID NOs: 71-78,
175-182, 201-208, 227-234, 643-650, 877-884, 1111-1118, 747-754,
981-988, 1215-1222, 773-780, 1007-1014, 1241-1248, 799-806,
1033-1040, 1267-1274 and as defined in columns "C, G-J" (opt1,
opt5, opt6, opt7) of Table 1 and columns "C, G-J" (opt1, opt5,
opt6, opt7) of Table 1B, or of a fragment or variant of any one of
these sequences.
[0307] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus, comprises
or consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (G/C modified) RNA sequences as
defined in columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 1 and
columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 1B, or of a
fragment or variant of any one of these sequences.
[0308] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Hendra virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (G/C modified) RNA
sequences according as defined in columns "C, G-J" ("opt1, opt5,
opt6, opt7") of Table 1 and columns "C, G-J" (opt1, opt5, opt6,
opt7) of Table 1B, or of a fragment or variant of any one of these
sequences.
[0309] G/C Content Modified Nipah Virus Sequences:
[0310] According to a preferred embodiment, the present invention
provides a nucleic acid sequence, encoding at least one antigenic
peptide or protein derived from Nipah virus, comprising at least
one coding sequence, wherein the coding sequence comprises or
consists of any one of the (G/C modified) RNA sequences as defined
in columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 2 and columns
"C, G-J" (opt1, opt5, opt6, opt7) of Table 2B, or of a fragment or
variant of any one of these sequences.
[0311] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (G/C modified) RNA sequences according to SEQ ID
NOs: 53-59, 157-163, 183-189, 209-215, 625-631, 859-865, 1093-1099,
729-735, 963-969, 1197-1203, 755-761, 989-995, 1223-1229, 781-787,
1015-1021, 1249-1255, 1519-1521, 1531-1539 and as defined in
columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 2 and columns
"C, G-J" (opt1, opt5, opt6, opt7) of Table 2B, or of a fragment or
variant of any one of these sequences.
[0312] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (G/C modified) RNA sequences according to SEQ ID NOs: 64-70,
168-174, 194-200, 220-226, 636-642, 870-876, 1104-1110, 740-746,
974-980, 1208-1214, 766-772, 1000-1006, 1234-1240 and as defined in
columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 2 and columns
"C, G-J" (opt1, opt5, opt6, opt7) of Table 2B, or of a fragment or
variant of any one of these sequences.
[0313] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Nipah virus, comprises or
consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (G/C modified) RNA sequences as
defined in columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 2 and
columns "C, G-J" (opt1, opt5, opt6, opt7) of Table 2B, or of a
fragment or variant of any one of these sequences.
[0314] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Nipah virus,
according to the invention comprises or consists of an nucleic acid
sequence having a sequence identity of at least 80% with any one of
the (G/C modified) RNA sequences as defined in columns "C, G-J"
(opt1, opt5, opt6, opt7) of Table 2 and columns "C, G-J" (opt1,
opt5, opt6, opt7) of Table 2B, or of a fragment or variant of any
one of these sequences.
[0315] Sequence Adaptation to Human Codon Usage:
[0316] According to the invention, a further preferred modification
of the nucleic acid sequence of the present invention is based on
the finding that codons encoding the same amino acid typically
occur at different frequencies. According to the invention, in the
modified nucleic acid sequence of the present invention, the coding
sequence as defined herein is preferably modified compared to the
corresponding coding sequence of the respective wild type nucleic
acid such that the frequency of the codons encoding the same amino
acid corresponds to the naturally occurring frequency of that codon
according to the human codon usage as e.g. shown in Table 4.
[0317] For example, in the case of the amino acid alanine (Ala)
present in an amino acid sequence encoded by the at least one
coding sequence of the a nucleic acid sequence according to the
invention, the wild type coding sequence is preferably adapted in a
way that the codon "GCC" is used with a frequency of 0.40, the
codon "GCT" is used with a frequency of 0.28, the codon "GCA" is
used with a frequency of 0.22 and the codon "GCG" is used with a
frequency of 0.10 etc. (see Table 4).
TABLE-US-00006 TABLE 4 Human codon usage table Amino Amino acid
codon fraction /1000 acid codon fraction /1000 Ala GCG 0.10 7.4 Pro
CCG 0.11 6.9 Ala GCA 0.22 15.8 Pro CCA 0.27 16.9 Ala GCT 0.28 18.5
Pro CCT 0.29 17.5 Ala GCC* 0.40 27.7 Pro CCC* 0.33 19.8 Cys TGT
0.42 10.6 Gln CAG* 0.73 34.2 Cys TGC* 0.58 12.6 Gln CAA 0.27 12.3
Asp GAT 0.44 21.8 Arg AGG 0.22 12.0 Asp GAC* 0.56 25.1 Arg AGA*
0.21 12.1 Glu GAG* 0.59 39.6 Arg CGG 0.19 11.4 Glu GAA 0.41 29.0
Arg CGA 0.10 6.2 Phe TTT 0.43 17.6 Arg CGT 0.09 4.5 Phe TTC* 0.57
20.3 Arg CGC 0.19 10.4 Gly GGG 0.23 16.5 Ser AGT 0.14 12.1 Gly GGA
0.26 16.5 Ser AGC* 0.25 19.5 Gly GGT 0.18 10.8 Ser TCG 0.06 4.4 Gly
GGC* 0.33 22.2 Ser TCA 0.15 12.2 His CAT 0.41 10.9 Ser TCT 0.18
15.2 His CAC* 0.59 15.1 Ser TCC 0.23 17.7 Ile ATA 0.14 7.5 Thr ACG
0.12 6.1 Ile ATT 0.35 16.0 Thr ACA 0.27 15.1 Ile ATC* 0.52 20.8 Thr
ACT 0.23 13.1 Lys AAG* 0.60 31.9 Thr ACC* 0.38 18.9 Lys AAA 0.40
24.4 Val GTG* 0.48 28.1 Leu TTG 0.12 12.9 Val GTA 0.10 7.1 Leu TTA
0.06 7.7 Val GTT 0.17 11.0 Leu CTG* 0.43 39.6 Val GTC 0.25 14.5 Leu
CTA 0.07 7.2 Trp TGG* 1 13.2 Leu CTT 0.12 13.2 Tyr TAT 0.42 12.2
Leu CTC 0.20 19.6 Tyr TAC* 0.58 15.3 Met ATG* 1 22.0 Stop TGA* 0.61
1.6 Asn AAT 0.44 17.0 Stop TAG 0.17 0.8 Asn AAC* 0.56 19.1 Stop TAA
0.22 1.0 *most frequent codon
[0318] Human Codon Usage Adapted Hendra Virus Sequences:
[0319] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Hendra virus, wherein the coding sequence comprises or
consists of any one of the (human codon usage adapted) RNA
sequences as defined in column "E" (opt3) of Table 1 and column "E"
(opt3) of Table 1B, or of a fragment or variant of any one of these
sequences.
[0320] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (human codon usage adapted) RNA sequences according
to SEQ ID NOs: 112-115, 684-687, 918-921, 1152-1155, and as defined
in column "E" (opt3) of Table 1 and column "E" (opt3) of Table 1B,
or of a fragment or variant of any one of these sequences.
[0321] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (human codon usage adapted) RNA sequences according to SEQ
ID NOs: 123-130, 695-702, 929-936, 1163-1170 and as defined in
column "E" (opt3) of Table 1 and column "E" (opt3) of Table 1B, or
of a fragment or variant of any one of these sequences.
[0322] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus, comprises
or consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (human codon usage adapted) RNA
sequences as defined in column "E" (opt3) of Table 1 and column "E"
(opt3) of Table 1B, or of a fragment or variant of any one of these
sequences.
[0323] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Hendra virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (human codon usage
adapted) RNA sequences as defined in column "E" (opt3) of Table 1
and column "E" (opt3) of Table 1B, or of a fragment or variant of
any one of these sequences.
[0324] Human Codon Usage Adapted Nipah Virus Sequences:
[0325] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Nipah virus, wherein the coding sequence comprises or
consists of any one of the (human codon usage adapted) RNA
sequences as defined in column "E" (opt3) of Table 2 and column "E"
(opt3) of Table 2B, or of a fragment or variant of any one of these
sequences.
[0326] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (human codon usage adapted) RNA sequences according
to SEQ ID NOs: 105-111, 677-683, 911-917, 1145-1151, 1525-1527 and
as defined in column "E" (opt3) of Table 2 and column "E" (opt3) of
Table 2B, or of a fragment or variant of any one of these
sequences.
[0327] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (human codon usage adapted) RNA sequences according to SEQ
ID NOs: 116-122, 688-694, 922-928, 1156-1162 and as defined in
column "E" (opt3) of Table 2 and column "E" (opt3) of Table 2B, or
of a fragment or variant of any one of these sequences.
[0328] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Nipah virus, comprises or
consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (human codon usage adapted) RNA
sequences as defined in column "E" (opt3) of Table 2 and column "E"
(opt3) of Table 2B, or of a fragment or variant of any one of these
sequences.
[0329] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Nipah virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (human codon usage
adapted) RNA sequences as defined in column "E" (opt3) of Table 2
and column "E" (opt3) of Table 2B, or of a fragment or variant of
any one of these sequences.
[0330] Codon-Optimization (CAI Maximization):
[0331] As described above it is preferred according to the
invention, that all codons of the wild type sequence which code for
a tRNA, which is relatively rare in the cell, are exchanged for a
codon which codes for a tRNA, which is relatively frequent in the
cell and which, in each case, carries the same amino acid as the
relatively rare tRNA. Therefore it is particularly preferred that
the most frequent codons are used for each encoded amino acid (see
Table 4, most frequent codons are marked with asterisks). Such an
optimization procedure increases the codon adaptation index (CAI)
and ultimately maximises the CAI. In the context of the invention,
sequences with increased or maximized CAI are typically referred to
as "codon-optimized" sequences and/or CAI increased and/or
maximized sequences. According to a preferred embodiment, the
nucleic acid sequence of the present invention comprises at least
one coding sequence, wherein the coding sequence/sequence is
codon-optimized as described herein. More preferably, the codon
adaptation index (CAI) of the at least one coding sequence is at
least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most
preferably, the codon adaptation index (CAI) of the at least one
coding sequence is 1.
[0332] For example, in the case of the amino acid alanine (Ala)
present in the amino acid sequence encoded by the at least one
coding sequence of the nucleic acid sequence according to the
invention, the wild type coding sequence is adapted in a way that
the most frequent human codon "GCC" is always used for said amino
acid, or for the amino acid Cysteine (Cys), the wild type sequence
is adapted in a way that the most frequent human codon "TGC" is
always used for said amino acid etc.
[0333] Codon Optimized Hendra Virus Sequences:
[0334] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Hendra virus, wherein the coding sequence comprises or
consists of any one of the (codon optimized) RNA sequences as
defined in column "F" (opt4) of Table 1 and column "F" (opt4) of
Table 1B, or of a fragment or variant of any one of these
sequences.
[0335] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (codon optimized) RNA sequences according to SEQ ID
NOs: 138-141, 710-713, 944-947, 1178-1181 and as defined in columns
"F" ("opt4") of Table 1 and and columns "F" ("opt4") of Table 1B,
or of a fragment or variant of any one of these sequences.
[0336] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (codon optimized) RNA sequences according to SEQ ID NOs:
149-156, 721-728, 955-962, 1189-1196 and as defined in columns "F"
("opt4") of Table 1 and columns "F" ("opt4") of Table 1B, or of a
fragment or variant of any one of these sequences.
[0337] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus, comprises
or consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (codon optimized) RNA sequences as
defined in column "F" (opt4) of Table 1 and column "F" (opt4) of
Table 1B, or of a fragment or variant of any one of these
sequences.
[0338] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Hendra virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (codon optimized) RNA
sequences as defined in column "F" (opt4) of Table 1 and column "F"
(opt4) of Table 1B, or of a fragment or variant of any one of these
sequences.
[0339] Codon Optimized Nipah Virus Sequences:
[0340] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Nipah virus, wherein the coding sequence comprises or
consists of any one of the (codon optimized) RNA sequences as
defined in column "F" (opt4) of Table 2 and column "F" (opt4) of
Table 2B, or of a fragment or variant of any one of these
sequences.
[0341] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (codon optimized) RNA sequences according to SEQ ID
NOs: 131-137, 703-709, 937-943, 1171-1177, 1528-1530 and as defined
in column "F" (opt4) of Table 2 and column "F" (opt4) of Table 2B,
or of a fragment or variant of any one of these sequences.
[0342] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (codon optimized) RNA sequences according to SEQ ID NOs:
142-148, 714-720, 948-954, 1182-1188 and as defined in column "F"
(opt4) of Table 2 and column "F" (opt4) of Table 2B, or of a
fragment or variant of any one of these sequences.
[0343] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Nipah virus, comprises or
consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (codon optimized) RNA sequences
according as defined in column "F" (opt4) of Table 2 and column "F"
(opt4) of Table 2B, or of a fragment or variant of any one of these
sequences.
[0344] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Nipah virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (codon optimized) RNA
sequences as defined in column "F" (opt4) of Table 2 and column "F"
(opt4) of Table 2B, or of a fragment or variant of any one of these
sequences.
[0345] Cytosine Optimization:
[0346] According to another embodiment, the nucleic acid sequence
of the present invention may be modified by modifying, preferably
increasing, the cytosine (C) content of the nucleic acid sequence,
preferably of the coding sequence of the nucleic acid sequence,
more preferably the coding sequence of the RNA sequence.
[0347] In a particularly preferred embodiment of the present
invention, the C content of the coding sequence of the nucleic acid
sequence of the present invention is modified, preferably
increased, compared to the C content of the coding sequence of the
respective wild type nucleic acid, i.e. the unmodified nucleic
acid. The amino acid sequence encoded by the at least one coding
sequence of the nucleic acid sequence of the present invention is
preferably not modified as compared to the amino acid sequence
encoded by the respective wild type nucleic acid.
[0348] In a preferred embodiment of the present invention, the
modified nucleic acid, particularly the modified RNA sequence is
modified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or
80%, or at least 90% of the theoretically possible maximum
cytosine-content or even a maximum cytosine-content is
achieved.
[0349] In further preferred embodiments, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or even 100% of the codons of the
target nucleic acid, particularly the modified RNA wild type
sequence, which are "cytosine content optimizable" are replaced by
codons having a higher cytosine-content than the ones present in
the wild type sequence.
[0350] In a further preferred embodiment, some of the codons of the
wild type coding sequence may additionally be modified such that a
codon for a relatively rare tRNA in the cell is exchanged by a
codon for a relatively frequent tRNA in the cell, provided that the
substituted codon for a relatively frequent tRNA carries the same
amino acid as the relatively rare tRNA of the original wild type
codon. Preferably, all of the codons for a relatively rare tRNA are
replaced by a codon for a relatively frequent tRNA in the cell,
except codons encoding amino acids, which are exclusively encoded
by codons not containing any cytosine, or except for glutamine
(Gin), which is encoded by two codons each containing the same
number of cytosines.
[0351] In a further preferred embodiment of the present invention,
the modified target nucleic acid, preferably the RNA is modified
such that at least 80%, or at least 90% of the theoretically
possible maximum cytosine-content or even a maximum
cytosine-content is achieved by means of codons, which code for
relatively frequent tRNAs in the cell, wherein the amino acid
sequence remains unchanged.
[0352] Due to the naturally occurring degeneracy of the genetic
code, more than one codon may encode a particular amino acid.
Accordingly, 18 out of 20 naturally occurring amino acids are
encoded by more than one codon (with Tryp and Met being an
exception), e.g. by 2 codons (e.g. Cys, Asp, Glu), by three codons
(e.g. Ile), by 4 codons (e.g. Al, Gly, Pro) or by 6 codons (e.g.
Leu, Arg, Ser). However, not all codons encoding the same amino
acid are utilized with the same frequency under in vivo conditions.
Depending on each single organism, a typical codon usage profile is
established.
[0353] The term "cytosine content-optimizable codon" as used within
the context of the present invention refers to codons, which
exhibit a lower content of cytosines than other codons encoding the
same amino acid. Accordingly, any wild type codon, which may be
replaced by another codon encoding the same amino acid and
exhibiting a higher number of cytosines within that codon, is
considered to be cytosine-optimizable (C-optimizable). Any such
substitution of a C-optimizable wild type codon by the specific
C-optimized codon within a wild type coding sequence increases its
overall C-content and reflects a C-enriched modified nucleic acid
sequence. According to a preferred embodiment, the nucleic acid
sequence, particularly the RNA sequence of the present invention,
preferably the at least one coding sequence of the nucleic acid
sequence of the present invention comprises or consists of a
C-maximized RNA sequence containing C-optimized codons for all
potentially C-optimizable codons. Accordingly, 100% or all of the
theoretically replaceable C-optimizable codons are preferably
replaced by C-optimized codons over the entire length of the coding
sequence.
[0354] In this context, cytosine-content optimizable codons are
codons, which contain a lower number of cytosines than other codons
coding for the same amino acid. Any of the codons GCG, GCA, GCU
codes for the amino acid Ala, which may be exchanged by the codon
GCC encoding the same amino acid, and/or the codon UGU that codes
for Cys may be exchanged by the codon UGC encoding the same amino
acid, and/or the codon GAU which codes for Asp may be exchanged by
the codon GAC encoding the same amino acid, and/or the codon that
UUU that codes for Phe may be exchanged for the codon UUC encoding
the same amino acid, and/or any of the codons GGG, GGA, GGU that
code Gly may be exchanged by the codon GGC encoding the same amino
acid, and/or the codon CAU that codes for His may be exchanged by
the codon CAC encoding the same amino acid, and/or any of the
codons AUA, AUU that code for Ile may be exchanged by the codon
AUC, and/or any of the codons UUG, UUA, CUG, CUA, CUU coding for
Leu may be exchanged by the codon CUC encoding the same amino acid,
and/or the codon AAU that codes for Asn may be exchanged by the
codon AAC encoding the same amino acid, and/or any of the codons
CCG, CCA, CCU coding for Pro may be exchanged by the codon CCC
encoding the same amino acid, and/or any of the codons AGG, AGA,
CGG, CGA, CGU coding for Arg may be exchanged by the codon CGC
encoding the same amino acid, and/or any of the codons AGU, AGC,
UCG, UCA, UCU coding for Ser may be exchanged by the codon UCC
encoding the same amino acid, and/or any of the codons ACG, ACA,
ACU coding for Thr may be exchanged by the codon ACC encoding the
same amino acid, and/or any of the codons GUG, GUA, GUU coding for
Val may be exchanged by the codon GUC encoding the same amino acid,
and/or the codon UAU coding for Tyr may be exchanged by the codon
UAC encoding the same amino acid.
[0355] In any of the above instances, the number of cytosines is
increased by 1 per exchanged codon. Exchange of all non C-optimized
codons (corresponding to C-optimizable codons) of the coding
sequence results in a C-maximized coding sequence. In the context
of the invention, at least 70%, preferably at least 80%, more
preferably at least 90%, of the non C-optimized codons within the
at least one coding sequence of the RNA sequence according to the
invention are replaced by C-optimized codons.
[0356] It may be preferred that for some amino acids the percentage
of C-optimizable codons replaced by C-optimized codons is less than
70%, while for other amino acids the percentage of replaced codons
is higher than 70% to meet the overall percentage of C-optimization
of at least 70% of all C-optimizable wild type codons of the coding
sequence.
[0357] Preferably, in a C-optimized RNA sequence of the invention,
at least 50% of the C-optimizable wild type codons for any given
amino acid are replaced by C-optimized codons, e.g. any modified
C-enriched RNA sequence preferably contains at least 50%
C-optimized codons at C-optimizable wild type codon positions
encoding any one of the above mentioned amino acids Ala, Cys, Asp,
Phe, Gly, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val and Tyr,
preferably at least 60%.
[0358] In this context codons encoding amino acids, which are not
cytosine content-optimizable and which are, however, encoded by at
least two codons, may be used without any further selection
process. However, the codon of the wild type sequence that codes
for a relatively rare tRNA in the cell, e.g. a human cell, may be
exchanged for a codon that codes for a relatively frequent tRNA in
the cell, wherein both code for the same amino acid. Accordingly,
the relatively rare codon GAA coding for Glu may be exchanged by
the relative frequent codon GAG coding for the same amino acid,
and/or the relatively rare codon AAA coding for Lys may be
exchanged by the relative frequent codon AAG coding for the same
amino acid, and/or the relatively rare codon CAA coding for Gln may
be exchanged for the relative frequent codon CAG encoding the same
amino acid.
[0359] In this context, the amino acids Met (AUG) and Trp (UGG),
which are encoded by only one codon each, remain unchanged. Stop
codons are not cytosine-content optimized, however, the relatively
rare stop codons amber, ochre (UAA, UAG) may be exchanged by the
relatively frequent stop codon opal (UGA).
[0360] The single substitutions listed above may be used
individually as well as in all possible combinations in order to
optimize the cytosine-content of the modified nucleic acid sequence
compared to the wild type nucleic acid sequence.
[0361] Accordingly, the at least one coding sequence as defined
herein may be changed compared to the coding sequence of the
respective wild type nucleic acid in such a way that an amino acid
encoded by at least two or more codons, of which one comprises one
additional cytosine, such a codon may be exchanged by the
C-optimized codon comprising one additional cytosine, wherein the
amino acid is preferably unaltered compared to the wild type
sequence.
[0362] C-Optimized Hendra Virus Sequences:
[0363] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Hendra virus, wherein the coding sequence comprises or
consists of any one of the (C-- optimized) RNA sequences as defined
in column "D" ("opt2") of Table 1 and in column "D" (opt2) of Table
1B, or of a fragment or variant of any one of these sequences.
[0364] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (C-- optimized) RNA sequences according to SEQ ID
NOs: 86-89, 658-661, 892-895, 1126-1129 and as defined in column
"D" ("opt2") of Table 1 and in column "D" (opt2) of Table 1B, or of
a fragment or variant of any one of these sequences.
[0365] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Hendra virus
antigenic peptide or protein derived from Hendra virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (C-- optimized) RNA sequences according to SEQ ID NOs:
97-104, 669-676, 903-910, 1137-1144 and as defined in column "D"
("opt2") of Table 1 and in column "D" (opt2) of Table 1B, or of a
fragment or variant of any one of these sequences.
[0366] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus, comprises
or consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (C-- optimized) RNA sequences as
defined in column "D" ("opt2") of Table 1 and in column "D" (opt2)
of Table 1B, or of a fragment or variant of any one of these
sequences.
[0367] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Hendra virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (C-- optimized) RNA
sequences as defined in column "D" ("opt2") of Table 1 and in
column "D" (opt2) of Table 1B, or of a fragment or variant of any
one of these sequences.
[0368] C-Optimized Nipah Virus Sequences:
[0369] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Nipah virus, wherein the coding sequence comprises or
consists of any one of the (C-optimized) RNA sequences as defined
in column "D" (opt2) of Table 2 and in column "D" (opt2) of Table
2B, or of a fragment or variant of any one of these sequences.
[0370] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus fusion
protein (F), wherein the coding sequence comprises or consists of
any one of the (C-optimized) RNA sequences according to SEQ ID NOs:
79-85, 651-657, 885-891, 1119-1125, 1522-1524 and as defined in
column "D" (opt2) of Table 2 and in column "D" (opt2) of Table 2B,
or of a fragment or variant of any one of these sequences.
[0371] According to a preferred embodiment, the present invention
provides an nucleic acid sequence as defined herein comprising at
least one coding sequence encoding at least one Nipah virus
antigenic peptide or protein derived from Nipah virus glycoprotein
(G), wherein the coding sequence comprises or consists of any one
of the (C-optimized) RNA sequences according to SEQ ID NOs: 90-96,
662-668, 896-902, 1130-1136 and as defined in column "D" (opt2) of
Table 2 and in column "D" (opt2) of Table 2B, or of a fragment or
variant of any one of these sequences.
[0372] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Nipah virus, comprises or
consists of an nucleic acid sequence identical to or having a
sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably
of at least 80%, even more preferably at least 85%, even more
preferably of at least 90% and most preferably of at least 95% or
even 97%, with any one of the (C-optimized) RNA sequences as
defined in column "D" (opt2) of Table 2 and in column "D" (opt2) of
Table 2B, or of a fragment or variant of any one of these
sequences.
[0373] According to a particularly preferred embodiment, the at
least one coding sequence of the RNA sequence, encoding at least
one antigenic peptide or protein derived from Nipah virus,
comprises or consists of an nucleic acid sequence having a sequence
identity of at least 80% with any one of the (C-optimized) RNA
sequences as defined in column "D" (opt2) of Table 2 and in column
"D" (opt2) of Table 2B, or of a fragment or variant of any one of
these sequences.
[0374] Sequence Modified Secretory Signal Peptides:
[0375] According to a preferred embodiment, the present invention
provides a nucleic acid sequence comprising at least one coding
sequence, encoding at least one antigenic peptide or protein
derived from Hendra virus and/or Nipah virus, and, additionally, an
N-terminal secretory signal peptide, wherein the coding sequence
comprises or consists of any one of the (modified) RNA sequences as
defined in the columns "C-J" of Table 3, or of a fragment or
variant of any one of these sequences.
[0376] In a further preferred embodiment, the at least one coding
sequence of the nucleic acid sequence, encoding at least one
antigenic peptide or protein derived from Hendra virus and/or Nipah
virus, and, additionally, an N-terminal secretory signal peptide,
wherein the coding sequence comprises or consists of an nucleic
acid sequence identical to or having a sequence identity of at
least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,
preferably of at least 70%, more preferably of at least 80%, even
more preferably at least 85%, even more preferably of at least 90%
and most preferably of at least 95% or even 97%, with any one of
the (modified) RNA sequences as defined in the columns "C-J" of
Table 3, or of a fragment or variant of any one of these
sequences.
[0377] According to a particularly preferred embodiment, the at
least one coding sequence of the nucleic acid sequence comprises or
consists of an RNA sequence having a sequence identity of at least
80% with any one of the (modified) RNA sequences as defined in the
columns "C-J" of Table 3, or of a fragment or variant of any one of
these sequences.
[0378] According to preferred embodiments, the present invention
provides a nucleic acid sequence, wherein the at least one coding
sequence comprises a (G/C modified) 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 any one
of the (modified) RNA sequences as defined in the columns "C, G-J"
(opt1, opt5, opt6, opt7) of Table 3.
[0379] 5'-Cap Structure:
[0380] According to another preferred embodiment of the invention,
a modified nucleic acid sequence as defined herein, particularly a
modified RNA as defined herein can be modified by the addition of a
so-called "5'-cap" structure, which preferably stabilizes the
nucleic acid as described herein. A 5''-cap is an entity, typically
a modified nucleotide entity, which generally "caps" the 5'-end of
a mature RNA. A 5'-cap may typically be formed by a modified
nucleotide, particularly by a derivative of a guanine nucleotide.
Preferably, the 5''-cap is linked to the 5'-terminus via a
5'-5''-triphosphate linkage. A 5'-cap may be methylated, e.g.
m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic
acid carrying the 5'-cap, typically the 5'-end of an RNA. m7GpppN
is the 5'-cap structure, which naturally occurs in RNA transcribed
by polymerase II and is therefore preferably not considered as
modification comprised in a modified RNA in this context.
Accordingly, a modified RNA sequence of the present invention may
comprise a m7GpppN as 5'-cap, but additionally the modified RNA
sequence typically comprises at least one further modification as
defined herein.
[0381] Further examples of 5'-cap structures include glyceryl,
inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide,
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,
L-nucleotides, alpha-nucleotide, modified base nucleotide,
threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide,
acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic
moiety, 3'-2'-inverted nucleotide moiety, 3% 2'-inverted abasic
moiety, 1,4-butanediol phosphate, 3'-phosphoramidate,
hexylphosphate, aminohexyl phosphate, 3''-phosphate,
3'phosphorothioate, phosphorodithioate, or bridging or non-bridging
methylphosphonate moiety. These modified 5'-cap structures are
regarded as at least one modification in this context.
[0382] Particularly preferred modified 5'-cap structures are cap1
(methylation of the ribose of the adjacent nucleotide of m7G), cap2
(additional methylation of the ribose of the 2nd nucleotide
downstream of the m7G), cap3 (additional methylation of the ribose
of the 3rd nucleotide downstream of the m7G), cap4 (methylation of
the ribose of the 4th nucleotide downstream of the m7G), ARCA
(anti-reverse cap analogue, modified ARCA (e.g. phosphothioate
modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine. Accordingly, the RNA
according to the invention preferably comprises a 5'-cap
structure.
[0383] In a preferred embodiment, the 5'-cap structure is added
co-transcriptionally using cap-analogues as defined herein in an
RNA in vitro transcription reaction as defined herein. In another
embodiment, the 5'-cap structure is added via enzymatic capping
using capping enzymes (e.g. vaccinia virus capping enzymes). In
another embodiment, the 5''-cap structure is added via enzymatic
capping using immobilized capping enzymes, e.g. in a capping
reactor (WO 2016/193226).
[0384] Accordingly, in preferred embodiments, the artificial
nucleic acid of the invention comprises a 5''-cap structure as
defined herein.
[0385] Poly(A) Sequence/Tail:
[0386] According to a further preferred embodiment, the nucleic
acid sequence, particularly the RNA sequence of the present
invention may contain a poly-A tail on the 3''-terminus of
typically about 10 to 200 adenosine nucleotides, preferably about
10 to 100 adenosine nucleotides, more preferably about 40 to 80
adenosine nucleotides or even more preferably about 50 to 70
adenosine nucleotides.
[0387] Preferably, the poly(A) sequence in the RNA sequence of the
present invention is derived from a DNA template by RNA in vitro
transcription. Alternatively, the poly(A) sequence may also be
obtained in vitro by common methods of chemical-synthesis without
being necessarily transcribed from a DNA-progenitor. Moreover,
poly(A) sequences, or poly(A) tails may be generated by enzymatic
polyadenylation of the RNA according to the present invention using
commercially available polyadenylation kits and corresponding
protocols known in the art, or using immobilized poly(A)polymerases
e.g. in a polyadenylation reactor (WO/2016/174271)
[0388] Alternatively, the RNA as described herein optionally
comprises a polyadenylation signal, which is defined herein as a
signal, which conveys polyadenylation to a (transcribed) RNA by
specific protein factors (e.g. cleavage and polyadenylation
specificity factor (CPSF), cleavage stimulation factor (CstF),
cleavage factors I and II (CF I and CF II), poly(A) polymerase
(PAP)). In this context, a consensus polyadenylation signal is
preferred comprising the NN(U/T)ANA consensus sequence. In a
particularly preferred aspect, the polyadenylation signal comprises
one of the following sequences: AA(U/T)AAA or A(U/T)(U/T)AAA
(wherein uridine is usually present in RNA and thymidine is usually
present in DNA).
[0389] Poly(C) Sequence:
[0390] According to a further preferred embodiment, the nucleic
acid sequence, particularly the RNA sequence of the present
invention may contain a poly(C) tail on the 3''-terminus of
typically about 10 to 200 cytosine nucleotides, preferably about 10
to 100 cytosine nucleotides, more preferably about 20 to 70
cytosine nucleotides or even more preferably about 20 to 60 or even
10 to 40 cytosine nucleotides. Preferably, the poly(C) sequence in
the RNA sequence of the present invention is derived from a DNA
template by RNA in vitro transcription.
[0391] UTRs:
[0392] In a preferred embodiment, the artificial nucleic acid of
the invention comprises at least one untranslated region (UTR.
[0393] In a preferred embodiment, the nucleic acid sequence,
particularly the RNA sequence according to the invention comprises
at least one 5% and/or 3'-UTR element. In this context, an UTR
element comprises or consists of a nucleic acid sequence, which is
derived from the 5''- or 3'-UTR of any naturally occurring gene or
which is derived from a fragment, a homolog or a variant of the
5''- or 3'-UTR of a gene. Preferably, the 5'- or 3''-UTR element
used according to the present invention is heterologous to the at
least one coding sequence of the RNA sequence of the invention.
Even if 5''- or 3''-UTR elements derived from naturally occurring
genes are preferred, also synthetically engineered UTR elements may
be used in the context of the present invention.
[0394] 3''-UTR Elements:
[0395] In preferred embodiment, the artificial nucleic acid of the
invention comprises at least one 3'-UTR.
[0396] In a particularly preferred embodiment, the artificial
nucleic acid of the invention comprises at least one heterologous
3''-UTR.
[0397] Preferably, the 3'-UTR comprises a poly(A) sequence and/or a
poly(C) sequence as defined above, 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.
[0398] The term "3'-UTR element" typically refers to a nucleic acid
sequence, which comprises or consists of a nucleic acid sequence
that is derived from a 3'-UTR or from a variant of a 3''-UTR. A
3''-UTR element in the sense of the present invention may represent
the 3''-UTR of a nucleic acid molecule, particularly of an RNA or
DNA, preferably an mRNA. Thus, in the sense of the present
invention, preferably, a 3''-UTR element may be the 3'-UTR of an
RNA, preferably of an mRNA, or it may be the transcription template
for a 3''-UTR of an RNA. Thus, a 3''-UTR element preferably is a
nucleic acid sequence which corresponds to the 3''-UTR of an RNA,
preferably to the 3'-UTR of an mRNA, such as an mRNA obtained by
transcription of a genetically engineered vector construct.
Preferably, the 3''-UTR element fulfils the function of a 3''-UTR
or encodes a sequence which fulfils the function of a 3''-UTR.
[0399] Preferably, the at least one 3'-UTR element comprises or
consists of a nucleic acid sequence derived from the 3''-UTR of a
chordate gene, preferably a vertebrate gene, more preferably a
mammalian gene, most preferably a human gene, or from a variant of
the 3''-UTR of a chordate gene, preferably a vertebrate gene, more
preferably a mammalian gene, most preferably a human gene.
[0400] Preferably, the nucleic acid sequence, particularly the RNA
sequence of the present invention comprises a 3'-UTR element, which
may be derivable from a gene that relates to an RNA with an
enhanced half-life (that provides a stable RNA), for example a
3'-UTR element as defined and described below. Preferably, the
3''-UTR element is a nucleic acid sequence derived from a 3'' UTR
of a gene, which preferably encodes a stable RNA, or from a
homolog, a fragment or a variant of said gene.
[0401] In a particularly preferred embodiment, the 3'-UTR element
comprises or consists of a nucleic acid sequence, which is 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, such as a collagen alpha 1(1) gene, or from a variant
of 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, such as a collagen alpha 1(1) gene according to SEQ ID
NOs: 1369-1390 of the patent application WO2013/143700, whose
disclosure is incorporated herein by reference, or from a homolog,
a fragment or a variant thereof. In a particularly preferred
embodiment, the 3''-UTR element comprises or consists of a nucleic
acid sequence which is derived from a 3'-UTR of an albumin gene,
preferably a vertebrate albumin gene, more preferably a mammalian
albumin gene, most preferably a human albumin gene according to SEQ
ID NOs: 247, 249, 251 or the corresponding RNA sequence SEQ ID NOs:
248, 250, 252.
[0402] In this context it is particularly preferred that the RNA
sequence according to the invention comprises a 3'-UTR element
comprising a corresponding RNA sequence derived from the nucleic
acids according to SEQ ID NOs: 1369-1390 of the patent application
WO2013/143700 or a fragment, homolog or variant thereof.
[0403] Most preferably the 3'-UTR element comprises the nucleic
acid sequence derived from a fragment of the human albumin gene
according to SEQ ID NOs: 249-252.
[0404] In this context, it is particularly preferred that the
3'-UTR element of the RNA sequence according to the present
invention comprises or consists of a corresponding RNA sequence of
the nucleic acid sequence according to SEQ ID NOs: 249 or 251 as
shown in SEQ ID NOs: 250 or 252.
[0405] In another particularly preferred embodiment, the 3'-UTR
element comprises or consists of a nucleic acid sequence which is
derived from a 3'-UTR of an alpha- or beta-globin gene, preferably
a vertebrate alpha- or beta-globin gene, more preferably a
mammalian alpha- or beta-globin gene, most preferably a human
alpha- or beta-globin gene according to SEQ ID NOs: 239, 241, 243,
245 or the corresponding RNA sequences SEQ ID NOs: 240, 242, 244,
246.
[0406] For example, the 3'-UTR element may comprise or consist of
the center, .alpha.-complex-binding portion of the 3'-UTR of an
.alpha.-globin gene, such as of a human .alpha.-globin gene, or a
homolog, a fragment, or a variant of an .alpha.-globin gene,
preferably according to SEQ ID NO: 245 or 246.
[0407] In this context it is particularly preferred that the
3''-UTR element of the RNA sequence according to the invention
comprises or consists of a corresponding RNA sequence of the
nucleic acid sequence according to SEQ ID NO: 245 as shown in SEQ
ID NO: 246, or a homolog, a fragment or variant thereof.
[0408] The term "a nucleic acid sequence which is derived from the
3'-UTR of a [ . . . ] gene" preferably refers to a nucleic acid
sequence which is based on the 3'-UTR sequence of a [ . . . ] gene
or on a part thereof, such as on the 3'-UTR of an albumin gene, an
alpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene,
a lipoxygenase gene, or a collagen alpha gene, such as a collagen
alpha 1(1) gene, preferably of an albumin gene or on a part
thereof. This term includes sequences corresponding to the entire
3'-UTR sequence, i.e. the full length 3'-UTR sequence of a gene,
and sequences corresponding to a fragment of the 3'-UTR sequence of
a gene, such as an albumin gene, alpha-globin gene, beta-globin
gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen
alpha gene, such as a collagen alpha 1(1) gene, preferably of an
albumin gene.
[0409] The term "a nucleic acid sequence which is derived from a
variant of the 3'-UTR of a [ . . . ] gene" preferably refers to a
nucleic acid sequence, which is based on a variant of the 3'-UTR
sequence of a gene, such as on a variant of the 3'-UTR of an
albumin gene, an .alpha.-globin gene, a .beta.-globin gene, a
tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha
gene, such as a collagen alpha 1(1) gene, or on a part thereof as
described above. This term includes sequences corresponding to the
entire sequence of the variant of the 3'-UTR of a gene, i.e. the
full length variant 3'-UTR sequence of a gene, and sequences
corresponding to a fragment of the variant 3'-UTR sequence of a
gene. A fragment in this context preferably consists of a
continuous stretch of nucleotides corresponding to a continuous
stretch of nucleotides in the full-length variant 3''-UTR, which
represents at least 20%, preferably at least 30%, more preferably
at least 40%, more preferably at least 50%, even more preferably at
least 60%, even more preferably at least 70%, even more preferably
at least 80%, and most preferably at least 90% of the full-length
variant 3''-UTR. Such a fragment of a variant, in the sense of the
present invention, is preferably a functional fragment of a variant
as described herein.
[0410] In further embodiments, the artificial nucleic acid as
defined herein, particularly the RNA as defined herein comprises a
3''-UTR element, which may be any 3''-UTR element described in
WO2016/107877. In this context, the disclosure of WO2016/107877
relating to 3''-UTR elements/sequences is herewith incorporated by
reference. Particularly preferred 3''-UTR elements are SEQ ID NOs:
1 to 24 and SEQ ID NOs: 49 to 318 of the patent application
WO2016/107877, or fragments or variants of these sequences. In this
context, it is particularly preferred that the 3''-UTR element of
the RNA sequence according to the present invention comprises or
consists of a corresponding RNA sequence of the nucleic acid
sequence according SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 of
the patent application WO2016/107877.
[0411] In embodiments, the artificial nucleic acid as defined
herein, particularly the RNA as defined herein comprises a 3''-UTR
element, which may be any 3''-UTR element as described in
WO2017/036580. In this context, the disclosure of WO2017/036580
relating to 3'-UTR elements/sequences is herewith incorporated by
reference. Particularly preferred 3'-UTR elements are nucleic acid
sequences according to SEQ ID NOs: 152 to 204 of the patent
application WO2017/036580, or fragments or variants of these
sequences. In this context, it is particularly preferred that the
3''-UTR element of the RNA sequence according to the present
invention comprises or consists of a corresponding RNA sequence of
the nucleic acid sequence according SEQ ID NOs: 152 to 204 of the
patent application WO2017/036580.
[0412] According to a preferred embodiment, the nucleic acid
sequence, particularly the RNA sequence according to the invention
comprises a 5''-cap structure and/or at least one 3'-untranslated
region element (3''-UTR element), preferably as defined herein.
More preferably, the RNA further comprises a 5''-UTR element as
defined herein.
[0413] In a preferred embodiment the RNA sequence comprises,
preferably in 5''- to 3''-direction: [0414] a.) a 5''-cap
structure, preferably m7GpppN; [0415] b1.) optionally, at least one
coding sequence encoding at least one heterologous secretory signal
sequence; [0416] b2.) at least one coding sequence encoding at
least one antigenic peptide or protein derived from a Henipavirus,
or a fragment or variant thereof, [0417] c.) a 3''-UTR element
comprising or consisting of a nucleic acid sequence which is
derived from an alpha globin gene, preferably comprising the
corresponding RNA sequence of the nucleic acid sequence according
to SEQ ID NO: 246, a homolog, a fragment or a variant thereof;
[0418] d.) optionally, a poly(A) sequence, preferably comprising 64
adenosines; [0419] e.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines;
[0420] In a particularly preferred embodiment the RNA sequence
comprises, preferably in 5''- to 3'-direction: [0421] a.) a 5'-cap
structure, preferably m7GpppN; [0422] b1.) optionally, at least one
coding sequence encoding at least one heterologous secretory signal
sequence, preferably selected from SEQ ID NOs 317-572; [0423] b2.)
at least one coding sequence encoding at least one antigenic
peptide or protein derived from a Hendra virus protein or peptide
or a fragment or variant thereof, preferably comprising or
consisting of any one of the nucleic acid sequences according 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, or a fragment or
variant thereof; [0424] c.) a 3''-UTR element comprising or
consisting of a nucleic acid sequence which is derived from an
alpha globin gene, preferably comprising the corresponding RNA
sequence of the nucleic acid sequence according to SEQ ID NO: 246,
a homolog, a fragment or a variant thereof; [0425] d.) optionally,
a poly(A) sequence, preferably comprising 64 adenosines; [0426] e.)
optionally, a poly(C) sequence, preferably comprising 30
cytosines;
[0427] In a further particularly preferred embodiment the RNA
sequence comprises, preferably in 5''- to 3''-direction: [0428] a)
a 5''-cap structure, preferably m7GpppN; [0429] b1.) optionally, at
least one coding sequence encoding at least one heterologous
secretory signal sequence, preferably selected from SEQ ID NOs
317-572; [0430] b2.) at least one coding sequence encoding at least
one antigenic peptide or protein derived from a Nipah virus protein
or peptide or a fragment or variant thereof, preferably comprising
or consisting of any one of the nucleic acid sequences according 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, 1516-1539 or a fragment
or variant thereof; [0431] c.) a 3''-UTR element comprising or
consisting of a nucleic acid sequence which is derived from an
alpha globin gene, preferably comprising the corresponding RNA
sequence of the nucleic acid sequence according to SEQ ID NO: 246,
a homolog, a fragment or a variant thereof; [0432] d.) optionally,
a poly(A) sequence, preferably comprising 64 adenosines; [0433] e.)
optionally, a poly(C) sequence, preferably comprising 30
cytosines;
[0434] 5''-UTR Elements:
[0435] In a particularly preferred embodiment, the at least one
nucleic acid sequence, in particular, the RNA sequence comprises at
least one 5''-untranslated region element (5''-UTR element).
Preferably, the at least one 5''-UTR element comprises or consists
of a nucleic acid sequence, which is derived from the 5''-UTR of a
TOP gene or which is derived from a fragment, homolog or variant of
the 5''-UTR of a TOP gene.
[0436] It is particularly preferred that the 5''-UTR element does
not comprise a TOP-motif or a 5''-TOP, as defined above.
[0437] In some embodiments, the nucleic acid sequence of the 5'-UTR
element, which is derived from a 5'-UTR of a TOP gene, terminates
at its 3'-end with a nucleotide located at position 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U/T)G) of the
gene or RNA it is derived from. Thus, the 5'-UTR element does not
comprise any part of the protein coding sequence. Thus, preferably,
the only protein coding part of the at least one nucleic acid
sequence, particularly of the RNA sequence, is provided by the
coding sequence.
[0438] The nucleic acid sequence derived from the 5'-UTR of a TOP
gene is preferably derived from a eukaryotic TOP gene, preferably a
plant or animal TOP gene, more preferably a chordate TOP gene, even
more preferably a vertebrate TOP gene, most preferably a mammalian
TOP gene, such as a human TOP gene.
[0439] For example, the 5'-UTR element is preferably selected from
5''-UTR elements comprising or consisting of a nucleic acid
sequence, which is derived from a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-1363, SEQ ID NO: 1395,
SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application
WO2013/143700, whose disclosure is incorporated herein by
reference, from the homologs of SEQ ID NOs: 1-1363, SEQ ID NO:
1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application
WO2013/143700, from a variant thereof, or preferably from a
corresponding RNA sequence. The term "homologs of SEQ ID NOs:
1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the
patent application WO2013/143700" refers to sequences of other
species than Homo sapiens, which are homologous to the sequences
according to SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO. 1421
and SEQ ID NO: 1422 of the patent application WO2013/143700.
[0440] In a preferred embodiment, the 5'-UTR element of the nucleic
acid sequence, particularly of the RNA sequence according to the
invention comprises or consists of a nucleic acid sequence, which
is derived from a nucleic acid sequence extending from nucleotide
position 5 (i.e. the nucleotide that is located at position 5 in
the sequence) to the nucleotide position immediately 5' to the
start codon (located at the 3'-end of the sequences), e.g. the
nucleotide position immediately 5' to the ATG sequence, of a
nucleic acid sequence selected from SEQ ID NOs: 1-1363, SEQ ID NO:
1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application
WO2013/143700, from the homologs of SEQ ID NOs: 1-1363, SEQ ID NO:
1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application
WO2013/143700 from a variant thereof, or a corresponding RNA
sequence. It is particularly preferred that the 5'-UTR element is
derived from a nucleic acid sequence extending from the nucleotide
position immediately 3' to the 5''-TOP to the nucleotide position
immediately 5' to the start codon (located at the 3'-end of the
sequences), e.g. the nucleotide position immediately 5' to the ATG
sequence, of a nucleic acid sequence selected from SEQ ID NOs:
1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the
patent application WO2013/143700, from the homologs of SEQ ID NOs:
1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the
patent application WO2013/143700, from a variant thereof, or a
corresponding RNA sequence.
[0441] In a particularly preferred embodiment, the 5'-UTR element
comprises or consists of a nucleic acid sequence, which is derived
from a 5'-UTR of a TOP gene encoding a ribosomal protein or from a
variant of a 5''-UTR of a TOP gene encoding a ribosomal protein.
For example, the 5'-UTR element comprises or consists of a nucleic
acid sequence, which is derived from a 5'-UTR of a nucleic acid
sequence according to any of SEQ ID NOs: 67, 170, 193, 244, 259,
554, 650, 675, 700, 721, 913, 1016, 1063, 1120, 1138, and 1284-1360
of the patent application WO2013/143700, a corresponding RNA
sequence, a homolog thereof, or a variant thereof as described
herein, preferably lacking the 5'-TOP motif. As described above,
the sequence extending from position 5 to the nucleotide
immediately 5' to the ATG (which is located at the 3'-end of the
sequences) corresponds to the 5'-UTR of said sequences.
[0442] Preferably, the 5'-UTR element comprises or consists of a
nucleic acid sequence, which is derived from a 5''-UTR of a TOP
gene encoding a ribosomal Large protein (RPL) or from a homolog or
variant of a 5'-UTR of a TOP gene encoding a ribosomal Large
protein (RPL). For example, the 5'-UTR element comprises or
consists of a nucleic acid sequence, which is derived from a 5'-UTR
of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259,
1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 of the
patent application WO2013/143700, a corresponding RNA sequence, a
homolog thereof, or a variant thereof as described herein,
preferably lacking the 5'-TOP motif.
[0443] In a particularly preferred embodiment, the 5'-UTR element
comprises or consists of a nucleic acid sequence which is derived
from the 5'-UTR of a ribosomal protein Large 32 gene, preferably
from a vertebrate ribosomal protein Large 32 (L32) gene, more
preferably from a mammalian ribosomal protein Large 32 (L32) gene,
most preferably from a human ribosomal protein Large 32 (L32) gene,
or from a variant of the 5'-UTR of a ribosomal protein Large 32
gene, preferably from a vertebrate ribosomal protein Large 32 (L32)
gene, more preferably from a mammalian ribosomal protein Large 32
(L32) gene, most preferably from a human ribosomal protein Large 32
(L32) gene, wherein preferably the 5'-UTR element does not comprise
the 5'-TOP of said gene.
[0444] Accordingly, in a particularly preferred embodiment, the
5'-UTR element comprises or consists of a nucleic acid sequence,
which has an identity of at least about 40%, preferably of at least
about 50%, preferably of at least about 60%, preferably of at least
about 70%, more preferably of at least about 80%, more preferably
of at least about 90%, even more preferably of at least about 95%,
even more preferably of at least about 99% to the nucleic acid
sequence according to SEQ ID NO: 3529 or 3530 (5'-UTR of human
ribosomal protein Large 32 lacking the 5'-terminal oligopyrimidine
tract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to
SEQ ID No. 1368 of the patent application WO2013/143700) or
preferably to a corresponding RNA sequence, or wherein the at least
one 5'-UTR element comprises or consists of a fragment of a nucleic
acid sequence which has an identity of at least about 40%,
preferably of at least about 50%, preferably of at least about 60%,
preferably of at least about 70%, more preferably of at least about
80%, more preferably of at least about 90%, even more preferably of
at least about 95%, even more preferably of at least about 99% to
the nucleic acid sequence according to SEQ ID NO: 235 or more
preferably to a corresponding RNA sequence (SEQ ID NO: 236),
wherein, preferably, the fragment is as described above, i.e. being
a continuous stretch of nucleotides representing at least 20% etc.
of the full-length 5'-UTR. Preferably, the fragment exhibits a
length of at least about 20 nucleotides or more, preferably of at
least about 30 nucleotides or more, more preferably of at least
about 40 nucleotides or more. Preferably, the fragment is a
functional fragment as described herein.
[0445] In some embodiments, the RNA sequence according to the
invention comprises a 5'-UTR element, which comprises or consists
of a nucleic acid sequence, which is derived from the 5'-UTR of a
vertebrate TOP gene, such as a mammalian, e.g. a human TOP gene,
selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7,
RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A,
RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25,
RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6,
RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13,
RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22,
RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30,
RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A,
RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0,
RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, ElF3E, ElF3F,
EIF3H, ElF2S3, EIF3C, ElF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1,
TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from a
homolog or variant thereof, wherein preferably the 5''-UTR element
does not comprise a TOP-motif or the 5''-TOP of said genes, and
wherein optionally the 5''-UTR element starts at its 5'-end with a
nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
downstream of the 5''-terminal oligopyrimidine tract (TOP) and
wherein further optionally the 5''-UTR element which is derived
from a 5'-UTR of a TOP gene terminates at its 3'-end with a
nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
upstream of the start codon (A(UIT)G) of the gene it is derived
from.
[0446] In further particularly preferred embodiments, the 5''-UTR
element comprises or consists of a nucleic acid sequence, which is
derived from the 5'-UTR of a ribosomal protein Large 32 gene
(RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal
protein Large 21 gene (RPL21), an ATP synthase, H+ transporting,
mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1)
gene, an hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4),
an androgen-induced 1 gene (AIG1), cytochrome c oxidase subunit Vic
gene (COX6C), or a N-acylsphingosine amidohydrolase (acid
ceramidase) 1 gene (ASAH1) or from a variant thereof, preferably
from a vertebrate ribosomal protein Large 32 gene (RPL32), a
vertebrate ribosomal protein Large 35 gene (RPL35), a vertebrate
ribosomal protein Large 21 gene (RPL21), a vertebrate ATP synthase,
H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac
muscle (ATP5A1) gene, a vertebrate hydroxysteroid (17-beta)
dehydrogenase 4 gene (HSD17B4), a vertebrate androgen-induced 1
gene (AIG1), a vertebrate cytochrome c oxidase subunit Vic gene
(COX6C), or a vertebrate N-acylsphingosine amidohydrolase (acid
ceramidase) 1 gene (ASAH1) or from a variant thereof, more
preferably from a mammalian ribosomal protein Large 32 gene
(RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal
protein Large 21 gene (RPL21), a mammalian ATP synthase, H+
transporting, mitochondrial F1 complex, alpha subunit 1, cardiac
muscle (ATP5A1) gene, a mammalian hydroxysteroid (17-beta)
dehydrogenase 4 gene (HSD17B4), a mammalian androgen-induced 1 gene
(AIG1), a mammalian cyto-chrome c oxidase subunit Vic gene (COX6C),
or a mammalian N-acylsphingosine ami-dohydrolase (acid ceramidase)
1 gene (ASAH1) or from a variant thereof, most preferably from a
human ribosomal protein Large 32 gene (RPL32), a human ribosomal
protein Large 35 gene (RPL35), a human ribosomal protein Large 21
gene (RPL21), a human ATP synthase, H+ transporting, mitochondrial
F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene, a human
hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a human
androgen-induced 1 gene (AIG1), a human cytochrome c oxidase
subunit Vic gene (COX6C), or a human N-acylsphingosine
amidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variant
thereof, wherein preferably the 5''-UTR element does not comprise
the 5'TOP of said gene.
[0447] Accordingly, in a particularly preferred embodiment, the
5''-UTR element comprises or consists of a nucleic acid sequence,
which has an identity of at least about 40%, preferably of at least
about 50%, preferably of at least about 60%, preferably of at least
about 70%, more preferably of at least about 80%, more preferably
of at least about 90%, even more preferably of at least about 95%,
even more preferably of at least about 99% to the nucleic acid
sequence according to SEQ ID NOs: 1412-1420 of the patent
application WO2013/143700, or a corresponding RNA sequence or
wherein the at least one 5'-UTR element comprises or consists of a
fragment of a nucleic acid sequence which has an identity of at
least about 40%, preferably of at least about 50%, preferably of at
least about 60%, preferably of at least about 70%, more preferably
of at least about 80%, more preferably of at least about 90%, even
more preferably of at least about 95%, even more preferably of at
least about 99% to the nucleic acid sequence according SEQ ID NOs:
1412-1420 of the patent application WO2013/143700, wherein,
preferably, the fragment is as described above, i.e. being a
continuous stretch of nucleotides representing at least 20% etc. of
the full-length 5''-UTR. Preferably, the fragment exhibits a length
of at least about 20 nucleotides or more, preferably of at least
about 30 nucleotides or more, more preferably of at least about 40
nucleotides or more. Preferably, the fragment is a functional
fragment as described herein.
[0448] Accordingly, in a particularly preferred embodiment, the
5''-UTR element comprises or consists of a nucleic acid sequence,
which has an identity of at least about 40%, preferably of at least
about 50%, preferably of at least about 60%, preferably of at least
about 70%, more preferably of at least about 80%, more preferably
of at least about 90%, even more preferably of at least about 95%,
even more preferably of at least about 99% to the nucleic acid
sequence according to SEQ ID NOs: 237 or 238 (5''-UTR of ATP5A1
lacking the 5''-terminal oligopyrimidine tract:
GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCGGAGTAAC
TGCAAAG; corresponding to SEQ ID NO: 1414 of the patent application
WO2013/143700) or preferably to a corresponding RNA sequence, or
wherein the at least one 5''-UTR element comprises or consists of a
fragment of a nucleic acid sequence which has an identity of at
least about 40%, preferably of at least about 50%, preferably of at
least about 60%, preferably of at least about 70%, more preferably
of at least about 80%, more preferably of at least about 90%, even
more preferably of at least about 95%, even more preferably of at
least about 99% to the nucleic acid sequence according to SEQ ID
NO: 237 or more preferably to a corresponding RNA sequence (SEQ ID
NO: 238), wherein, preferably, the fragment is as described above,
i.e. being a continuous stretch of nucleotides representing at
least 20% etc. of the full-length 5''-UTR. Preferably, the fragment
exhibits a length of at least about 20 nucleotides or more,
preferably of at least about 30 nucleotides or more, more
preferably of at least about 40 nucleotides or more. Preferably,
the fragment is a functional fragment as described herein.
[0449] In embodiments, the artificial nucleic acid as defined
herein, particularly the RNA as defined herein comprises a 5''-UTR
element, which may be any 5''-UTR element described in
WO2016/107877. In this context, the disclosure of WO2016/107877
relating to 5''-UTR elements/sequences is herewith incorporated by
reference. Particularly preferred 5''-UTR elements are nucleic acid
sequences according to SEQ ID NOs: 25 to 30 and SEQ ID NOs: 319 to
382 of the patent application WO2016/107877, or fragments or
variants of these sequences. In this context, it is particularly
preferred that the 5'-UTR element of the RNA sequence according to
the present invention comprises or consists of a corresponding RNA
sequence of the nucleic acid sequence according SEQ ID NOs: 25 to
30 and SEQ ID NOs: 319 to 382 of the patent application
WO2016/107877.
[0450] In embodiments, the artificial nucleic acid sequence as
defined herein, particularly the RNA as defined herein comprises a
5''-UTR element, which may be any 5'-UTR element as described in
WO2017/036580. In this context, the disclosure of WO2017/036580
relating to 5''-UTR elements/sequences is herewith incorporated by
reference. Particularly preferred 5''-UTR elements are nucleic acid
sequences according to SEQ ID NOs: 1 to 151 of the patent
application WO2017/036580, or fragments or variants of these
sequences. In this context, it is particularly preferred that the
5''-UTR element of the RNA sequence according to the present
invention comprises or consists of a corresponding RNA sequence of
the nucleic acid sequence according to SEQ ID NOs: 1 to 151 of the
patent application WO2017/036580.
[0451] Preferably, the at least one 5''-UTR element and the at
least one 3''-UTR element act synergistically to increase protein
production from the at least one RNA sequence as described
above.
[0452] According to a preferred embodiment the RNA sequence
according to the invention comprises, preferably in 5''- to
3''-direction: [0453] a.) a 5''-cap structure, preferably m7GpppN;
[0454] b.) a 5''-UTR element which comprises or consists of a
nucleic acid sequence which is derived from the 5''-UTR of a TOP
gene (SEQ ID NOs: 235-238), a homolog, a fragment or a variant
thereof; [0455] c1.) optionally, at least one coding sequence
encoding at least one heterologous secretory signal sequence;
[0456] c2.) at least one coding sequence encoding at least one
antigenic peptide or protein derived from a Henipavirus protein or
peptide as defined herein or a fragment or variant thereof. [0457]
d.) a 3'-UTR element comprising or consisting of a nucleic acid
sequence which is derived from a gene providing a stable RNA,
preferably comprising or consisting of the corresponding RNA
sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or
252, a homolog, a fragment or a variant thereof; [0458] e.)
optionally, a poly(A) sequence preferably comprising 64 adenosines;
and [0459] f.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines.
[0460] According to a particularly preferred embodiment the RNA
sequence according to the invention comprises, preferably in 5''-
to 3''-direction: [0461] a.) a 5''-cap structure, preferably
m7GpppN; [0462] b.) a 5'-UTR element which comprises or consists of
a nucleic acid sequence which is derived from the 5''-UTR of a TOP
gene (SEQ ID NOs: 235-238), a homolog, a fragment or a variant
thereof; [0463] c1.) optionally, at least one coding sequence
encoding at least one heterologous secretory signal sequence,
preferably selected from SEQ ID NOs 317-572; [0464] c2.) at least
one coding sequence encoding at least one antigenic peptide or
protein derived from a Hendra virus protein or peptide or a
fragment or variant thereof, preferably comprising or consisting of
any one of the nucleic acid sequences according 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, or a fragment or variant thereof;
[0465] d.) a 3'-UTR element comprising or consisting of a nucleic
acid sequence which is derived from a gene providing a stable RNA,
preferably comprising or consisting of the corresponding RNA
sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or
252, a homolog, a fragment or a variant thereof; [0466] e.)
optionally, a poly(A) sequence preferably comprising 64 adenosines;
and [0467] f.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines.
[0468] According to a particularly preferred embodiment the RNA
sequence according to the invention comprises, preferably in 5''-
to 3''-direction: [0469] a.) a 5''-cap structure, preferably
m7GpppN; [0470] b.) a 5''-UTR element which comprises or consists
of a nucleic acid sequence which is derived from the 5''-UTR of a
TOP gene (SEQ ID NOs: 235-238), a homolog, a fragment or a variant
thereof; [0471] c1.) optionally, at least one coding sequence
encoding at least one heterologous secretory signal sequence,
preferably selected from SEQ ID NOs 317-572; [0472] c2.) at least
one coding sequence encoding at least one antigenic peptide or
protein derived from a Nipah virus protein or peptide or a fragment
or variant thereof, preferably comprising or consisting of any one
of the nucleic acid sequences according 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, 1516-1539 or a fragment or variant thereof;
[0473] d.) a 3''-UTR element comprising or consisting of a nucleic
acid sequence which is derived from a gene providing a stable RNA,
preferably comprising or consisting of the corresponding RNA
sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or
252, a homolog, a fragment or a variant thereof; [0474] e.)
optionally, a poly(A) sequence preferably comprising 64 adenosines;
and [0475] f.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines.
[0476] Histone Stem-Loop:
[0477] In a particularly preferred embodiment, the nucleic acid
sequence, particularity the RNA sequence according to the invention
comprises a histone stem-loop sequence/structure. Such histone
stem-loop sequences are preferably selected from histone stem-loop
sequences as disclosed in WO 2012/019780, the disclosure of which
is incorporated herewith by reference.
[0478] A histone stem-loop sequence suitable to be used within the
present invention is preferably derived from formulae (I) or (II)
of the patent application WO2012/019780, herewith incorporated by
reference. According to a further preferred embodiment the RNA as
defined herein may comprise at least one histone stem-loop sequence
derived from at least one of the specific formulae (Ia) or (IIa) of
the patent application WO2012/019780.
[0479] A particular preferred histone stem-loop sequence is the
sequence according to SEQ ID NO: 253 or more preferably the
corresponding RNA sequence according to SEQ ID NO: 254.
[0480] Accordingly, in preferred embodiments, the artificial
nucleic acid of the invention comprises at least one histone
stem-loop as defined herein. Preferably, 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.
[0481] In particularly preferred embodiments, the artificial
nucleic acid, preferably the artificial mRNA of the invention
comprises a 3''-terminal sequence element comprising a
poyl(A)sequence as defined herein and a histons-stem-loop sequence
as defined herein, wherein the 3''-terminal sequence element may be
selected from SEQ ID NOs: 1509, 1510, 1511 or 1512.
[0482] mRNA Structures:
[0483] Any of the above modifications may be applied to the nucleic
acid sequence, in particular, to the DNA and/or RNA sequence of the
present invention, and further to any DNA or RNA as used in the
context of the present invention and may be, if suitable or
necessary, be combined with each other in any combination,
provided, these combinations of modifications do not interfere with
each other in the respective nucleic acid sequence. A person
skilled in the art will be able to take his choice accordingly.
[0484] The artificial nucleic acid sequence according to the
invention, particularly the RNA sequence according to the present
invention which comprises at least one coding sequence as defined
herein, may preferably comprise a 5''-UTR and/or a 3''-UTR
preferably containing at least one histone stem-loop. The 3''-UTR
of the RNA sequence according to the invention preferably comprises
also a poly(A) and/or a poly(C) sequence as defined herein. The
single elements of the 3''-UTR may occur therein in any order from
5'' to 3' along the sequence of the RNA sequence of the present
invention. In addition, further elements as described herein, may
also be contained, such as a stabilizing sequence as defined herein
(e.g. derived from the UTR of a globin gene), IRES sequences, etc.
Each of the elements may also be repeated in the RNA sequence
according to the invention at least once (particularly in di- or
multicistronic constructs), preferably twice or more. As an
example, the single elements may be present in the nucleic acid
sequence, particularly in the RNA sequence according to the
invention in the following order:
5''-coding sequence-histone stem-loop-poly(A)/(C) sequence-3''; or
5''-coding sequence-poly(A)/(C) sequence-histone stem-loop-3''; or
5''-coding sequence-histone stem-loop-polyadenylation signal-3'';
or 5''-coding sequence-polyadenylation signal-histone
stem-loop-3''; or 5''-coding sequence-histone stem-loop-histone
stem-loop-poly(A)/(C) sequence-3''; or 5''-coding sequence-histone
stem-loop-histone stem-loop-polyadenylation signal-3'; or
5''-coding sequence-stabilizing sequence-poly(A)/(C)
sequence-histone stem-loop-3''; or 5''-coding sequence-stabilizing
sequence-poly(A)/(C) sequence-poly(A)/(C) sequence-histone
stem-loop-3''; etc.
[0485] According to a further embodiment, the nucleic acid sequence
of the present invention, particularly the RNA sequence of the
present invention, preferably comprises at least one of the
following structural elements: a 5'- and/or 3''-untranslated region
element (UTR element), particularly a 5''-UTR element, which
preferably comprises or consists of a nucleic acid sequence which
is derived from the 5''-UTR of a TOP gene or from a fragment,
homolog or a variant thereof, or a 5'- and/or 3''-UTR element which
may preferably be derivable from a gene that provides a stable RNA
or from a homolog, fragment or variant thereof; a histone-stem-loop
structure, preferably a histone-stem-loop in its 3' untranslated
region; a 5'-cap structure; a poly-A tail; or a poly(C)
sequence.
[0486] In preferred embodiments the nucleic acid sequence, in
particular, the RNA sequence comprises, preferably in 5''- to
3''-direction, the following elements a)-h): [0487] a) 5''-cap
structure, preferably as defined herein; [0488] b) optionally,
5''-UTR element, preferably as defined herein; [0489] c) at least
one coding sequence, preferably as defined herein; [0490] d) a
3''-UTR element, preferably as defined herein; [0491] e)
optionally, poly(A) sequence, preferably as defined herein; [0492]
f) optionally, poly(C) sequence, preferably as defined herein;
[0493] g) optionally, a histone stem-loop, preferably as defined
herein; and [0494] h) optionally, a 3''-terminal sequence element
as defined herein.
[0495] In a preferred embodiment the nucleic acid sequence, in
particular, the RNA sequence comprises, preferably in 5''- to
3''-direction: [0496] a) a 5'-cap structure, preferably m7GpppN;
[0497] b1.) optionally, at least one coding sequence encoding at
least one heterologuous secretory signal sequence; [0498] b2.) at
least one coding sequence encoding at least one antigenic peptide
or protein derived from a Henipavirus protein or peptide as defined
herein or a fragment or variant thereof; [0499] c.) a 3''-UTR
element comprising or consisting of a nucleic acid sequence which
is derived from an alpha globin gene, preferably comprising the
corresponding RNA sequence of the nucleic acid sequence according
to SEQ ID NO: 246, a homolog, a fragment or a variant thereof;
[0500] d.) optionally, a poly(A) sequence, preferably comprising 64
adenosines; [0501] e.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines; [0502] f.) optionally, a histone
stem-loop, preferably comprising the RNA sequence according to SEQ
ID NO: 254; [0503] g.) optionally, a poly(A) sequence and a histone
stem-loop comprising the RNA sequence according to SEQ ID NOs:
1509, 1510, 1511 or 1512
[0504] In a particularly preferred embodiment the nucleic acid
sequence, in particular, the RNA sequence comprises, preferably in
5''- to 3'-direction: [0505] a.) a 5''-cap structure, preferably
m7GpppN; [0506] b1.) optionally, at least one coding sequence
encoding at least one heterologuous secretory signal sequence
according to SEQ ID NOs: 317-572; [0507] b2.) at least one coding
sequence encoding at least one antigenic peptide or protein derived
from a Hendra virus protein or peptide or a fragment or variant
thereof, preferably comprising or consisting of any one of the
nucleic acid according to 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 or a fragment
or variant thereof; [0508] c.) a 3''-UTR element comprising or
consisting of a nucleic acid sequence which is derived from an
alpha globin gene, preferably comprising the corresponding RNA
sequence of the nucleic acid sequence according to SEQ ID NO: 246,
a homolog, a fragment or a variant thereof; [0509] d.) optionally,
a poly(A) sequence, preferably comprising 64 adenosines; [0510] e.)
optionally, a poly(C) sequence, preferably comprising 30 cytosines;
[0511] f.) optionally, a histone stem-loop, preferably comprising
the RNA sequence according to SEQ ID NO: 254; [0512] g.)
optionally, a poly(A) sequence and a histone stem-loop comprising
the RNA sequence according to SEQ ID NOs: 1509, 1510, 1511 or
1512
[0513] In a further particularly preferred embodiment the nucleic
acid sequence, in particular, the RNA sequence comprises,
preferably in 5''- to 3''-direction: [0514] a.) a 5''-cap
structure, preferably m7GpppN; [0515] b1.) optionally, at least one
coding sequence encoding at least one heterologuous secretory
signal sequence according to SEQ ID NOs: 317-572; [0516] b2.) at
least one coding sequence encoding at least one antigenic peptide
or protein derived from a Nipah virus protein or peptide or a
fragment or variant thereof, preferably comprising or consisting of
any one of the nucleic acid according 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, 1516-1539 or a fragment or variant thereof; [0517] c.) a
3''-UTR element comprising or consisting of a nucleic acid sequence
which is derived from an alpha globin gene, preferably comprising
the corresponding RNA sequence of the nucleic acid sequence
according to SEQ ID NO: 246, a homolog, a fragment or a variant
thereof; [0518] d.) optionally, a poly(A) sequence, preferably
comprising 64 adenosines; [0519] e.) optionally, a poly(C)
sequence, preferably comprising 30 cytosines; [0520] f.)
optionally, a histone stem-loop, preferably comprising the RNA
sequence according to SEQ ID NO: 254; [0521] g.) optionally, a
poly(A) sequence and a histone stem-loop comprising the RNA
sequence according to SEQ ID NOs: 1509, 1510, 1511 or 1512
[0522] According to another particularly preferred embodiment the
nucleic acid sequence, in particular, the RNA sequence according to
the invention comprises, preferably in 5'- to 3''-direction: [0523]
a.) a 5''-cap structure, preferably m7GpppN; [0524] b.) a 5'-UTR
element which comprises or consists of a nucleic acid sequence
which is derived from the 5'-UTR of a TOP gene (SEQ ID NOs:
235-238), a homolog, a fragment or a variant thereof; [0525] c1.)
optionally, at least one coding sequence encoding at least one
heterologuous secretory signal sequence according to SEQ ID NOs:
317-572; [0526] c2.) at least one coding sequence encoding at least
one antigenic peptide or protein derived from a Hendra virus
protein or peptide or a fragment or variant thereof, preferably
comprising or consisting of any one of the nucleic acid sequences
according 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 or a fragment or variant
thereof; [0527] d.) a 3'-UTR element comprising or consisting of a
nucleic acid sequence which is derived from a gene providing a
stable RNA, preferably comprising or consisting of the
corresponding RNA sequence of a nucleic acid sequence according to
SEQ ID NOs: 250 or 252, a homolog, a fragment or a variant thereof;
[0528] e.) optionally, a poly(A) sequence preferably comprising 64
adenosines; [0529] f.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines; [0530] g.) optionally, a histone
stem-loop, preferably comprising the RNA sequence according to SEQ
ID NO: 254; [0531] h.) optionally, a poly(A) sequence and a histone
stem-loop comprising the RNA sequence according to SEQ ID NOs:
1509, 1510, 1511 or 1512.
[0532] According to another particularly preferred embodiment the
nucleic acid sequence, in particular, the RNA sequence according to
the invention comprises, preferably in 5'- to 3'-direction: [0533]
a.) a 5''-cap structure, preferably m7GpppN; [0534] b.) a 5''-UTR
element which comprises or consists of a nucleic acid sequence
which is derived from the 5''-UTR of a TOP gene (SEQ ID NOs:
235-238), a homolog, a fragment or a variant thereof; [0535] c1.)
optionally, at least one coding sequence encoding at least one
heterologuous secretory signal sequence according to SEQ ID NOs:
317-572; [0536] c2.) at least one coding sequence encoding at least
one antigenic peptide or protein derived from a Nipah virus protein
or peptide or a fragment or variant thereof, preferably comprising
or consisting of any one of the nucleic acid sequences according 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, 1516-1539 or a fragment
or variant thereof; [0537] d.) a 3''-UTR element comprising or
consisting of a nucleic acid sequence which is derived from a gene
providing a stable RNA, preferably comprising or consisting of the
corresponding RNA sequence of a nucleic acid sequence according to
SEQ ID NOs: 250 or 252, a homolog, a fragment or a variant thereof;
[0538] e.) optionally, a poly(A) sequence preferably comprising 64
adenosines; [0539] f.) optionally, a poly(C) sequence, preferably
comprising 30 cytosines; [0540] g.) optionally, a histone
stem-loop, preferably comprising the RNA sequence according to SEQ
ID NO:
[0541] 254; [0542] h.) optionally, a poly(A) sequence and a histone
stem-loop comprising the RNA sequence according to SEQ ID NOs:
1509, 1510, 1511 or 1512.
[0543] Preferred Hendra and Nipah Constructs of the Invention:
[0544] In the following, preferred and particularly suitable Hendra
virus and Nipah virus mRNA sequences of the invention are
provided.
[0545] Preferred Hendra polypeptide, nucleic acid and mRNA
sequences are provided in Table 5. Therein, each row (row 1-36)
represents a specific suitable Hendra virus construct of the
invention. The protein design/name is indicated for each row
(column "Name"). Accession numbers are provided in the <223>
identifier of the respective SEQ ID NOs in the sequence listing.
Column "SEQ ID NO: Protein" provides the respective SEQ ID NOs of
the protein constructs as provided in the sequence listing. mRNA
constructs comprising coding sequences encoding said proteins are
provided in column "SEQ ID NO: mRNA design 1" column "SEQ ID NO:
mRNA design 2" and column "SEQ ID NO: mRNA design 3". Additional
information regarding each of the sequences provided in Table 5 may
also be derived from the sequence listing, in particular from the
details provided therein under identifier <223>.
TABLE-US-00007 TABLE 5 Preferred Hendra virus polypeptide, nucleic
acid and mRNA sequences SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
Row Name Protein mRNA design 1 mRNA design 2 mRNA design 3 1 F 8
1282 1360 1438 2 F 9 1283 1361 1439 3 F 10 1284 1362 1440 4 F 11
1285 1363 1441 5 HsIgE(1-18)_F(27-546) 814 1308 1386 1464 6
HsIgE(1-18)_F(27-546) 815 1309 1387 1465 7 HsIgE(1-18)_F(26-546)
816 1310 1388 1466 8 HsIgE(1-18)_F(27-546) 817 1311 1389 1467 9
H1N1-HA(1-17)_F(27-546) 1048 1334 1412 1490 10
H1N1-HA(1-17)_F(27-546) 1049 1335 1413 1491 11
H1N1-HA(1-17)_F(26-546) 1050 1336 1414 1492 12
H1N1-HA(1-17)_F(27-546) 1051 1337 1415 1493 13 G 19 1293 1371 1449
14 G 20 1294 1372 1450 15 G 21 1295 1373 1451 16 G 22 1296 1374
1452 17 G 23 1297 1375 1453 18 G 24 1298 1376 1454 19 G 25 1299
1377 1455 20 G 26 1300 1378 1456 21 HsIgE(1-18)_G(70-604) 825 1319
1397 1475 22 HsIgE(1-18)_G(70-604) 826 1320 1398 1476 23
HsIgE(1-18)_G(70-604) 827 1321 1399 1477 24 HsIgE(1-18)_G(70-604)
828 1322 1400 1478 25 HsIgE(1-18)_G(70-604) 829 1323 1401 1479 26
HsIgE(1-18)_G(70-604) 830 1324 1402 1480 27 HsIgE(1-18)_G(70-604)
831 1325 1403 1481 28 HsIgE(1-18)_G(70-604) 832 1326 1404 1482 29
H1N1-HA(1-17)_G(70-604) 1059 1345 1423 1501 30
H1N1-HA(1-17)_G(70-604) 1060 1346 1424 1502 31
H1N1-HA(1-17)_G(70-604) 1061 1347 1425 1503 32
H1N1-HA(1-17)_G(70-604) 1062 1348 1426 1504 33
H1N1-HA(1-17)_G(70-604) 1063 1349 1427 1505 34
H1N1-HA(1-17)_G(70-604) 1064 1350 1428 1506 35
H1N1-HA(1-17)_G(70-604) 1065 1351 1429 1507 36
H1N1-HA(1-17)_G(70-604) 1066 1352 1430 1508
[0546] Preferred Nipah polypeptide, nucleic acid and mRNA sequences
are provided in Table 6. Therein, each row (row 1-42) represents a
specific suitable Nipah virus construct of the invention. The
protein design/name is indicated for each row (column "Name").
Accession numbers are provided in the <223> identifier of the
respective SEQ ID NOs in the sequence listing. Column "SEQ ID NO:
Protein" provides the respective SEQ ID NOs of the protein
constructs as provided in the sequence listing. mRNA constructs
comprising coding sequences encoding said proteins are provided in
column "SEQ ID NO: mRNA design 1" column "SEQ ID NO: mRNA design 2"
and column "SEQ ID NO: mRNA design 3". Additional information
regarding each of the sequences provided in Table 5 may also be
derived from the sequence listing, in particular from the details
provided therein under identifier <223>.
TABLE-US-00008 TABLE 6 Preferred Nipah virus polypeptide, nucleic
acid and mRNA sequences SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
Row Name Protein mRNA design 1 mRNA design 2 mRNA design 3 1 F 1
1275 1353 1431 2 F 2 1276 1354 1432 3 F 3 1277 1355 1433 4 F 4 1278
1356 1434 5 F 5 1279 1357 1435 6 F 6 1280 1358 1436 7 F 7 1281 1359
1437 8 HsIgE(1-18)_F(27-546) 807 1301 1379 1457 9
HsIgE(1-18)_F(27-546) 808 1302 1380 1458 10 HsIgE(1-18)_F(27-546)
809 1303 1381 1459 11 HsIgE(1-18)_F(27-546) 810 1304 1382 1460 12
HsIgE(1-18)_F(27-546) 811 1305 1383 1461 13 HsIgE(1-18)_F(27-546)
812 1306 1384 1462 14 HsIgE(1-18)_F(27-546) 813 1307 1385 1463 15
H1N1-HA(1-17)_F(27-546) 1041 1327 1405 1483 16
H1N1-HA(1-17)_F(27-546) 1042 1328 1406 1484 17
H1N1-HA(1-17)_F(27-546) 1043 1329 1407 1485 18
H1N1-HA(1-17)_F(27-546) 1044 1330 1408 1486 19
H1N1-HA(1-17)_F(27-546) 1045 1331 1409 1487 20
H1N1-HA(1-17)_F(27-546) 1046 1332 1410 1488 21
H1N1-HA(1-17)_F(27-546) 1047 1333 1411 1489 22 G 12 1286 1364 1442
23 G 13 1287 1365 1443 24 G 14 1288 1366 1444 25 G 15 1289 1367
1445 26 G 16 1290 1368 1446 27 G 17 1291 1369 1447 28 G 18 1292
1370 1448 29 HsIgE(1-18)_G(70-602) 818 1312 1390 1468 30
HsIgE(1-18)_G(70-602) 819 1313 1391 1469 31 HsIgE(1-18)_G(70-602)
820 1314 1392 1470 32 HsIgE(1-18)_G(70-602) 821 1315 1393 1471 33
HsIgE(1-18)_G(70-602) 822 1316 1394 1472 34 HsIgE(1-18)_G(70-602)
823 1317 1395 1473 35 HsIgE(1-18)_G(70-602) 824 1318 1396 1474 36
H1N1-HA(1-17)_G(70-602) 1052 1338 1416 1494 37
H1N1-HA(1-17)_G(70-602) 1053 1339 1417 1495 38
H1N1-HA(1-17)_G(70-602) 1054 1340 1418 1496 39
H1N1-HA(1-17)_G(70-602) 1055 1341 1419 1497 40
H1N1-HA(1-17)_G(70-602) 1056 1342 1420 1498 41
H1N1-HA(1-17)_G(70-602) 1057 1343 1421 1499 42
H1N1-HA(1-17)_G(70-602) 1058 1344 1422 1500 43
HsSPARC(1-17)_F(27-546) 1513 1540 1543 1546 44
HsCTRB2(1-18)_F(27-546) 1514 1541 1544 1547 45 Nipah 1515 1542 1545
1548 henipavirus_AAK50553_F(1- 26)F(27-546)
[0547] Accordingly, it is particularly preferred that the nucleic
acid sequence according to the invention 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.
[0548] Accordingly, it is particularly preferred that the nucleic
acid sequence according to the invention 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.
[0549] Accordingly, it is particularly preferred that the nucleic
acid sequence according to the invention 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.
[0550] Composition:
[0551] In a further aspect, the present invention concerns a
composition comprising at least one artificial nucleic acid
comprising at least one coding sequence as defined herein and a
pharmaceutically acceptable carrier. The composition according to
the invention is preferably provided as a pharmaceutical
composition or as a vaccine.
[0552] According to a preferred embodiment, the (pharmaceutical)
composition or the vaccine according to the invention comprises at
least one nucleic acid of the present invention, wherein the at
least one coding sequence of the at least one nucleic acid sequence
encodes at least one Henipavirus peptide or protein selected from
Henipavirus RNA-directed RNA polymerase (L), Henipavirus fusion
protein (F), Henipavirus non-structural protein (V), Henipavirus
glycoprotein (G), Henipavirus nucleoprotein (N), Henipavirus matrix
protein (M), Henipavirus phosphoprotein (P), Henipavirus protein C,
and Henipavirus protein W, as well as to fragments or variants of
all these proteins.
[0553] The (pharmaceutical) composition or vaccine according to the
invention may thus comprise at least one nucleic acid comprising at
least one nucleic acid sequence comprising at least one coding
region, encoding at least one Henipavirus antigenic peptide or
protein, particularly, at least one Henipavirus protein selected
from Henipavirus RNA-directed RNA polymerase (L), Henipavirus
fusion protein (F), Henipavirus non-structural protein (V),
Henipavirus glycoprotein (G), Henipavirus nucleoprotein (N),
Henipavirus matrix protein (M), Henipavirus phosphoprotein (P),
Henipavirus protein C, and Henipavirus protein W, a fragment or
variant thereof, wherein the at least one coding region of the at
least one nucleic acid sequence encodes one specific Henipavirus
antigenic peptide or protein as defined herein or a fragment or a
variant thereof.
[0554] According to a further preferred embodiment, the
(pharmaceutical) composition or the vaccine according to the
invention comprises at least one nucleic acid of the present
invention, wherein the at least one coding sequence of the at least
one nucleic acid sequence encodes at least one Hendra virus peptide
or protein selected from Hendra virus RNA-directed RNA polymerase
(L), Hendra virus fusion protein (F), Hendra virus non-structural
protein (V), Hendra virus glycoprotein (G), Hendra virus
nucleoprotein (N), Hendra virus matrix protein (M), Hendra virus
phosphoprotein (P), Hendra virus protein C, and Hendra virus
protein W, as well as to fragments or variants of all these
proteins.
[0555] In a particularly preferred embodiment, the (pharmaceutical)
composition or the vaccine according to the invention comprises at
least one nucleic acid of the present invention, wherein the at
least one coding sequence of the at least one nucleic acid sequence
encodes at least one Hendra virus peptide or protein selected from
Hendra virus fusion protein (F) and Hendra virus glycoprotein (G)
(and optionally a secretory signal sequence) as well as to
fragments or variants of all these proteins, preferably proteins or
peptides according to SEQ ID NOs: 8-11, 19-26, 580-583, 591-598,
814-817, 825-832, 1048-1051, 1059-1066 or a homolog, fragment or
variant of any of these sequences (see Table 1 and Table 1B, column
"A").
[0556] Preferably, the (pharmaceutical) composition or the vaccine
according to the invention comprises at least one nucleic acid of
the present invention, wherein the at least one coding sequence of
the at least one nucleic acid sequence encodes at least one Hendra
virus peptide or protein selected from Hendra virus fusion protein
(F) and Hendra virus glycoprotein (G) (and, optionally, a secretory
signal sequence) as well as to fragments or variants of all these
proteins, wherein the Hendra virus peptide or protein preferably
comprises or consists of an amino acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with any one of the amino acid sequences according to SEQ ID NOs:
8-11, 19-26, 580-583, 591-598, 814-817, 825-832, 1048-1051,
1059-1066, or a homolog, fragment or variant of any of these
sequences (see Table 1 and Table 1B, column "A") fragment or
variant of any one of these sequences.
[0557] More preferably, the (pharmaceutical) composition or the
vaccine according to the invention comprises at least one nucleic
acid comprising at least one nucleic acid sequence of the present
invention, wherein the at least one coding sequence of the at least
one nucleic acid sequence comprises or consists of a nucleic acid
sequence encoding at least one Hendra virus peptide or protein
(and, optionally, a secretory signal sequence) preferably comprises
or consists of an amino acid sequence having a sequence identity of
at least 80% with any one of the amino acid sequences according to
SEQ ID NOs: 8-11, 19-26, 580-583, 591-598, 814-817, 825-832,
1048-1051, 1059-1066, or a homolog, fragment or variant of any of
these sequences (see Table 1 and Table 1B, column "A") fragment or
variant of any one of these sequences.
[0558] In preferred embodiments, the (pharmaceutical) composition
or the vaccine according to the invention comprises at least one
nucleic acid comprising at least one nucleic acid sequence of the
present invention, wherein the at least one coding sequence
(encoding at least one Hendra virus antigenic peptide or protein,
and, optionally, a secretory signal sequence) of the at least one
nucleic acid sequence comprises or consists of any one of the
nucleic acid sequences according 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 (as defined in Table 1 and Table 1B) or a fragment or
variant of any one of these sequences.
[0559] According to another embodiment, the (pharmaceutical)
composition or the vaccine according to the invention comprises at
least one nucleic acid comprising at least one nucleic acid
sequence of the present invention, wherein the at least one coding
sequence (encoding at least one Hendra virus antigenic peptide or
protein, and, optionally, a secretory signal sequence) of the at
least one nucleic acid sequence comprises or consists of a nucleic
acid sequence having a sequence identity of at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least
70%, more preferably of at least 80%, even more preferably at least
85%, even more preferably of at least 90% and most preferably of at
least 95% or even 97%, with any one of the nucleic acid sequences
according to SEQ ID 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 (as defined in Table 1
and Table 1B) or a fragment or variant of any one of these
sequences.
[0560] According to another preferred embodiment, the
(pharmaceutical) composition or the vaccine according to the
invention comprises at least one nucleic acid comprising at least
one nucleic acid sequence of the present invention, wherein the at
least one coding sequence (encoding at least one Hendra virus
antigenic peptide or protein, and, optionally, a secretory signal
sequence) of the at least one nucleic acid sequence comprises or
consists of a nucleic acid sequence having a sequence identity of
at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,
preferably of at least 70%, more preferably of at least 80%, even
more preferably at least 85%, even more preferably of at least 90%
and most preferably of at least 95% or even 97%, with any one of
the 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 (as defined in Table 5)
or a fragment or variant of any one of these sequences.
[0561] In the context of the present invention, the
(pharmaceutical) composition or vaccine may encode one or more of
the Hendra virus antigenic proteins or peptides as defined herein
or a fragment or variant thereof.
[0562] The (pharmaceutical) composition or vaccine according to the
invention may thus comprise at least one nucleic acid comprising at
least one nucleic acid sequence comprising at least one coding
region, encoding at least one Hendra virus antigenic peptide or
protein, particularly, at least one Hendra virus protein selected
from Hendra virus RNA-directed RNA polymerase (L), Hendra virus
fusion protein (F), Hendra virus non-structural protein (V), Hendra
virus glycoprotein (G), Hendra virus nucleoprotein (N), Hendra
virus matrix protein (M), Hendra virus phosphoprotein (P), Hendra
virus protein C, and Hendra virus protein W, a fragment or variant
thereof, wherein the at least one coding region of the at least one
nucleic acid sequence encodes one specific Hendra virus antigenic
peptide or protein as defined herein or a fragment or a variant
thereof.
[0563] According to a further preferred embodiment, the
(pharmaceutical) composition or the vaccine according to the
invention comprises at least one nucleic acid of the present
invention, wherein the at least one coding sequence of the at least
one nucleic acid sequence encodes at least one Nipah virus peptide
or protein selected from Nipah virus RNA-directed RNA polymerase
(L), Nipah virus fusion protein (F), Nipah virus non-structural
protein (V), Nipah virus glycoprotein (G), Nipah virus
nucleoprotein (N), Nipah virus matrix protein (M), Nipah virus
phosphoprotein (P), Nipah virus protein C, and Nipah virus protein
W, as well as to fragments or variants of all these proteins.
[0564] In a particularly preferred embodiment, the (pharmaceutical)
composition or the vaccine according to the invention comprises at
least one nucleic acid of the present invention, wherein the at
least one coding sequence of the at least one nucleic acid sequence
encodes at least one Nipah virus peptide or protein selected from
Nipah virus fusion protein (F) and Nipah virus glycoprotein (G)
(and, optionally, a secretory signal sequence) as well as to
fragments or variants of all these proteins, preferably proteins or
peptides according to SEQ ID NOs: 1-7, 12-18, 573-579, 584-590,
807-813, 818-824, 1041-1047, 1052-1058, 1513-1515 or a homolog,
fragment or variant of any of these sequences (see Table 2 and
Table 2B, column "A").
[0565] Preferably, the (pharmaceutical) composition or the vaccine
according to the invention comprises at least one nucleic acid of
the present invention, wherein the at least one coding sequence of
the at least one nucleic acid sequence encodes at least one Nipah
virus peptide or protein selected from Nipah virus fusion protein
(F) and Nipah virus glycoprotein (G) (and, optionally, a secretory
signal sequence) as well as to fragments or variants of all these
proteins, wherein the Nipah virus peptide or protein preferably
comprises or consists of an amino acid sequence having a sequence
identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, preferably of at least 70%, more preferably of at
least 80%, even more preferably at least 85%, even more preferably
of at least 90% and most preferably of at least 95% or even 97%,
with any one of the amino acid sequences according to SEQ ID NOs:
1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047,
1052-1058, or a homolog, fragment or variant of any of these
sequences (see Table 2 and Table 2B, column "A") fragment or
variant of any one of these sequences.
[0566] More preferably, the (pharmaceutical) composition or the
vaccine according to the invention comprises at least one nucleic
acid comprising at least one nucleic acid sequence of the present
invention, wherein the at least one coding sequence of the at least
one nucleic acid sequence comprises or consists of a nucleic acid
sequence encoding at least one Nipah virus peptide or protein (and,
optionally, a secretory signal sequence) preferably comprises or
consists of an amino acid sequence having a sequence identity of at
least 80% with any one of the amino acid sequences according to SEQ
ID NOs: 1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047,
1052-1058, 1513-1515 or a homolog, fragment or variant of any of
these sequences (see Table 2 and Table 2B, column "A") fragment or
variant of any one of these sequences.
[0567] In preferred embodiments, the (pharmaceutical) composition
or the vaccine according to the invention comprises at least one
nucleic acid comprising at least one nucleic acid sequence of the
present invention, wherein the at least one coding sequence
(encoding at least one Nipah virus antigenic peptide or protein,
and, optionally, a secretory signal sequence) of the at least one
nucleic acid sequence comprises or consists of any one of the
nucleic acid sequences according 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, 1516-1539 (as defined in Table 2 and Table 2B) or a
fragment or variant of any one of these sequences.
[0568] According to another embodiment, the (pharmaceutical)
composition or the vaccine according to the invention comprises at
least one nucleic acid comprising at least one nucleic acid
sequence of the present invention, wherein the at least one coding
sequence (encoding at least one Nipah virus antigenic peptide or
protein, and, optionally, a secretory signal sequence) of the at
least one nucleic acid sequence comprises or consists of a nucleic
acid sequence having a sequence identity of at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least
70%, more preferably of at least 80%, even more preferably at least
85%, even more preferably of at least 90% and most preferably of at
least 95% or even 97%, with any one of the nucleic acid sequences
according to SEQ ID 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, 1516-1539 (as defined
in Table 2 and Table 2B) or a fragment or variant of any one of
these sequences.
[0569] According to another preferred embodiment, the
(pharmaceutical) composition or the vaccine according to the
invention comprises at least one nucleic acid comprising at least
one nucleic acid sequence of the present invention, wherein the at
least one coding sequence (encoding at least one Nipah virus
antigenic peptide or protein and, optionally, a secretory signal
sequence) of the at least one nucleic acid sequence comprises or
consists of a nucleic acid sequence having a sequence identity of
at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,
preferably of at least 70%, more preferably of at least 80%, even
more preferably at least 85%, even more preferably of at least 90%
and most preferably of at least 95% or even 97%, with any one of
the 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 (as defined
in Table 6) or a fragment or variant of any one of these
sequences
[0570] In the context of the present invention, the
(pharmaceutical) composition or vaccine may encode one or more of
the Nipah virus antigenic proteins or peptides as defined herein or
a fragment or variant thereof.
[0571] The (pharmaceutical) composition or vaccine according to the
invention may thus comprise at least one nucleic acid comprising at
least one nucleic acid sequence comprising at least one coding
region, encoding at least one Nipah virus antigenic peptide or
protein, particularly, at least one Nipah virus protein selected
from Nipah virus RNA-directed RNA polymerase (L), Nipah virus
fusion protein (F), Nipah virus non-structural protein (V), Nipah
virus glycoprotein (G), Nipah virus nucleoprotein (N), Nipah virus
matrix protein (M), Nipah virus phosphoprotein (P), Nipah virus
protein C, and Nipah virus protein W, a fragment or variant
thereof, wherein the at least one coding region of the at least one
nucleic acid sequence encodes one specific Nipah virus antigenic
peptide or protein as defined herein or a fragment or a variant
thereof.
[0572] Alternatively, the (pharmaceutical) composition or vaccine
of the present invention may comprise at least one nucleic acid
comprising at least one nucleic acid sequence according to the
invention, wherein the at least one nucleic acid sequence encodes
at least two, three, four, five, six, seven, eight, nine, ten,
eleven or twelve distinct Henipavirus and/or Hendra virus and/or
Nipah virus antigenic peptides or proteins as defined herein or a
fragment or variant thereof.
[0573] In this context it is particularly preferred that the at
least one nucleic acid comprised in the (pharmaceutical)
composition or vaccine is a bi- or multicistronic nucleic acid,
particularly a bi- or multicistronic nucleic acid as defined
herein, which encodes the at least two, three, four, five, six,
seven, eight, nine, ten, eleven or twelve distinct Henipavirus
and/or Hendra virus and/or Nipah virus peptides or proteins derived
from a protein of a Henipavirus and/or Hendra virus and/or Nipah
virus. Mixtures between these embodiments are also envisaged, such
as compositions comprising more than one nucleic acid sequences,
wherein at least one nucleic acid sequence may be monocistronic,
while at least one other nucleic acid sequence may be bi- or
multicistronic.
[0574] The (pharmaceutical) composition or vaccine according to the
present invention, preferably the at least one coding sequence of
the nucleic acid sequence comprised therein, may thus comprise any
combination of the nucleic acid sequences as defined herein.
[0575] Preferably, the (pharmaceutical) composition or vaccine
comprises a plurality or more than one of the nucleic sequences
according to the invention, wherein each nucleic acid sequence
comprises at least one coding region encoding at least one
antigenic peptide or protein derived from a protein of a
Henipavirus and/or Hendra virus and/or Nipah virus or a fragment or
variant thereof.
[0576] In a particularly preferred embodiment, the composition
comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more
different artificial nucleic acids each encoding at least one
antigenic peptide or protein derived from genetically the same
Henipavirus and/or Hendra virus and/or Nipah virus or a fragment or
variant thereof.
[0577] In another preferred embodiment each nucleic acid sequence
encodes at least one different Henipavirus and/or Hendra virus
and/or Nipah virus antigenic peptide or protein derived from
proteins of different Henipavirus and/or Hendra virus and/or Nipah
virus or a fragment or variant thereof.
[0578] In a particularly preferred embodiment, the composition
comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more
different artificial nucleic acids each encoding at least one
peptide or protein derived from a different Henipavirus and/or
Hendra virus and/or Nipah virus or a fragment or variant
thereof.
[0579] In an embodiment, the composition comprises at least one
artificial nucleic acid encoding at least one antigenic peptide or
protein derived from Hendra virus fusion protein (F) and/or at
least one artificial nucleic acid encoding at least one antigenic
peptide or protein derived from Hendra virus glycoprotein (G)
and/or at least one artificial nucleic acid encoding at least one
antigenic peptide or protein derived from Nipah virus fusion
protein (F) and/or at least one artificial nucleic acid encoding at
least one antigenic peptide or protein derived from Nipah virus
glycoprotein (G) or a fragment or variant thereof.
[0580] In a specific embodiment, the composition comprises at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Hendra virus fusion protein (F) and at
least one artificial nucleic acid encoding at least one antigenic
peptide or protein derived from Hendra virus glycoprotein (G) or a
fragment or variant thereof.
[0581] In a specific embodiment, the composition comprises at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Nipah virus fusion protein (F) and at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Nipah virus glycoprotein (G) or a fragment
or variant thereof.
[0582] In a specific embodiment, the composition comprises at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Nipah virus fusion protein (F) and at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Hendra virus glycoprotein (G) or a fragment
or variant thereof.
[0583] In a specific embodiment, the composition comprises at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Nipah virus fusion protein (F) and at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Hendra virus fusion protein (F) or a
fragment or variant thereof.
[0584] In a specific embodiment, the composition comprises at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Nipah virus glycoprotein (G) and at least
one artificial nucleic acid encoding at least one antigenic peptide
or protein derived from Hendra virus glycoprotein (G) or a fragment
or variant thereof.
[0585] Complexation and Formulation:
[0586] In a preferred embodiment of the composition according to
the invention, the at least one nucleic acid comprising at least
one nucleic acid sequence according to the invention is complexed
with one or more cationic or polycationic compounds, preferably
with cationic or polycationic polymers, cationic or polycationic
peptides or proteins, e.g. protamine, cationic or polycationic
polysaccharides and/or cationic or polycationic lipids.
[0587] According to a preferred embodiment, the at least one
nucleic acid of the composition according to the present invention
may be complexed with lipids to form one or more liposomes,
lipoplexes, or lipid nanoparticles. Therefore, in one embodiment,
the inventive composition comprises liposomes, lipoplexes, and/or
lipid nanoparticles comprising the at least one nucleic acid,
preferably RNA, more preferably mRNA.
[0588] Lipid-based formulations have been increasingly recognized
as one of the most promising delivery systems for nucleic acids,
particularly of RNA, due to their biocompatibility and their ease
of large-scale production. Cationic lipids have been widely studied
as synthetic materials for delivery of RNA. After mixing together,
nucleic acids are condensed by cationic lipids to form
lipid/nucleic acid complexes known as lipoplexes. These lipid
complexes are able to protect genetic material from the action of
nucleases and deliver it into cells by interacting with the
negatively charged cell membrane. Lipoplexes can be prepared by
directly mixing positively charged lipids at physiological pH with
negatively charged nucleic acids.
[0589] Conventional liposomes consist of a lipid bilayer that can
be composed of cationic, anionic, or neutral (phospho)lipids and
cholesterol, which encloses an aqueous core. Both the lipid bilayer
and the aqueous space can incorporate hydrophobic or hydrophilic
compounds, respectively. Liposome characteristics and behaviour in
vivo can be modified by addition of a hydrophilic polymer coating,
e.g. polyethylene glycol (PEG), to the liposome surface to confer
steric stabilization. Furthermore, liposomes can be used for
specific targeting by attaching ligands (e.g., antibodies,
peptides, and carbohydrates) to its surface or to the terminal end
of the attached PEG chains.
[0590] Liposomes are colloidal lipid-based and surfactant-based
delivery systems composed of a phospholipid bilayer surrounding an
aqueous compartment. They may present as spherical vesicles and can
range in size from 20 nm to a few microns. Cationic lipid-based
liposomes are able to complex with negatively charged nucleic acids
via electrostatic interactions, resulting in complexes that offer
biocompatibility, low toxicity, and the possibility of the
large-scale production required for in vivo clinical applications.
Liposomes can fuse with the plasma membrane for uptake; once inside
the cell, the liposomes are processed via the endocytic pathway and
the genetic material is then released from the endosome/carrier
into the cytoplasm. Liposomes have long been perceived as drug
delivery vehicles because of their superior biocompatibility, given
that liposomes are basically analogs of biological membranes, and
can be prepared from both natural and synthetic phospholipids.
[0591] Cationic liposomes have been traditionally the most commonly
used non-viral delivery systems for oligonucleotides, including
plasmid DNA, antisense oligos, and siRNA/small hairpin RNA-shRNA).
Cationic lipids, such as DOTAP,
(1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl
sulfate) can form complexes or lipoplexes with negatively charged
nucleic acids to form nanoparticles by electrostatic interaction,
providing high in vitro transfection efficiency. Furthermore,
neutral lipid-based nanoliposomes for RNA delivery as e.g. neutral
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based
nanoliposomes were developed.
[0592] Therefore, in one embodiment the at least one nucleic acid,
preferably the RNA of the composition according to the present
invention is complexed with cationic lipids and/or neutral lipids
and thereby forms liposomes, lipid nanoparticles, lipoplexes or
neutral lipid-based nanoliposomes.
[0593] In the context of the present invention, the term "lipid
nanoparticle", also referred to as "LNP", is not restricted to any
particular morphology, and includes any morphology generated when a
cationic lipid and optionally one or more further lipids are
combined, e.g. in an aqueous environment and/or in the presence of
an RNA. For example, a liposome, a lipid complex, a lipoplex, an
emulsion, a micelle, a lipidic nanocapsule, a nanosuspension and
the like are within the scope of a lipid nanoparticle (LNP).
[0594] LNPs typically comprise a cationic lipid and one or more
excipient selected from neutral lipids, charged lipids, steroids
and polymer conjugated lipids (e.g. PEGylated lipid). The nucleic
acid may be encapsulated in the lipid portion of the LNP or an
aqueous space enveloped by some or the entire lipid portion of the
LNP. The RNA or a portion thereof may also be associated and
complexed with the LNP. An LNP may comprise any lipid capable of
forming a particle to which the nucleic acids are attached, or in
which the one or more nucleic acids are encapsulated. Preferably,
the LNP comprising nucleic acids comprises one or more cationic
lipids, and one or more stabilizing lipids. Stabilizing lipids
include neutral lipids and PEGylated lipids.
[0595] In one embodiment, the LNP consists essentially of (i) at
least one cationic lipid; (ii) a neutral lipid; (iii) a sterol,
e.g., cholesterol; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA,
in a molar ratio of about 20-60% cationic lipid: 5-25% neutral
lipid: 25-55% sterol; 0.5-15% PEG-lipid.
[0596] In that context, a preferred sterol is cholesterol. The
sterol can be about 10 mol % to about 60 mol % or about 25 mol % to
about 40 mol % of the lipid particle. In one embodiment, the sterol
is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol %
of the total lipid present in the lipid particle. In another
embodiment, the LNPs include from about 5% to about 50% on a molar
basis of the sterol, e.g., about 15% to about 45%, about 20% to
about 40%, about 48%, about 40%, about 38.5%, about 35%, about
34.4%, about 31.5% or about 31% on a molar basis (based upon 100%
total moles of lipid in the lipid nanoparticle).
[0597] The cationic lipid of an LNP may be cationisable, i.e. it
becomes protonated as the pH is lowered below the pK of the
ionizable group of the lipid, but is progressively more neutral at
higher pH values. At pH values below the pK, the lipid is then able
to associate with negatively charged nucleic acids. In certain
embodiments, the cationic lipid comprises a zwitterionic lipid that
assumes a positive charge on pH decrease.
[0598] The LNP may comprise any further cationic or cationisable
lipid, i.e. any of a number of lipid species which carry a net
positive charge at a selective pH, such as physiological pH.
[0599] Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);
3-(N--(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),
N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl
carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane
(DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and
N-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). In some aspects, the lipid is selected from the
group consisting of 98N12-5, C12-200, and ckk-E12.
[0600] In some embodiments, the lipid is selected from the group
consisting of 98N12-5, C12-200, and ckk-E12.
[0601] In one embodiment, the nucleic acids may be formulated in an
aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in
the present invention may be prepared by the methods described in
U.S. Pat. No. 8,450,298, herein incorporated by reference in its
entirety. Suitable ionizable lipids can also be the compounds as
disclosed in Tables 1, 2 and 3 and claims 1-24 of International
Publication No. WO 2017/075531 A1, hereby incorporated by reference
in its entirety. In another embodiment, ionizable lipids can also
be the compounds as disclosed in International Publication No. WO
2015/074085 A1 (i.e. ATX-001 to ATX-032 or the compounds as
mentioned in claims 1-26), U.S. Appl. Nos. 61/905,724 and Ser. No.
15/614,499 or U.S. Pat. Nos. 9,593,077 and 9,567,296 hereby
incorporated by reference in their entirety.
[0602] Additionally, a number of commercial preparations of
cationic lipids are available which can be used in the present
invention. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from
Promega Corp., Madison, Wis.). The following lipids are cationic
and have a positive charge at below physiological pH: DODAP, DODMA,
DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
[0603] The further cationic lipid may also be an amino lipid.
Representative amino lipids include, but are not limited to,
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanediol (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate
(DLin-MC3-DMA); MC3 (US20100324120).
[0604] Other suitable (cationic) lipids are disclosed in
WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406,
WO2010/088537, WO2010/129709, WO2011/153493, US2011/0256175,
U52012/0128760, US2012/0027803, and U.S. Pat. No. 8,158,601. In
that context, the disclosures of WO2009/086558, WO2009/127060,
WO2010/048536, WO2010/054406, WO2010/088537, WO2010/129709,
WO2011/153493, US2011/0256175, US2012/0128760, US2012/0027803, and
U.S. Pat. No. 8,158,601 are incorporated herewith by reference.
[0605] The amount of the permanently cationic lipid or lipidoid may
be selected taking the amount of the nucleic acid cargo into
account. In one embodiment, these amounts are selected such as to
result in an N/P ratio of the nanoparticle(s) or of the composition
in the range from about 0.1 to about 20. In this context, the N/P
ratio is defined as the mole ratio of the nitrogen atoms ("N") of
the basic nitrogen-containing groups of the lipid or lipidoid to
the phosphate groups ("P") of the RNA which is used as cargo. The
N/P ratio may be calculated on the basis that, for example, 1 .mu.g
RNA typically contains about 3 nmol phosphate residues, provided
that the RNA exhibits a statistical distribution of bases. The
"N"-value of the lipid or lipidoid may be calculated on the basis
of its molecular weight and the relative content of permanently
cationic and--if present--cationisable groups.
[0606] In certain embodiments, the LNP comprises one or more
additional lipids which stabilize the formation of particles during
their formation.
[0607] Suitable stabilizing lipids include neutral lipids and
anionic lipids. The term "neutral lipid" refers to any one of a
number of lipid species that exist in either an uncharged or
neutral zwitterionic form at physiological pH.
[0608] Representative neutral lipids include
diacylphosphatidylcholines, diacylphosphatidylethanolamines,
ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and
cerebrosides.
[0609] Exemplary neutral lipids include, for example,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one
embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3phosphocholine (DSPC).
[0610] In some embodiments, the LNPs comprise a neutral lipid
selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various
embodiments, the molar ratio of the cationic lipid to the neutral
lipid ranges from about 2:1 to about 8:1.
[0611] LNP in vivo characteristics and behavior can be modified by
addition of a hydrophilic polymer coating, e.g. polyethylene glycol
(PEG), to the LNP surface to confer steric stabilization.
Furthermore, LNPs can be used for specific targeting by attaching
ligands (e.g. antibodies, peptides, and carbohydrates) to its
surface or to the terminal end of the attached PEG chains (e.g. via
PEGylated lipids).
[0612] In some embodiments, the LNPs comprise a polymer conjugated
lipid. The term "polymer conjugated lipid" refers to a molecule
comprising both a lipid portion and a polymer portion. An example
of a polymer conjugated lipid is a PEGylated lipid. The term
"PEGylated lipid" refers to a molecule comprising both a lipid
portion and a polyethylene glycol portion. PEGylated lipids are
known in the art and include
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-s-DMG) and the like.
[0613] In certain embodiments, the LNP comprises an additional,
stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated
lipid). Suitable polyethylene glycol-lipids include PEG-modified
phosphatidylethanolamine, PEG-modified phosphatidic acid,
PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20),
PEG-modified dialkylamines, PEG-modified diacylglycerols,
PEG-modified dialkylglycerols. Representative polyethylene
glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one
embodiment, the polyethylene glycol-lipid is N-[(methoxy
poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is
PEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylated
diacylglycerol (PEG-DAG) such as
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG
succinate diacylglycerol (PEG-S-DAG) such as
4-O-(2',3''-di(tetradecanoyloxy)propyl-1-O-(.omega.-methoxy(polyethoxy)et-
hyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a
PEG dialkoxypropylcarbamate such as
.omega.-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate
or
2,3-di(tetradecanoxy)propyl-N-(.omega.-methoxy(polyethoxy)ethyl)carbam-
ate.
[0614] Further examples of PEG-lipids suitable in that context are
provided in US20150376115A1 and WO2015199952, each of which is
incorporated by reference in its entirety.
[0615] In some embodiments, LNPs include less than about 3, 2, or 1
mole percent of PEG or PEG-modified lipid, based on the total moles
of lipid in the LNP. In further embodiments, LNPs comprise from
about 0.1% to about 20% of the PEG-modified lipid on a molar basis,
e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 10%,
about 5%, about 3.5%, about 3%, about 2,5%, about 2%, about 1.5%,
about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100%
total moles of lipids in the LNP).
[0616] In various embodiments, the molar ratio of the cationic
lipid to the PEGylated lipid ranges from about 100:1 to about
25:1.
[0617] The total amount of nucleic acid, particularly the RNA in
the lipid nanoparticles varies and may be defined depending on the
e.g. RNA to total lipid w/w ratio. In one embodiment of the
invention the RNA to total lipid ratio is less than 0.06 w/w,
preferably between 0.03 w/w and 0.04 w/w.
[0618] In a preferred embodiment, the composition according to the
invention comprises the nucleic acid comprising at least one
nucleic acid sequence according to the invention that is formulated
together with a cationic or polycationic compound and/or with a
polymeric carrier. Accordingly, in a further embodiment of the
invention, it is preferred that the nucleic acid as defined herein
or any other nucleic acid comprised in the inventive
(pharmaceutical) composition or vaccine is associated with or
complexed with a cationic or polycationic compound or a polymeric
carrier, optionally in a weight ratio selected from a range of
about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about
5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1
(w/w) to about 1:1 (w/w) or of about 3:1 (w/w) to about 1:1 (w/w),
and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w)
of mRNA or nucleic acid to cationic or polycationic compound and/or
with a polymeric carrier; or optionally in a nitrogen/phosphate
(N/P) ratio of mRNA or nucleic acid to cationic or polycationic
compound and/or polymeric carrier in the range of about 0.1-10,
preferably in a range of about 0.3-4 or 0.3-1, and most preferably
in a range of about 0.5-1 or 0.7-1, and even most preferably in a
range of about 0.3-0.9 or 0.5-0.9. More preferably, the N/P ratio
of the at least one mRNA to the one or more polycations is in the
range of about 0.1 to 10, 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.
[0619] Therein, the nucleic acid as defined herein or any other
nucleic acid comprised in the (pharmaceutical) composition or
vaccine according to the invention can also be associated with a
vehicle, transfection or complexation agent for increasing the
transfection efficiency and/or the immunostimulatory properties of
the nucleic acid according to the invention or of optionally
comprised further included nucleic acids.
[0620] Cationic or polycationic compounds, being particularly
preferred agents in this context include protamine, nucleoline,
spermine or spermidine, or other cationic peptides or proteins,
such as poly-L-lysine (PLL), poly-arginine, basic polypeptides,
cell penetrating peptides (CPPs), including HIV-binding peptides,
HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or
analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein
transduction domains (PTDs), PpT620, prolin-rich peptides,
arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),
Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived
peptides (particularly from Drosophila antennapedia), pAntp, plsl,
FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1),
pVEC, hCT-derived peptides, SAP, or histones. More preferably, the
mRNA according to the invention is complexed with one or more
polycations, preferably with protamine or oligofectamine, most
preferably with protamine. In this context protamine is
particularly preferred.
[0621] Additionally, preferred cationic or polycationic proteins or
peptides may be selected from the following proteins or peptides
having the following total formula (III):
(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x, (formula (III))
wherein l+m+n+o+x=8-15, and l, m, n or o independently of each
other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or 15, provided that the overall content of Arg,
Lys, His and Orn represents at least 50% of all amino acids of the
oligopeptide; and Xaa may be any amino acid selected from native
(=naturally occurring) or non-native amino acids except of Arg,
Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3
or 4, provided, that the overall content of Xaa does not exceed 50%
of all amino acids of the oligopeptide. Particularly preferred
cationic peptides in this context are e.g. Arg7, Arg8, Arg9, H3R9,
R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc. In this context the
disclosure of WO 2009/030481 is incorporated herewith by
reference.
[0622] Preferred cationic or polycationic proteins or peptides may
be derived from formula
Cys{(Arg).sub.l;(Lys)m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys or
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x} of
the patent application WO2009/030481 or WO2011/026641, the
disclosure of WO2009/030481 and WO2011/026641 relating thereto are
incorporated herewith by reference. In a preferred embodiment, the
cationic or polycationic proteins or peptides comprises
CHHHHHHRRRRHHHHHHC (SEQ ID NO: 309), CR.sub.12C (SEQ ID NO: 306),
CR.sub.12 (SEQ ID NO: 307) or WR.sub.12C (SEQ ID NO: 308).
[0623] Further preferred cationic or polycationic compounds, which
can be used as transfection or complexation agent may include
cationic polysaccharides, for example chitosan, polybrene, cationic
polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium
chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,
DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,
DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:
Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:
dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:
O,O-ditetradecanoyl-N-(.alpha.-trimethylammonioacetyl)diethanolamine
chloride, CLIP1:
rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,
CLIP9:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylamm-
onium, oligofectamine, or cationic or polycationic polymers, e.g.
modified polyaminoacids, such as .beta.-aminoacid-polymers or
reversed polyamides, etc., modified polyethylenes, such as PVP
(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified
acrylates, such as pDMAEMA (poly(dimethylaminoethyl
methylacrylate)), etc., modified amidoamines such as pAMAM
(poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such
as diamine end modified 1,4 butanediol
diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such
as polypropylamine dendrimers or pAMAM based dendrimers, etc.,
polyimine(s), such as PEI: poly(ethyleneimine),
poly(propyleneimine), etc., polyallylamine, sugar backbone based
polymers, such as cyclodextrin based polymers, dextran based
polymers, chitosan, etc., silan backbone based polymers, such as
PMOXA-PDMS copolymers, etc., blockpolymers consisting of a
combination of one or more cationic blocks (e.g. selected from a
cationic polymer as mentioned above) and of one or more hydrophilic
or hydrophobic blocks (e.g. polyethyleneglycole); etc.
[0624] According to a preferred embodiment, the composition of the
present invention comprises the nucleic acid as defined herein,
preferably an RNA, and a polymeric carrier. A polymeric carrier
used according to the invention might be a polymeric carrier formed
by disulfide-crosslinked cationic components. The
disulfide-crosslinked cationic components may be the same or
different from each other. The polymeric carrier can also contain
further components. It is also particularly preferred that the
polymeric carrier used according to the present invention comprises
mixtures of cationic peptides, proteins or polymers and optionally
further components as defined herein, which are crosslinked by
disulfide bonds as described herein. In this context, the
disclosure of WO 2012/013326 is incorporated herewith by
reference.
[0625] In this context, the cationic components, which form basis
for the polymeric carrier by disulfide-crosslinkage, are typically
selected from any suitable cationic or polycationic peptide,
protein or polymer suitable for this purpose, particular any
cationic or polycationic peptide, protein or polymer capable of
complexing the mRNA as defined herein or a further nucleic acid
comprised in the composition, and thereby preferably condensing the
mRNA or the nucleic acid. The cationic or polycationic peptide,
protein or polymer, is preferably a linear molecule, however,
branched cationic or polycationic peptides, proteins or polymers
may also be used.
[0626] Every disulfide-crosslinking cationic or polycationic
protein, peptide or polymer of the polymeric carrier, which may be
used to complex the mRNA according to the invention or any further
nucleic acid comprised in the (pharmaceutical) composition or
vaccine of the present invention contains at least one--SH moiety,
most preferably at least one cysteine residue or any further
chemical group exhibiting an --SH moiety, capable of forming a
disulfide linkage upon condensation with at least one further
cationic or polycationic protein, peptide or polymer as cationic
component of the polymeric carrier as mentioned herein.
[0627] As defined above, the polymeric carrier, which may be used
to complex the nucleic acid of the present invention or any further
nucleic acid comprised in the (pharmaceutical) composition or
vaccine according to the invention may be formed by
disulfide-crosslinked cationic (or polycationic) components.
Preferably, such cationic or polycationic peptides or proteins or
polymers of the polymeric carrier, which comprise or are
additionally modified to comprise at least one --SH moiety, are
selected from, proteins, peptides and polymers as defined herein
for complexation agent.
[0628] In a further particular embodiment, the polymeric carrier
which may be used to complex the nucleic acid as defined herein or
any further nucleic acid comprised in the (pharmaceutical)
composition or vaccine according to the invention may be selected
from a polymeric carrier molecule according to generic formula
(IV):
L-P.sup.1--S[S--P.sup.2--S].sub.n--S--P.sup.3-L formula (IV)
wherein, [0629] P.sup.1 and P.sup.3 are different or identical to
each other and represent a linear or branched hydrophilic polymer
chain, each P.sup.1 and P.sup.3 exhibiting at least one
--SH-moiety, capable to form a disulfide linkage upon condensation
with component P.sup.2, or alternatively with (AA), (AA).sub.x, or
[(AA).sub.x].sub.z if such components are used as a linker between
P.sup.1 and P.sup.2 or P.sup.3 and P.sup.2) and/or with further
components (e.g. (AA), (AA).sub.x, [(AA).sub.x].sub.z or L), the
linear or branched hydrophilic polymer chain selected independent
from each other from polyethylene glycol (PEG),
poly-N-(2-hydroxypropyl)methacrylamide,
poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl
L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),
hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the
hydrophilic polymer chain exhibits a molecular weight of about 1
kDa to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or
more preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to
about 25 kDa or 5 kDa to about 10 kDa; [0630] P.sup.2 is a cationic
or polycationic peptide or protein, e.g. as defined above for the
polymeric carrier formed by disulfide-crosslinked cationic
components, and preferably having a length of about 3 to about 100
amino acids, more preferably having a length of about 3 to about 50
amino acids, even more preferably having a length of about 3 to
about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15, 10
to 20 or 15 to 25 amino acids, more preferably a length of about 5
to about 20 and even more preferably a length of about 10 to about
20; or [0631] is a cationic or polycationic polymer, e.g. as
defined above for the polymeric carrier formed by
disulfide-crosslinked cationic components, typically having a
molecular weight of about 0.5 kDa to about 30 kDa, including a
molecular weight of about 1 kDa to about 20 kDa, even more
preferably of about 1.5 kDa to about 10 kDa, or having a molecular
weight of about 0.5 kDa to about 100 kDa, including a molecular
weight of about 10 kDa to about 50 kDa, even more preferably of
about 10 kDa to about 30 kDa; [0632] each P.sup.2 exhibiting at
least two --SH-moieties, capable to form a disulfide linkage upon
condensation with further components P.sup.2 or component(s)
P.sup.1 and/or P.sup.3 or alternatively with further components
(e.g. (AA), (AA).sub.x, or [(AA).sub.x].sub.z); [0633] --S--S-- is
a (reversible) disulfide bond (the brackets are omitted for better
readability), wherein S preferably represents sulphur or a --SH
carrying moiety, which has formed a (reversible) disulfide bond.
The (reversible) disulfide bond is preferably formed by
condensation of --SH-moieties of either components P.sup.1 and
P.sup.2, P.sup.2 and P.sup.2, or P.sup.2 and P.sup.3, or optionally
of further components as defined herein (e.g. L, (AA), (AA).sub.x,
[(AA).sub.x].sub.z, etc); The --SH-moiety may be part of the
structure of these components or added by a modification as defined
below; [0634] L is an optional ligand, which may be present or not,
and may be selected independent from the other from RGD,
Transferrin, Folate, a signal peptide or signal sequence, a
localization signal or sequence, a nuclear localization signal or
sequence (NLS), an antibody, a cell penetrating peptide, (e.g. TAT
or KALA), a ligand of a receptor (e.g. cytokines, hormones, growth
factors etc), small molecules (e.g. carbohydrates like mannose or
galactose or synthetic ligands), small molecule agonists,
inhibitors or antagonists of receptors (e.g. RGD peptidomimetic
analogues), or any further protein as defined herein, etc.; [0635]
n is an integer, typically selected from a range of about 1 to 50,
preferably from a range of about 1, 2 or 3 to 30, more preferably
from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about
1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15,
or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range
of about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20,
or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a
range of about 6 to 11 or 7 to 10. Most preferably, n is in a range
of about 1, 2, 3, 4, or 5 to 10, more preferably in a range of
about 1, 2, 3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8,
or in a range of about 1, 2, or 3 to 7.
[0636] In this context, the disclosure of WO 2011/026641 is
incorporated herewith by reference. Each of hydrophilic polymers P1
and P3 typically exhibits at least one --SH-moiety, wherein the at
least one --SH-moiety is capable to form a disulfide linkage upon
reaction with component P2 or with component (AA) or (AA)x, if used
as linker between P1 and P2 or P3 and P2 as defined below and
optionally with a further component, e.g. L and/or (AA) or (AA)x,
e.g. if two or more --SH-moieties are contained. The following
subformulae "P1-S--S--P2" and "P2-S--S--P3" within generic formula
(IV) above (the brackets are omitted for better readability),
wherein any of S, P1 and P3 are as defined herein, typically
represent a situation, wherein one --SH-moiety of hydrophilic
polymers P1 and P3 was condensed with one --SH-moiety of component
P2 of generic formula (IV) above, wherein both sulphurs of these
--SH-moieties form a disulfide bond --S--S-- as defined herein in
formula (IV). These --SH-moieties are typically provided by each of
the hydrophilic polymers P1 and P3, e.g. via an internal cysteine
or any further (modified) amino acid or compound which carries a
--SH moiety. Accordingly, the subformulae "P1-S--S--P2" and
"P2-S--S--P3" may also be written as "P1-Cys-Cys-P2" and
"P2-Cys-Cys-P3", if the --SH-- moiety is provided by a cysteine,
wherein the term "Cys-Cys" represents two cysteines coupled via a
disulfide bond, not via a peptide bond. In this case, the term
"--S--S--" in these formulae may also be written as "--S-Cys", as
"-Cys-S" or as "-Cys-Cys-". In this context, the term "-Cys-Cys-"
does not represent a peptide bond but a linkage of two cysteines
via their --SH-moieties to form a disulfide bond. Accordingly, the
term "-Cys-Cys-" also may be understood generally as
"-(Cys-S)-(S-Cys)-", wherein in this specific case S indicates the
sulphur of the --SH-moiety of cysteine. Likewise, the terms
"--S-Cys" and "--Cys-S" indicate a disulfide bond between a --SH
containing moiety and a cysteine, which may also be written as
"--S--(S-Cys)" and "-(Cys-S)--S". Alternatively, the hydrophilic
polymers P1 and P3 may be modified with a --SH moiety, preferably
via a chemical reaction with a compound carrying a --SH moiety,
such that each of the hydrophilic polymers P1 and P3 carries at
least one such --SH moiety. Such a compound carrying a --SH moiety
may be e.g. an (additional) cysteine or any further (modified)
amino acid, which carries a --SH moiety. Such a compound may also
be any non-amino compound or moiety, which contains or allows to
introduce a --SH moiety into hydrophilic polymers P1 and P3 as
defined herein. Such non-amino compounds may be attached to the
hydrophilic polymers P1 and P3 of formula (IV) of the polymeric
carrier according to the present invention via chemical reactions
or binding of compounds, e.g. by binding of a 3-thio propionic acid
or thioimolane, by amide formation (e.g. carboxylic acids,
sulphonic acids, amines, etc), by Michael addition (e.g maleinimide
moieties, .alpha.,.beta.-unsatured carbonyls, etc), by click
chemistry (e.g. azides or alkines), by alkene/alkine methatesis
(e.g. alkenes or alkines), imine or hydrozone formation (aldehydes
or ketons, hydrazins, hydroxylamins, amines), complexation
reactions (avidin, biotin, protein G) or components which allow
Sn-type substitution reactions (e.g halogenalkans, thiols,
alcohols, amines, hydrazines, hydrazides, sulphonic acid esters,
oxyphosphonium salts) or other chemical moieties which can be
utilized in the attachment of further components. A particularly
preferred PEG derivate in this context is
alpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case,
the SH-moiety, e.g. of a cysteine or of any further (modified)
amino acid or compound, may be present at the terminal ends or
internally at any position of hydrophilic polymers P1 and P3. As
defined herein, each of hydrophilic polymers P1 and P3 typically
exhibits at least one --SH-moiety preferably at one terminal end,
but may also contain two or even more --SH-moieties, which may be
used to additionally attach further components as defined herein,
preferably further functional peptides or proteins e.g. a ligand,
an amino acid component (AA) or (AA)x, antibodies, cell penetrating
peptides or enhancer peptides (e.g. TAT, KALA), etc.
[0637] In a particularly preferred embodiment, the polymeric
carrier is a peptide polymer, preferably a polyethylene
glycol/peptide polymer comprising
HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S-).sub.7-S-PEG.sub.5000-OH
(peptide monomer: SEQ ID NO: 309) and a lipid component, preferably
a lipidoid component, more preferably lipidoid 3-012-OH.
[0638] The lipidoid 3-012-OH
##STR00001##
(as shown above) may be 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 3-012-OH is 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 (cf. compound C12
and compound 110 in FIG. 1 of Love et al.). In preferred
embodiments, the peptide polymer comprising lipidoid 3-C12-0H as
specified above is used to complex the artificial nucleic acid of
the invention, in particular RNA, to form complexes having an N/P
ratio from about 0.1 to about 20, or from about 0.2 to about 15, or
from about 2 to about 15, or from about 2 to about 12, wherein the
N/P ratio is defined as the mole ratio of the nitrogen atoms of the
basic groups of the cationic peptide or polymer to the phosphate
groups of the artificial nucleic acid.
[0639] In another embodiment, the polymeric carrier comprises a
lipidoid compound according to formula Ia
##STR00002##
wherein [0640] R.sub.A is independently selected for each
occurrence an unsubstituted, cyclic or acyclic, branched or
unbranched C.sub.1-20 aliphatic group; a substituted or
unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20
heteroaliphatic group; a substituted or unsubstituted aryl; a
substituted or unsubstituted heteroaryl;
##STR00003##
[0640] wherein at least one R.sub.A is
##STR00004## [0641] R.sub.5 is independently selected for each
occurrence of from an unsubstituted, cyclic or acyclic, branched or
unbranched C.sub.8-16 aliphatic; a substituted or unsubstituted
aryl; or a substituted or unsubstituted heteroaryl; [0642] each
occurrence of x is an integer from 1 to 10; [0643] each occurrence
of y is an integer from 1 to 10; or a pharmaceutically acceptable
salt thereof.
[0644] In that context, the disclosure of the PCT patent
application PCT/EP2017/064059 is herewith incorporated by
reference.
[0645] In other embodiments, the composition, which is preferably a
(pharmaceutical) composition comprises at least one artificial
nucleic acid as described herein, wherein the at least one
artificial nucleic acid is complexed or associated with polymeric
carriers and, optionally, with at least one lipid component as
described in the PCT applications PCT/EP2017/064065,
PCT/EP2017/064058. In this context, the disclosures of
PCT/EP2017/064065, and PCT/EP2017/064058 is herewith incorporated
by reference.
[0646] Preferably, the inventive composition comprises at least one
nucleic acid as defined herein, which is complexed with one or more
polycations, and at least one free nucleic acid, wherein the at
least one complexed nucleic acid is preferably identical to the at
least one free nucleic acid. In this context, it is particularly
preferred that the composition of the present invention comprises
the nucleic acid, preferably the RNA, according to the invention
that is complexed at least partially with a cationic or
polycationic compound and/or a polymeric carrier, preferably
cationic proteins or peptides. In this context, the disclosure of
WO 2010/037539 and WO 2012/113513 is incorporated herewith by
reference. Partially means that only a part of the nucleic acid as
defined herein is complexed in the composition according to the
invention with a cationic compound and that the rest of the nucleic
acid as defined herein is (comprised in the inventive
(pharmaceutical) composition or vaccine) in uncomplexed form
("free"). Preferably, 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. More
preferably the ratio of complexed nucleic acid to free nucleic acid
(in the (pharmaceutical) composition or vaccine of the present
invention) is selected from a range of about 5:1 (w/w) to about
1:10 (w/w), more preferably from a range of about 4:1 (w/w) to
about 1:8 (w/w), even more preferably from a range of about 3:1
(w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the
ratio of complexed nucleic acid to free nucleic acid in the
inventive pharmaceutical composition or vaccine is selected from a
ratio of about 1:1 (w/w).
[0647] The complexed nucleic acid in the (pharmaceutical)
composition or vaccine according to the present invention, is
preferably prepared according to a first step by complexing the
nucleic acid according to the invention with a cationic or
polycationic compound and/or with a polymeric carrier, preferably
as defined herein, in a specific ratio to form a stable complex. In
this context, it is highly preferable, that no free cationic or
polycationic compound or polymeric carrier or only a negligibly
small amount thereof remains in the component of the complexed
nucleic acid after complexing the nucleic acid. Accordingly, the
ratio of the nucleic acid and the cationic or polycationic compound
and/or the polymeric carrier in the component of the complexed RNA
is typically selected in a range so that the nucleic acid is
entirely complexed and no free cationic or polycationic compound or
polymeric carrier or only a negligibly small amount thereof remains
in the composition.
[0648] Preferably the ratio of the nucleic acid, preferably the RNA
as defined herein to the cationic or polycationic compound and/or
the polymeric carrier, preferably as defined herein, is 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). Alternatively, the ratio of the nucleic
acid as defined herein to the cationic or polycationic compound
and/or the polymeric carrier, preferably as defined herein, in the
component of the complexed nucleic acid, may also be calculated on
the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire
complex. In the context of the present invention, an N/P-ratio is
preferably in the range of about 0.1-10, preferably in a range of
about 0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2
regarding the ratio of mRNA: cationic or polycationic compound
and/or polymeric carrier, preferably as defined herein, in the
complex, and most preferably in a range of about 0.7-1.5, 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, preferably provided that the cationic or polycationic
compound in the complex is a cationic or polycationic cationic or
polycationic protein or peptide and/or the polymeric carrier as
defined above. In this specific embodiment the complexed mRNA as
defined herein is also emcompassed in the term "adjuvant
component".
[0649] In other embodiments, the composition according to the
invention comprising the nucleic acid, preferably the mRNA as
defined herein may be administered naked without being associated
with any further vehicle, transfection or complexation agent.
[0650] It has to be understood and recognized, that according to
the present invention, the inventive composition may comprise at
least one naked nucleic acid, particularly naked mRNA as defined
herein and/or at least one formulated/complexed mRNA as defined
herein, wherein every formulation and/or complexation as disclosed
above may be used.
[0651] Adjuvants:
[0652] According to another embodiment, the (pharmaceutical)
composition or vaccine according to the invention may comprise an
adjuvant, which is preferably added in order to enhance the
immunostimulatory properties of the composition. In this context,
an adjuvant may be understood as any compound, which is suitable to
support administration and delivery of the composition according to
the invention. Furthermore, such an adjuvant may, without being
bound thereto, initiate or increase an immune response of the
innate immune system, i.e. a non-specific immune response. In other
words, when administered, the composition according to the
invention typically initiates an adaptive immune response due to an
NIPAH virus antigen as defined herein or a fragment or variant
thereof, which is encoded by the at least one coding sequence of
the inventive mRNA contained in the composition of the present
invention. Additionally, the composition according to the invention
may generate an (supportive) innate immune response due to addition
of an adjuvant as defined herein to the composition according to
the invention.
[0653] Such an adjuvant may be selected from any adjuvant known to
a skilled person and suitable for the present case, i.e. supporting
the induction of an immune response in a mammal. Preferably, the
adjuvant may be selected from the group consisting of, without
being limited thereto, TDM, MDP, muramyl dipeptide, pluronics, alum
solution, aluminium hydroxide, ADJUMER.TM. (polyphosphazene);
aluminium phosphate gel; glucans from algae; algammulin; aluminium
hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide
gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of
squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%),
phosphate-buffered saline, pH 7.4); AVRIDINE.TM. (propanediamine);
BAY R1005.TM.
((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-a-
mide hydroacetate); CALCITRIOL.TM. (1-alpha,25-dihydroxy-vitamin
D3); calcium phosphate gel; CAP.TM. (calcium phosphate
nanoparticles); cholera holotoxin,
cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of
the cholera toxin; CRL 1005 (block copolymer P1205);
cytokine-containing liposomes; DDA (dimethyldioctadecylammonium
bromide); DHEA (dehydroepiandrosterone); DMPC
(dimyristoylphosphatidylcholine); DMPG
(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic
acid sodium salt); Freund's complete adjuvant; Freund's incomplete
adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i)
N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine
(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)
zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP
(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);
imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);
ImmTherm.TM.
(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol
dipalmitate); DRVs (immunoliposomes prepared from
dehydration-rehydration vesicles); interferon-gamma;
interleukin-1beta; interleukin-2; interleukin-7; interleukin-12;
ISCOMS.TM.; ISCOPREP 7.0.3..TM.; liposomes; LOXORIBINE.TM.
(7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile
enterotoxin-protoxin); microspheres and microparticles of any
composition; MF59.TM.; (squalene-water emulsion); MONTANIDE ISA
51.TM. (purified incomplete Freund's adjuvant); MONTANIDE ISA
720.TM. (metabolisable oil adjuvant); MPL.TM.
(3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE
liposomes
((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glyce-
ro-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt);
MURAMETIDE.TM. (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE.TM. and
D-MURAPALMITINE.TM.
(Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO
(neuraminidase-galactose oxidase); nanospheres or nanoparticles of
any composition; NISVs (non-ionic surfactant vesicles); PLEURAN.TM.
(.beta.-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic
acid and glycolic acid; microspheres/nanospheres); PLURONIC
L121.TM.; PMMA (polymethyl methacrylate); PODDS.TM. (proteinoid
microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU
(polyadenylic acid-polyuridylic acid complex); polysorbate 80
(Tween 80); protein cochleates (Avanti Polar Lipids, Inc.,
Alabaster, Ala.); STIMULON.TM. (QS-21); Quil-A (Quil-A saponin);
S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5
c]quinoline-1-ethanol); SAF-1.TM. ("Syntex adjuvant formulation");
Sendai proteoliposomes and Sendai-containing lipid matrices;
Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span
85 and Tween 85); squalene or Robane.RTM.
(2,6,10,15,19,23-hexamethyltetracosan and
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);
stearyltyrosine (octadecyltyrosine hydrochloride); Theramid.RTM.
(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypro-
pylamide); Theronyl-MDP (Termurtide.TM. or [thr 1]-MDP;
N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs
or virus-like particles); Walter-Reed liposomes (liposomes
containing lipid A adsorbed on aluminium hydroxide), and
lipopeptides, including Pam3Cys, in particular aluminium salts,
such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including
CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin;
copolymers, including Optivax (CRL1005), L121, Poloaxmer4010),
etc.; liposomes, including Stealth, cochleates, including BIORAL;
plant derived adjuvants, including QS21, Quil A, lscomatrix, ISCOM;
adjuvants suitable for costimulation including Tomatine,
biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants,
including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid
sequences, CpG7909, ligands of human TLR 1-10, ligands of murine
TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529,
IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,
Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,
UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as
antagonists including CGRP neuropeptide.
[0654] Particularly preferred, an adjuvant may be selected from
adjuvants, which support induction of a Th1-immune response or
maturation of naive T-cells, such as GM-CSF, IL-12, IFN.gamma., any
immunostimulatory nucleic acid as defined above, preferably an
immunostimulatory RNA, CpG DNA, etc.
[0655] In a further preferred embodiment it is also possible that
the inventive composition contains besides the antigen-providing
nucleic acid as disclosed herein further components which are
selected from the group comprising: further antigens (e.g. in the
form of a peptide or protein) or further antigen-encoding nucleic
acids; a further immunotherapeutic agent; one or more auxiliary
substances; or any further compound, which is known to be immune
stimulating due to its binding affinity (as ligands) to human
Toll-like receptors; and/or an adjuvant nucleic acid, preferably an
immunostimulatory RNA (isRNA).
[0656] The composition of the present invention can additionally
contain one or more auxiliary substances in order to increase its
immunogenicity or immunostimulatory capacity, if desired. A
synergistic action of the nucleic acid or preferably the mRNA as
defined herein and of an auxiliary substance, which may be
optionally contained in the inventive composition, is preferably
achieved thereby. Depending on the various types of auxiliary
substances, various mechanisms can come into consideration in this
respect. For example, compounds that permit the maturation of
dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha
or CD40 ligand, form a first class of suitable auxiliary
substances. In general, it is possible to use as auxiliary
substance any agent that influences the immune system in the manner
of a "danger signal" (LPS, GP96, etc.) or cytokines, such as
GM-CFS, which allow an immune response to be enhanced and/or
influenced in a targeted manner. Particularly preferred auxiliary
substances are cytokines, such as monokines, lymphokines,
interleukins or chemokines, that further promote the innate immune
response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,
IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth
factors, such as hGH.
[0657] Suitable adjuvants may also be selected from cationic or
polycationic compounds wherein the adjuvant is preferably prepared
upon complexing the mRNA of the composition according to the
invention with the cationic or polycationic compound. Associating
or complexing the mRNA of the composition with cationic or
polycationic compounds as defined herein preferably provides
adjuvant properties and confers a stabilizing effect to the mRNA of
the composition. In particular, such preferred cationic or
polycationic compounds are selected from cationic or polycationic
peptides or proteins, including protamine, nucleoline, spermin or
spermidine, or other cationic peptides or proteins, such as
poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell
penetrating peptides (CPPs), including HIV-binding peptides, Tat,
HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or
analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein
transduction domains (PTDs, PpT620, prolin-rich peptides,
arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),
Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived
peptides (particularly from Drosophila antennapedia), pAntp, plsl,
FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1),
pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine,
or histones. Further preferred cationic or polycationic compounds
may include cationic polysaccharides, for example chitosan,
polybrene, cationic polymers, e.g. polyethyleneimine (PEI),
cationic lipids, e.g. DOTMA:
[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,
DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC,
DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC,
DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:
Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:
dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:
O,O-ditetradecanoyl-N-(.alpha.-trimethylammonioacetyl)diethanolamine
chloride, CLIP1:
rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,
CLIP9:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylamm-
onium, oligofectamine, or cationic or polycationic polymers, e.g.
modified polyaminoacids, such as .beta.-aminoacid-polymers or
reversed polyamides, etc., modified polyethylenes, such as PVP
(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified
acrylates, such as pDMAEMA (poly(dimethylaminoethyl
methylacrylate)), etc., modified Amidoamines such as pAMAM
(poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such
as diamine end modified 1,4 butanediol
diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such
as polypropylamine dendrimers or pAMAM based dendrimers, etc.,
polyimine(s), such as PEI: poly(ethyleneimine),
poly(propyleneimine), etc., polyallylamine, sugar backbone based
polymers, such as cyclodextrin based polymers, dextran based
polymers, Chitosan, etc., silan backbone based polymers, such as
PMOXA-PDMS copolymers, etc., blockpolymers consisting of a
combination of one or more cationic blocks (e.g. selected of a
cationic polymer as mentioned above) and of one or more
hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole);
etc.
[0658] Additionally, preferred cationic or polycationic proteins or
peptides, which can be used as an adjuvant by complexing the mRNA
of the composition according to the invention, may be selected from
following proteins or peptides having the following total formula
(III): (Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x, wherein l+m+n+o+x=8-15,
and l, m, n or o independently of each other may be any number
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or
15, provided that the overall content of Arg, Lys, His and Orn
represents at least 50% of all amino acids of the oligopeptide; and
Xaa may be any amino acid selected from native (=naturally
occurring) or non-native amino acids except of Arg, Lys, His or
Orn; and x may be any number selected from 0, 1, 2, 3 or 4,
provided, that the overall content of Xaa does not exceed 50% of
all amino acids of the oligopeptide. Particularly preferred
oligoarginines in this context are e.g. Arg7, Arg8, Arg9, Arg7,
H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc.
[0659] The ratio of the nucleic acid, particularly of mRNA to the
cationic or polycationic compound in the adjuvant component may be
calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio)
of the entire mRNA complex, i.e. the ratio of positively charged
(nitrogen) atoms of the cationic or polycationic compound to the
negatively charged phosphate atoms of the nucleic acids. For
example, 1 .mu.g of RNA typically contains about 3 nmol phosphate
residues, provided the RNA exhibits a statistical distribution of
bases. Additionally, 1 .mu.g of peptide typically contains about x
nmol nitrogen residues, dependent on the molecular weight and the
number of basic amino acids. When exemplarily calculated for (Arg)9
(molecular weight 1424 g/mol, 9 nitrogen atoms), 1 .mu.g (Arg)9
contains about 700 pmol (Arg)9 and thus 700.times.9=6300 pmol basic
amino acids=6.3 nmol nitrogen atoms. For a mass ratio of about 1:1
RNA/(Arg)9 an N/P ratio of about 2 can be calculated. When
exemplarily calculated for protamine (molecular weight about 4250
g/mol, 21 nitrogen atoms, when protamine from salmon is used) with
a mass ratio of about 2:1 with 2 .mu.g RNA, 6 nmol phosphate are to
be calulated for the RNA; 1 .mu.g protamine contains about 235 pmol
protamine molecues and thus 235.times.21=4935 pmol basic nitrogen
atoms=4.9 nmol nitrogen atoms. For a mass ratio of about 2:1
RNA/protamine an N/P ratio of about 0.81 can be calculated. For a
mass ratio of about 8:1 RNA/protamine an N/P ratio of about 0.2 can
be calculated. In the context of the present invention, an
N/P-ratio is preferably in the range of about 0.1-10, preferably in
a range of about 0.3-4 and most preferably in a range of about
0.5-2 or 0.7-2 regarding the ratio of RNA: peptide in the complex,
and most preferably in the range of about 0.7-1.5.
[0660] In a preferred embodiment, the composition of the present
invention is obtained in two separate steps in order to obtain
both, an efficient immunostimulatory effect and efficient
translation of the nucleic acid, particularly the mRNA according to
the invention. Therein, a so called "adjuvant component" is
prepared by complexing--in a first step--an mRNA as defined herein
of the adjuvant component with a cationic or polycationic compound
in a specific ratio to form a stable complex. In this context, it
is important, that no free cationic or polycationic compound or
only a negligibly small amount remains in the adjuvant component
after complexing the mRNA. Accordingly, the ratio of the mRNA and
the cationic or polycationic compound in the adjuvant component is
typically selected in a range that the mRNA is entirely complexed
and no free cationic or polycationic compound or only a negligible
small amount remains in the composition. Preferably the ratio of
the adjuvant component, i.e. the ratio of the mRNA to the cationic
or polycationic compound is 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).
[0661] According to a preferred embodiment, the nucleic acid,
particularly the mRNA of the invention comprising at least one mRNA
sequence comprising at least one coding region as defined herein is
added in a second step to the complexed mRNA of the adjuvant
component in order to form the (immunostimulatory) composition of
the invention. Therein, the mRNA of the composition according to
the invention is added as free mRNA, which is not complexed by
other compounds. Prior to addition, the free mRNA is not complexed
and will preferably not undergo any detectable or significant
complexation reaction upon the addition of the adjuvant component.
This is due to the strong binding of the cationic or polycationic
compound to the above described mRNA according to the invention
comprised in the adjuvant component. In other words, when the mRNA
comprising at least one coding region as defined herein is added to
the "adjuvant component", preferably no free or substantially no
free cationic or polycationic compound is present, which could form
a complex with the free mRNA. Accordingly, an efficient translation
of the mRNA of the composition is possible in vivo. Therein, the
free mRNA, may occur as a mono-, di-, or multicistronic mRNA, i.e.
an mRNA which carries the coding sequences of one or more proteins.
Such coding sequences in di-, or even multicistronic mRNA may be
separated by at least one IRES sequence, e.g. as defined
herein.
[0662] In a particularly preferred embodiment, the free nucleic
acid, particularly the mRNA as defined herein, which is comprised
in the composition of the present invention, may be identical or
different to the RNA as defined herein, which is comprised in the
adjuvant component of the composition, depending on the specific
requirements of therapy. Even more preferably, the free RNA, which
is comprised in the composition according to the invention, is
identical to the RNA of the adjuvant component of the inventive
composition.
[0663] In a particularly preferred embodiment, the composition
according to the invention comprises the nucleic acid, particularly
the mRNA of the invention, which encodes at least one NIPAH virus
antigenic peptide or protein as defined herein and wherein said
mRNA is present in the composition partially as free mRNA and
partially as complexed mRNA. Preferably, the mRNA as defined herein
is complexed as described above and the same mRNA is then added as
free mRNA, wherein preferably the compound, which is used for
complexing the mRNA is not present in free form in the composition
at the moment of addition of the free mRNA component.
[0664] The ratio of the first component (i.e. the adjuvant
component comprising or consisting of the nucleic acid,
particularly the mRNA as defined herein complexed with a cationic
or polycationic compound) and the second component (i.e. the free
mRNA as defined herein) may be selected in the inventive
composition according to the specific requirements of a particular
therapy. Typically, the ratio of the mRNA in the adjuvant component
and the at least one free mRNA (mRNA in the adjuvant component:free
mRNA) of the composition according to the invention is selected
such that a significant stimulation of the innate immune system is
elicited due to the adjuvant component. In parallel, the ratio is
selected such that a significant amount of the free mRNA can be
provided in vivo leading to an efficient translation and
concentration of the expressed protein in vivo, e.g. the at least
one NIPAH virus antigenic peptide or protein as defined herein.
Preferably the ratio of the mRNA in the adjuvant component:free
mRNA in the inventive composition is selected from a range of about
5:1 (w/w) to about 1:10 (w/w), more preferably from a range of
about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a
range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most
preferably the ratio of mRNA in the adjuvant component: free mRNA
in the inventive composition is selected from a ratio of about 1:1
(w/w).
[0665] Additionally or alternatively, the ratio of the first
component (i.e. the adjuvant component comprising or consisting of
the nucleic acid, particularly the mRNA complexed with a cationic
or polycationic compound) and the second component (i.e. the free
mRNA) may be calculated on the basis of the nitrogen/phosphate
ratio (N/P-ratio) of the entire mRNA complex. In the context of the
present invention, an N/P-ratio is preferably in the range of about
0.1-10, preferably in a range of about 0.3-4 and most preferably in
a range of about 0.5-2 or 0.7-2 regarding the ratio of mRNA:
peptide in the complex, and most preferably in the range of about
0.7-1.5.
[0666] Additionally or alternatively, the ratio of the first
component (i.e. the adjuvant component comprising or consisting of
the nucleic acid, particularly the mRNA complexed with a cationic
or polycationic compound) and the second component (i.e. the free
mRNA) may also be selected in the composition according to the
invention on the basis of the molar ratio of both mRNAs to each
other, i.e. the mRNA of the adjuvant component, being complexed
with a cationic or polycationic compound and the free mRNA of the
second component. Typically, the molar ratio of the mRNA of the
adjuvant component to the free mRNA of the second component may be
selected such, that the molar ratio suffices the above (w/w) and/or
N/P-definitions. More preferably, the molar ratio of the mRNA of
the adjuvant component to the free mRNA of the second component may
be selected e.g. from a molar ratio of about 0.001:1, 0.01:1,
0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1,
1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1,
1:0.01, 1:0.001, etc. or from any range formed by any two of the
above values, e.g. a range selected from about 0.001:1 to 1:0.001,
including a range of about 0.01:1 to 1:0.001, 0.1:1 to 1:0.001,
0.2:1 to 1:0.001, 0.3:1 to 1:0.001, 0.4:1 to 1:0.001, 0.5:1 to
1:0.001, 0.6:1 to 1:0.001, 0.7:1 to 1:0.001, 0.8:1 to 1:0.001,
0.9:1 to 1:0.001, 1:1 to 1:0.001, 1:0.9 to 1:0.001, 1:0.8 to
1:0.001, 1:0.7 to 1:0.001, 1:0.6 to 1:0.001, 1:0.5 to 1:0.001,
1:0.4 to 1:0.001, 1:0.3 to 1:0.001, 1:0.2 to 1:0.001, 1:0.1 to
1:0.001, 1:0.01 to 1:0.001, or a range of about 0.01:1 to 1:0.01,
0.1:1 to 1:0.01, 0.2:1 to 1:0.01, 0.3:1 to 1:0.01, 0.4:1 to 1:0.01,
0.5:1 to 1:0.01, 0.6:1 to 1:0.01, 0.7:1 to 1:0.01, 0.8:1 to 1:0.01,
0.9:1 to 1:0.01, 1:1 to 1:0.01, 1:0.9 to 1:0.01, 1:0.8 to 1:0.01,
1:0.7 to 1:0.01, 1:0.6 to 1:0.01, 1:0.5 to 1:0.01, 1:0.4 to 1:0.01,
1:0.3 to 1:0.01, 1:0.2 to 1:0.01, 1:0.1 to 1:0.01, 1:0.01 to
1:0.01, or including a range of about 0.001:1 to 1:0.01, 0.001:1 to
1:0.1, 0.001:1 to 1:0.2, 0.001:1 to 1:0.3, 0.001:1 to 1:0.4,
0.001:1 to 1:0.5, 0.001:1 to 1:0.6, 0.001:1 to 1:0.7, 0.001:1 to
1:0.8, 0.001:1 to 1:0.9, 0.001:1 to 1:1, 0.001 to 0.9:1, 0.001 to
0.8:1, 0.001 to 0.7:1, 0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to
0.4:1, 0.001 to 0.3:1, 0.001 to 0.2:1, 0.001 to 0.1:1, or a range
of about 0.01:1 to 1:0.01, 0.01:1 to 1:0.1, 0.01:1 to 1:0.2, 0.01:1
to 1:0.3, 0.01:1 to 1:0.4, 0.01:1 to 1:0.5, 0.01:1 to 1:0.6, 0.01:1
to 1:0.7, 0.01:1 to 1:0.8, 0.01:1 to 1:0.9, 0.01:1 to 1:1, 0.001 to
0.9:1, 0.001 to 0.8:1, 0.001 to 0.7:1, 0.001 to 0.6:1, 0.001 to
0.5:1, 0.001 to 0.4:1, 0.001 to 0.3:1, 0.001 to 0.2:1, 0.001 to
0.1:1, etc.
[0667] Even more preferably, the molar ratio of the nucleic acid,
particularly the mRNA of the adjuvant component to the free mRNA of
the second component may be selected e.g. from a range of about
0.01:1 to 1:0.01. Most preferably, the molar ratio of the mRNA of
the adjuvant component to the free mRNA of the second component may
be selected e.g. from a molar ratio of about 1:1. Any of the above
definitions with regard to (w/w) and/or N/P ratio may also
apply.
[0668] Suitable adjuvants may furthermore be selected from nucleic
acids having the formula (Va): GlXmGn, wherein: G is guanosine,
uracil or an analogue of guanosine or uracil; X is guanosine,
uracil, adenosine, thymidine, cytosine or an analogue of the
above-mentioned nucleotides; l is an integer from 1 to 40, wherein
when l=1 G is guanosine or an analogue thereof, when l>1 at
least 50% of the nucleotides are guanosine or an analogue thereof;
m is an integer and is at least 3; wherein when m=3 X is uracil or
an analogue thereof, when m>3 at least 3 successive uracils or
analogues of uracil occur; n is an integer from 1 to 40, wherein
when n=1 G is guanosine or an analogue thereof, when n>1 at
least 50% of the nucleotides are guanosine or an analogue thereof,
or formula (Vb): (NuGIXmGnNv)a, wherein: G is guanosine (guanine),
uridine (uracil) or an analogue of guanosine (guanine) or uridine
(uracil), preferably guanosine (guanine) or an analogue thereof; X
is guanosine (guanine), uridine (uracil), adenosine (adenine),
thymidine (thymine), cytidine (cytosine), or an analogue of these
nucleotides (nucleosides), preferably uridine (uracil) or an
analogue thereof; N is a nucleic acid sequence having a length of
about 4 to 50, preferably of about 4 to 40, more preferably of
about 4 to 30 or 4 to 20 nucleic acids, each N independently being
selected from guanosine (guanine), uridine (uracil), adenosine
(adenine), thymidine (thymine), cytidine (cytosine) or an analogue
of these nucleotides (nucleosides); a is an integer from 1 to 20,
preferably from 1 to 15, most preferably from 1 to 10; l is an
integer from 1 to 40, wherein when I=1, G is guanosine (guanine) or
an analogue thereof, when l>1, at least 50% of these nucleotides
(nucleosides) are guanosine (guanine) or an analogue thereof; m is
an integer and is at least 3; wherein when m=3, X is uridine
(uracil) or an analogue thereof, and when m>3, at least 3
successive uridines (uracils) or analogues of uridine (uracil)
occur; n is an integer from 1 to 40, wherein when n=1, G is
guanosine (guanine) or an analogue thereof, when n>1, at least
50% of these nucleotides (nucleosides) are guanosine (guanine) or
an analogue thereof; u,v may be independently from each other an
integer from 0 to 50, preferably wherein when u=0, v.gtoreq.1, or
when v=0, u 1; wherein the nucleic acid molecule of formula (Vb)
has a length of at least 50 nucleotides, preferably of at least 100
nucleotides, more preferably of at least 150 nucleotides, even more
preferably of at least 200 nucleotides and most preferably of at
least 250 nucleotides.
[0669] Other suitable adjuvants may furthermore be selected from
nucleic acids having the formula (VI): CIXmCn, wherein: C is
cytosine, uracil or an analogue of cytosine or uracil; X is
guanosine, uracil, adenosine, thymidine, cytosine or an analogue of
the above-mentioned nucleotides; l is an integer from 1 to 40,
wherein when I=1 C is cytosine or an analogue thereof, when l>1
at least 50% of the nucleotides are cytosine or an analogue
thereof; m is an integer and is at least 3; wherein when m=3 X is
uracil or an analogue thereof, when m>3 at least 3 successive
uracils or analogues of uracil occur; n is an integer from 1 to 40,
wherein when n=1 C is cytosine or an analogue thereof, when n>1
at least 50% of the nucleotides are cytosine or an analogue
thereof.
[0670] In this context the disclosure of WO2008014979 and
WO2009095226 is also incorporated herein by reference.
[0671] Vaccine:
[0672] In a further aspect, the present invention provides a
vaccine, which is based on the nucleic acid, particularly the mRNA
sequence according to the invention comprising at least one coding
region as defined herein. The vaccine according to the invention is
preferably a (pharmaceutical) composition as defined herein.
[0673] The vaccine according to the invention is based on the same
components as the (pharmaceutical) composition described herein.
Insofar, it may be referred to the description of the
(pharmaceutical) composition as provided herein. Preferably, the
vaccine according to the invention comprises at least one nucleic
acid comprising at least one nucleic acid sequence as defined
herein and a pharmaceutically acceptable carrier. In embodiments,
where the vaccine comprises more than one nucleic acid,
particularly more than one mRNA sequence (such as a plurality of
RNA sequences according to the invention, wherein each preferably
encodes a distinct antigenic peptide or protein), the vaccine may
be provided in physically separate form and may be administered by
separate administration steps. The vaccine according to the
invention may correspond to the (pharmaceutical) composition as
described herein, especially where the mRNA sequences are provided
by one single composition. However, the inventive vaccine may also
be provided physically separated. For instance, in embodiments,
wherein the vaccine comprises more than one mRNA sequences/species,
these RNA species may be provided such that, for example, two,
three, four, five or six separate compositions, which may contain
at least one mRNA species/sequence each (e.g, three distinct mRNA
species/sequences), each encoding distinct antigenic peptides or
proteins, are provided, which may or may not be combined. Also, the
inventive vaccine may be a combination of at least two distinct
compositions, each composition comprising at least one mRNA
encoding at least one of the antigenic peptides or proteins defined
herein. Alternatively, the vaccine may be provided as a combination
of at least one mRNA, preferably at least two, three, four, five,
six or more mRNAs, each encoding one of the antigenic peptides or
proteins defined herein. The vaccine may be combined to provide one
single composition prior to its use or it may be used such that
more than one administration is required to administer the distinct
mRNA sequences/species encoding any of the antigenic peptides or
proteins as defined herein. If the vaccine contains at least one
mRNA sequence, typically at least two mRNA sequences, encoding the
antigen combinations defined herein, it may e.g. be administered by
one single administration (combining all mRNA species/sequences),
by at least two separate administrations. Accordingly, any
combination of mono-, bi- or multicistronic mRNAs encoding the at
least one antigenic peptide or protein or any combination of
antigens as defined herein (and optionally further antigens),
provided as separate entities (containing one mRNA species) or as
combined entity (containing more than one mRNA species), is
understood as a vaccine according to the present invention.
[0674] As with the (pharmaceutical) composition according to the
present invention, the entities of the vaccine may be provided in
liquid and or in dry (e.g. lyophilized) form. They may contain
further components, in particular further components allowing for
its pharmaceutical use. The vaccine or the (pharmaceutical)
composition may, e.g., additionally contain a pharmaceutically
acceptable carrier and/or further auxiliary substances and
additives and/or adjuvants.
[0675] The vaccine or (pharmaceutical) composition typically
comprises a safe and effective amount of the nucleic acid,
particularly mRNA according to the invention as defined herein,
encoding an antigenic peptide or protein as defined herein or a
fragment or variant thereof or a combination of antigens,
preferably as defined herein. As used herein, "safe and effective
amount" means an amount of the mRNA that is sufficient to
significantly induce a positive modification of cancer or a disease
or disorder related to cancer. At the same time, however, a "safe
and effective amount" is small enough to avoid serious
side-effects, that is to say to permit a sensible relationship
between advantage and risk. The determination of these limits
typically lies within the scope of sensible medical judgment. In
relation to the vaccine or (pharmaceutical) composition of the
present invention, the expression "safe and effective amount"
preferably means an amount of the mRNA (and thus of the encoded
virus antigen) that is suitable for stimulating the adaptive immune
system in such a manner that no excessive or damaging immune
reactions are achieved but, preferably, also no such immune
reactions below a measurable level. Such a "safe and effective
amount" of the mRNA of the (pharmaceutical) composition or vaccine
as defined herein may furthermore be selected in dependence of the
type of mRNA, e.g. monocistronic, bi- or even multicistronic mRNA,
since a bi- or even multicistronic mRNA may lead to a significantly
higher expression of the encoded virus antigen(s) than the use of
an equal amount of a monocistronic mRNA. A "safe and effective
amount" of the mRNA of the (pharmaceutical) composition or vaccine
as defined above will furthermore vary in connection with the
particular condition to be treated and also with the age and
physical condition of the patient to be treated, the severity of
the condition, the duration of the treatment, the nature of the
accompanying therapy, of the particular pharmaceutically acceptable
carrier used, and similar factors, within the knowledge and
experience of the accompanying doctor. The vaccine or composition
according to the invention can be used according to the invention
for human and also for veterinary medical purposes (mammals,
vertebrates), as a pharmaceutical composition or as a vaccine.
[0676] In a preferred embodiment, the artificial nucleic acid,
vaccine or composition according to the invention is used as
pharmaceutical composition or as a vaccine in the prophylaxis or
treatment of disorders related to Henipavirus and/or Nipah virus
and/or Hendra virus in mammals, wherein the mammal may be selected
from canines (e.g., dogs), felines (e.g., cats), equines (e.g.,
horses), bovines (e.g., cattle) porcine (e.g., pigs), as well as
bats, flying foxes, rodents etc.
[0677] In a preferred embodiment, the nucleic acid, particularly
the mRNA of the (pharmaceutical) composition, vaccine or kit of
parts according to the invention is provided in lyophilized form.
Preferably, the lyophilized mRNA is reconstituted in a suitable
buffer, advantageously based on an aqueous carrier, prior to
administration, e.g. Ringer-Lactate solution, which is preferred,
Ringer solution, a phosphate buffer solution. In a preferred
embodiment, the (pharmaceutical) composition, the vaccine or the
kit of parts according to the invention contains at least one, two,
three, four, five, six or more mRNAs, preferably mRNAs which are
provided separately in lyophilized form (optionally together with
at least one further additive) and which are preferably
reconstituted separately in a suitable buffer (such as
Ringer-Lactate solution) prior to their use so as to allow
individual administration of each of the (monocistronic) mRNAs.
[0678] The vaccine or (pharmaceutical) composition according to the
invention may typically contain a pharmaceutically acceptable
carrier. The expression "pharmaceutically acceptable carrier" as
used herein preferably includes the liquid or non-liquid basis of
the inventive vaccine. If the inventive vaccine is provided in
liquid form, the carrier will be water, typically pyrogen-free
water; isotonic saline or buffered (aqueous) solutions, e.g
phosphate, citrate etc. buffered solutions. Particularly for
injection of the inventive vaccine, water or preferably a buffer,
more preferably an aqueous buffer, may be used, containing a sodium
salt, preferably at least 50 mM of a sodium salt, a calcium salt,
preferably at least 0.01 mM of a calcium salt, and optionally a
potassium salt, preferably at least 3 mM of a potassium salt.
According to a preferred embodiment, the sodium, calcium and,
optionally, potassium salts may occur in the form of their
halogenides, e.g. chlorides, iodides, or bromides, in the form of
their hydroxides, carbonates, hydrogen carbonates, or sulfates,
etc. Without being limited thereto, examples of sodium salts
include e.g. NaCl, Nal, NaBr, Na.sub.2CO.sub.3, NaHCO.sub.3,
Na.sub.2SO.sub.4, examples of the optional potassium salts include
e.g. KCl, Kl, KBr, K.sub.2CO.sub.3, KHCO.sub.3, K.sub.2SO.sub.4,
and examples of calcium salts include e.g. CaCl.sub.2), Cal.sub.2,
CaBr.sub.2, CaCO.sub.3, CaSO.sub.4, Ca(OH).sub.2. Furthermore,
organic anions of the aforementioned cations may be contained in
the buffer. According to a more preferred embodiment, the buffer
suitable for injection purposes as defined above, may contain salts
selected from sodium chloride (NaCl), calcium chloride
(CaCl.sub.2)) and optionally potassium chloride (KCl), wherein
further anions may be present additional to the chlorides.
CaCl.sub.2 can also be replaced by another salt like KCl.
Typically, the salts in the injection buffer are present in a
concentration of at least 50 mM sodium chloride (NaCl), at least 3
mM potassium chloride (KCl) and at least 0.01 mM calcium chloride
(CaCl.sub.2)). The injection buffer may be hypertonic, isotonic or
hypotonic with reference to the specific reference medium, i.e. the
buffer may have a higher, identical or lower salt content with
reference to the specific reference medium, wherein preferably such
concentrations of the afore mentioned salts may be used, which do
not lead to damage of cells due to osmosis or other concentration
effects. Reference media are e.g. in "in vivo" methods occurring
liquids such as blood, lymph, cytosolic liquids, or other body
liquids, or e.g. liquids, which may be used as reference media in
"in vitro" methods, such as common buffers or liquids. Such common
buffers or liquids are known to a skilled person. Ringer-Lactate
solution is particularly preferred as a liquid basis.
[0679] However, one or more compatible solid or liquid fillers or
diluents or encapsulating compounds may be used as well, which are
suitable for administration to a person. The term "compatible" as
used herein means that the constituents of the inventive vaccine
are capable of being mixed with the nucleic acid, particularly the
mRNA according to the invention as defined herein, in such a manner
that no interaction occurs, which would substantially reduce the
pharmaceutical effectiveness of the inventive vaccine under typical
use conditions. Pharmaceutically acceptable carriers, fillers and
diluents must, of course, have sufficiently high purity and
sufficiently low toxicity to make them suitable for administration
to a person to be treated. Some examples of compounds which can be
used as pharmaceutically acceptable carriers, fillers or
constituents thereof are sugars, such as, for example, lactose,
glucose, trehalose and sucrose; starches, such as, for example,
corn starch or potato starch; dextrose; cellulose and its
derivatives, such as, for example, sodium carboxymethylcellulose,
ethylcellulose, cellulose acetate; powdered tragacanth; malt;
gelatin; tallow; solid glidants, such as, for example, stearic
acid, magnesium stearate; calcium sulfate; vegetable oils, such as,
for example, groundnut oil, cottonseed oil, sesame oil, olive oil,
corn oil and oil from theobroma; polyols, such as, for example,
polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene
glycol; alginic acid.
[0680] The choice of a pharmaceutically acceptable carrier is
determined, in principle, by the manner, in which the
pharmaceutical composition or vaccine according to the invention is
administered. The composition or vaccine can be administered, for
example, systemically or locally. Routes for systemic
administration in general include, for example, transdermal, oral,
parenteral routes, including subcutaneous, intravenous,
intramuscular, intraarterial, intradermal and intraperitoneal
injections and/or intranasal administration routes. Routes for
local administration in general include, for example, topical
administration routes but also intradermal, transdermal,
subcutaneous, or intramuscular injections or intralesional,
intracranial, intrapulmonal, intracardial, and sublingual
injections. More preferably, composition or vaccines according to
the present invention may be administered by an intradermal,
subcutaneous, or intramuscular route, preferably by injection,
which may be needle-free and/or needle injection.
Compositions/vaccines are therefore preferably formulated in liquid
or solid form. The suitable amount of the vaccine or composition
according to the invention to be administered can be determined by
routine experiments, e.g. by using animal models. Such models
include, without implying any limitation, rabbit, sheep, mouse,
rat, dog and non-human primate models. Preferred unit dose forms
for injection include sterile solutions of water, physiological
saline or mixtures thereof. The pH of such solutions should be
adjusted to about 7.4. Suitable carriers for injection include
hydrogels, devices for controlled or delayed release, polylactic
acid and collagen matrices. Suitable pharmaceutically acceptable
carriers for topical application include those which are suitable
for use in lotions, creams, gels and the like. If the inventive
composition or vaccine is to be administered perorally, tablets,
capsules and the like are the preferred unit dose form. The
pharmaceutically acceptable carriers for the preparation of unit
dose forms which can be used for oral administration are well known
in the prior art. The choice thereof will depend on secondary
considerations such as taste, costs and storability, which are not
critical for the purposes of the present invention, and can be made
without difficulty by a person skilled in the art.
[0681] The inventive vaccine or composition can additionally
contain one or more auxiliary substances in order to further
increase the immunogenicity. A synergistic action of the nucleic
acid contained in the inventive composition and of an auxiliary
substance, which may be optionally be co-formulated (or separately
formulated) with the inventive vaccine or composition as described
above, is preferably achieved thereby. Depending on the various
types of auxiliary substances, various mechanisms may play a role
in this respect. For example, compounds that permit the maturation
of dendritic cells (DCs), for example lipopolysaccharides,
TNF-alpha or CD40 ligand, form a first class of suitable auxiliary
substances. In general, it is possible to use as auxiliary
substance any agent that influences the immune system in the manner
of a "danger signal" (LPS, GP96, etc.) or cytokines, such as
GM-CFS, which allow an immune response produced by the
immune-stimulating adjuvant according to the invention to be
enhanced and/or influenced in a targeted manner. Particularly
preferred auxiliary substances are cytokines, such as monokines,
lymphokines, interleukins or chemokines, that--additional to
induction of the adaptive immune response by the encoded at least
one antigen--promote the innate immune response, such as IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,
IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,
IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,
IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,
M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
Preferably, such immunogenicity increasing agents or compounds are
provided separately (not co-formulated with the inventive vaccine
or composition) and administered individually.
[0682] Further additives which may be included in the inventive
vaccine or composition are emulsifiers, such as, for example,
Tween; wetting agents, such as, for example, sodium lauryl sulfate;
colouring agents; taste-imparting agents, pharmaceutical carriers;
tablet-forming agents; stabilizers; antioxidants;
preservatives.
[0683] The inventive vaccine or composition can also additionally
contain any further compound, which is known to be
immune-stimulating due to its binding affinity (as ligands) to
human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to
murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
[0684] Another class of compounds, which may be added to an
inventive vaccine or composition in this context, may be CpG
nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or
CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a
double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss
CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic
acid is preferably in the form of CpG-RNA, more preferably in the
form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid
preferably contains at least one or more (mitogenic)
cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According
to a first preferred alternative, at least one CpG motif contained
in these sequences, that is to say the C (cytosine) and the G
(guanine) of the CpG motif, is unmethylated. All further cytosines
or guanines optionally contained in these sequences can be either
methylated or unmethylated. According to a further preferred
alternative, however, the C (cytosine) and the G (guanine) of the
CpG motif can also be present in methylated form.
[0685] In a further aspect, the present invention concerns a
polypeptide encoded by the inventive artificial nucleic acid as
described herein, or a fragment of said polypeptide.
[0686] In a further aspect, the present invention provides a
composition comprising at least one of the inventive polypeptides
as described herein. In a preferred embodiment, the inventive
composition comprises one type of polypeptide as described herein.
Alternatively, the inventive composition may comprise at least two
different inventive polypeptides as described herein.
[0687] In a preferred embodiment, the at least one of the inventive
polypeptides comprises at least one protein or peptides according
to SEQ ID NOs: 1-7, 12-18, 573-579, 584-590, 807-813, 818-824,
1041-1047, 1052-1058, 1513-1515 a fragment or variant thereof.
[0688] Preferably, the inventive composition comprises or consists
of at least one of the inventive polypeptides described herein and
a pharmaceutically acceptable carrier. In this context, the
pharmaceutically acceptable carrier as well as optional further
components of the composition preferably as described herein, with
respect to the inventive composition, comprises at least one
inventive artificial nucleic acid.
[0689] In another embodiment, the inventive composition comprises
or consists of at least one of the inventive polypeptides described
herein and a pharmaceutically acceptable carrier and at least one
adjuvant.
[0690] In another embodiment, the inventive composition comprises
or consists of at least one of the inventive polypeptides described
herein and a pharmaceutically acceptable carrier and at least one
adjuvant and at least one inventive nucleic acid as defined
herein.
[0691] In a further aspect, the invention concerns a vaccine
comprising the inventive composition comprising at least one of the
polypeptides according to the invention. Therein, the at least one
of the inventive polypeptides preferably elicits an adaptive immune
response upon administration to a subject. More preferably, the
vaccine according to the invention comprising at least one of the
inventive polypeptides or the inventive composition comprising at
least one of the polypeptides according to the invention is
preferably a vaccine as described herein. Reference is made to the
respective description herein.
[0692] As used herein, the term "inventive composition" may refer
to the inventive composition comprising at least one artificial
nucleic acid according to the invention. Likewise, the term
"inventive vaccine", as used in this context, may refer to an
inventive vaccine, which is based on the inventive artificial
nucleic acid, i.e. which comprises at least one artificial nucleic
acid according to the invention or which comprises the inventive
composition comprising said artificial nucleic acid.
[0693] Kit:
[0694] According to another embodiment, the present invention also
provides kits, particularly kits of parts, comprising the
artificial nucleic acid according to the invention, the inventive
composition comprising at least one artificial nucleic acid
according to the invention, the inventive polypeptides as described
herein, the inventive composition comprising at least one inventive
polypeptide or the inventive vaccine as described herein,
optionally a liquid vehicle for solubilising and optionally
technical instructions with information on the administration and
dosage of the artificial nucleic acid according as described
herein, the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein, the inventive composition
comprising at least one inventive polypeptide or the inventive
vaccine. The technical instructions may contain information about
administration and dosage. Such kits, preferably kits of parts, may
be applied e.g. for any of the applications or uses mentioned
herein, preferably for the use of the artificial nucleic acid
according as described herein, the inventive composition comprising
at least one artificial nucleic acid according to the invention,
the inventive polypeptides as described herein, the inventive
composition comprising at least one inventive polypeptide or the
inventive vaccine for the treatment or prophylaxis of a Henipvirus
and/or a Nipah virus and/or a Hendra virus infection or diseases or
disorders related thereto. The kits may also be applied for the use
of the artificial nucleic acid according as described herein, the
inventive composition comprising at least one artificial nucleic
acid according to the invention, the inventive polypeptides as
described herein, the inventive composition comprising at least one
inventive polypeptide or the inventive vaccine for the treatment or
prophylaxis of Henipvirus and/or a Nipah virus and/or a Hendra
virus infection or diseases or disorders related thereto, wherein
the artificial nucleic acid according as described herein, the
inventive composition comprising at least one artificial nucleic
acid according to the invention, the inventive polypeptides as
described herein, the inventive composition comprising at least one
inventive polypeptide or the inventive vaccine may induce or
enhance an immune response in a mammal as defined above.
Preferably, the artificial nucleic acid according as described
herein, the inventive composition comprising at least one
artificial nucleic acid according to the invention, or the
inventive vaccine is provided in a separate part of the kit,
wherein the artificial nucleic acid according as described herein,
the inventive composition comprising at least one artificial
nucleic acid according to the invention, or the inventive vaccine
are preferably lyophilised. More preferably, the kit further
contains as a part a vehicle for solubilising the artificial
nucleic acid according as described herein, the inventive
composition comprising at least one artificial nucleic acid
according to the invention, or the inventive vaccine, the vehicle
preferably being Ringer-lactate solution. Any of the above kits may
be used in a treatment or prophylaxis as defined above. More
preferably, any of the above kits may be used as a vaccine,
preferably a vaccine against Henipvirus and/or a Nipah virus and/or
a Hendra virus infection or a related disease or disorder.
[0695] Any of the above kits may be used in a treatment or
prophylaxis as defined above. More preferably, any of the above
kits may be used as a vaccine, preferably a vaccine against
Henipvirus and/or a Nipah virus and/or a Hendra virus infection or
a related disease or disorder.
[0696] Application and Medical Use:
[0697] According to one aspect of the present invention, the
nucleic sequence, the (pharmaceutical) composition or the vaccine
may be used according to the invention (for the preparation of a
medicament) as a medicament.
[0698] The present invention furthermore provides several
applications and uses of the artificial nucleic acid according to
the invention, the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein, the inventive composition
comprising at least one inventive polypeptide or the inventive
vaccine or of kits comprising same. In particular, the inventive
(pharmaceutical) composition(s) or the inventive vaccine may be
used for human and also for veterinary medical purposes, preferably
for human medical purposes, as a pharmaceutical composition in
general or as a vaccine.
[0699] In a further aspect, the invention provides the artificial
nucleic acid according to the invention, the inventive composition
comprising at least one artificial nucleic acid according to the
invention, the inventive polypeptides as described herein, the
inventive composition comprising at least one inventive
polypeptide, the inventive vaccine or the inventive kit or kit of
parts for use in a method of prophylactic (pre-exposure prophylaxis
or post-exposure prophylaxis) and/or therapeutic treatment of
Henipvirus and/or a Nipah virus and/or a Hendra virus infections.
Consequently, in a further aspect, the present invention is
directed to the first medical use of the artificial nucleic acid
according to the invention, the inventive composition comprising at
least one artificial nucleic acid according to the invention, the
inventive polypeptides as described herein, the inventive
composition comprising at least one inventive polypeptide, the
inventive vaccine or the inventive kit or kit of parts as defined
herein as a medicament. Particularly, the invention provides the
use of an artificial nucleic acid comprising at least one coding
region encoding at least one polypeptide comprising at least one
Henipvirus and/or a Nipah virus and/or a Hendra virus protein or
peptide as defined herein, or a fragment or variant thereof as
described herein for the preparation of a medicament.
[0700] According to another aspect, the present invention is
directed to the second medical use of the artificial nucleic acid
according to the invention, the inventive composition comprising at
least one artificial nucleic acid according to the invention, the
inventive polypeptides as described herein, the inventive
composition comprising at least one inventive polypeptide, the
inventive vaccine or the inventive kit or kit of parts for the
treatment of an infection with Henipvirus and/or a Nipah virus
and/or a Hendra virus or a disease or disorders related to an
infection with Henipvirus and/or a Nipah virus and/or a Hendra
virus as defined herein.
[0701] Particularly, the artificial nucleic acid comprising at
least one coding region encoding at least one polypeptide
comprising at least one antigenic protein or peptide as defined
herein, or a fragment or variant thereof as described herein to be
used in a method as said above is an artificial nucleic acid
formulated together with a pharmaceutically acceptable vehicle and
an optionally additional adjuvant and an optionally additional
further component as defined herein.
[0702] The invention provides the artificial nucleic acid according
to the invention, the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein, the inventive composition
comprising at least one inventive polypeptide, the inventive
vaccine or the inventive kit or kit of parts for medical use, in
particular for the treatment of an infection with Henipvirus and/or
a Nipah virus and/or a Hendra virus or a disease or disorders
related to an infection with Henipvirus and/or a Nipah virus and/or
a Hendra virus, wherein preferably an infection with Henipvirus
and/or a Nipah virus and/or a Hendra virus may involve any
Henipvirus and/or a Nipah virus and/or a Hendra virus as defined
herein.
[0703] As used herein, "a disorder related to a Henipvirus and/or a
Nipah virus and/or a Hendra virus infection" or "a disease related
to a Henipvirus and/or a Nipah virus and/or a Hendra virus
infection" may preferably comprise a complication of Henipvirus
and/or a Nipah virus and/or a Hendra virus infection. Complications
and disease related disorders associated with Nipah virus infection
include fever and headache, followed by drowsiness, disorientation
and mental confusion, respiratory illness and encephalitis
(inflammation of the brain). Complications and disease related
disorders associated with Hendra virus include various respiratory
and neurologic complications and symptoms.
[0704] In a preferred embodiment, the inventive composition or
vaccine is thus used for treatment or prophylaxis, preferably
prophylaxis, of complications associated with a Nipah virus
infection.
[0705] In a preferred embodiment, the inventive composition or
vaccine is thus used for treatment or prophylaxis, preferably
prophylaxis, of complications associated with a Hendra virus
infection.
[0706] In a preferred embodiment, the inventive composition or
vaccine is thus used for treatment or prophylaxis, preferably
prophylaxis, of complications associated with a Henipavirus
infection.
[0707] The inventive composition or the inventive vaccine, in
particular the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein or the inventive composition
comprising at least one inventive polypeptide, can be administered,
for example, systemically or locally. Routes for systemic
administration in general include, for example, transdermal, oral,
parenteral routes, including subcutaneous, intravenous,
intramuscular, intraarterial, intradermal and intraperitoneal
injections and/or intranasal administration routes. Routes for
local administration in general include, for example, topical
administration routes but also intradermal, transdermal,
subcutaneous, or intramuscular injections or intralesional,
intracranial, intrapulmonal, intracardial, and sublingual
injections. More preferably, vaccines may be administered by an
intradermal, subcutaneous, or intramuscular route. Inventive
vaccines are therefore preferably formulated in liquid (or
sometimes in solid) form. Preferably, the inventive vaccine may be
administered by conventional needle injection or needle-free jet
injection. In a preferred embodiment the inventive vaccine or
composition may be administered by jet injection as defined herein,
preferably intramuscularly or intradermally, more preferably
intradermally.
[0708] In a preferred embodiment, a single dose of the inventive
artificial nucleic acid, composition or vaccine comprises a
specific amount of the artificial nucleic acid according to the
invention.
[0709] In embodiments, the inventive artificial nucleic acid is
provided in an amount of 5 .mu.g, 10 .mu.g, 15 .mu.g, 20 .mu.g, 25
.mu.g, 30 .mu.g, 35 .mu.g, 40 .mu.g, 45 .mu.g, 50 .mu.g, 60 .mu.g,
70 .mu.g, 80 .mu.g, 90 .mu.g, or 100 .mu.g. Preferably, the
inventive artificial nucleic acid is provided in an amount of at
least 5 .mu.g per dose, preferably in an amount of from 10 to 500
.mu.g per dose, more preferably in an amount of from 20 to 200
.mu.g per dose. More specifically, in the case of intradermal
injection, which is preferably carried out by using a conventional
needle, the amount of the inventive artificial nucleic acid
comprised in a single dose is typically at least 5 .mu.g,
preferably from 10 .mu.g to 500 .mu.g, more preferably from 20
.mu.g to 200 .mu.g, even more preferably from 30 .mu.g to 100
.mu.g. In the case of intradermal injection, which is preferably
carried out via jet injection (e.g. using a Tropis device), the
amount of the inventive artificial nucleic acid comprised in a
single dose is typically at least 10 .mu.g, preferably from 20
.mu.g to 200 .mu.g, more preferably from 30 .mu.g to 100 .mu.g.
Moreover, in the case of intramuscular injection, which is
preferably carried out by using a conventional needle or via jet
injection, the amount of the inventive artificial nucleic acid
comprised in a single dose is typically at least 1 .mu.g,
preferably from 1 .mu.g to 500 .mu.g, more preferably from 5 .mu.g
to 500 .mu.g, even more preferably from 10 .mu.g to 200 .mu.g.
[0710] The immunization protocol for the treatment or prophylaxis
of a Henipvirus and/or a Nipah virus and/or a Hendra virus
infection, i.e the immunization of a subject against Henipvirus
and/or a Nipah virus and/or a Hendra virus, typically comprises a
series of single doses or dosages of the inventive composition or
the inventive vaccine. A single dosage, as used herein, refers to
the initial/first dose, a second dose or any further doses,
respectively, which are preferably administered in order to "boost"
the immune reaction.
[0711] According to a preferred embodiment, the artificial nucleic
acid according to the invention, the inventive composition
comprising at least one artificial nucleic acid according to the
invention, the inventive polypeptides as described herein, the
inventive composition comprising at least one inventive
polypeptide, the inventive vaccine or the inventive kit or kit of
parts is provided for use in treatment or prophylaxis, preferably
treatment or prophylaxis of a Henipvirus and/or a Nipah virus
and/or a Hendra virus infection or a related disorder or disease,
wherein the treatment or prophylaxis comprises the administration
of a further active pharmaceutical ingredient. More preferably, in
the case of the inventive vaccine or composition, which is based on
the inventive artificial nucleic acid, a polypeptide may be
co-administered as a further active pharmaceutical ingredient. For
example, at least one Henipvirus and/or a Nipah virus and/or a
Hendra virus protein or peptide as described herein, or a fragment
or variant thereof, may be co-administered in order to induce or
enhance an immune response. Likewise, in the case of the inventive
vaccine or composition, which is based on the inventive polypeptide
as described herein, an artificial nucleic acid as described herein
may be co-administered as a further active pharmaceutical
ingredient. For example, an artificial nucleic acid as described
herein encoding at least one polypeptide as described herein may be
co-administered in order to induce or enhance an immune
response.
[0712] A further component of the inventive vaccine or composition
may be an immunotherapeutic agent that can be selected from
immunoglobulins, preferably IgGs, monoclonal or polyclonal
antibodies, polyclonal serum or sera, etc., most preferably
immunoglobulins directed against a Henipvirus and/or a Nipah virus
and/or a Hendra virus.
[0713] Preferably, such a further immunotherapeutic agent may be
provided as a peptide/protein or may be encoded by a nucleic acid,
preferably by a DNA or an RNA, more preferably an mRNA. Such an
immunotherapeutic agent allows providing passive vaccination
additional to active vaccination triggered by the inventive
artificial nucleic acid or by the inventive polypeptide.
[0714] In a further aspect the invention provides a method of
treating or preventing a disorder, wherein the disorder is
preferably an infection with Henipvirus and/or a Nipah virus and/or
a Hendra virus or a disorder related to an infection with
Henipvirus and/or a Nipah virus and/or a Hendra virus, wherein the
method comprises administering to a subject in need thereof the
artificial nucleic acid according to the invention, the inventive
composition comprising at least one artificial nucleic acid
according to the invention, the inventive polypeptides as described
herein, the inventive composition comprising at least one inventive
polypeptide, the inventive vaccine or the inventive kit or kit of
parts.
[0715] In particular, such a method may preferably comprise the
steps of: [0716] a) providing the artificial nucleic acid according
to the invention, the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein, the inventive composition
comprising at least one inventive polypeptide, the inventive
vaccine or the inventive kit or kit of parts; [0717] b) applying or
administering the artificial nucleic acid according to the
invention, the inventive composition comprising at least one
artificial nucleic acid according to the invention, the inventive
polypeptides as described herein, the inventive composition
comprising at least one inventive polypeptide, the inventive
vaccine or the inventive kit or kit of parts to a tissue or an
organism; [0718] c) optionally administering immunoglobuline (IgGs)
against Henipvirus and/or a Nipah virus and/or a Hendra virus.
[0719] According to a further aspect, the present invention also
provides a method for expression of at least one polypeptide
comprising at least one Henipvirus and/or a Nipah virus and/or a
Hendra virus, or a fragment or variant thereof, wherein the method
preferably comprises the following steps:
a) providing the inventive artificial nucleic acid comprising at
least one coding region encoding at least one polypeptide
comprising at least one Henipvirus and/or a Nipah virus and/or a
Hendra virus, or a fragment or variant thereof, preferably as
defined herein, or a composition comprising said artificial nucleic
acid; and b) applying or administering the inventive artificial
nucleic acid or the inventive composition comprising said
artificial nucleic acid to an expression system, e.g. to a
cell-free expression system, a cell (e.g. an expression host cell
or a somatic cell), a tissue or an organism.
[0720] The method may be applied for laboratory, for research, for
diagnostic, for commercial production of peptides or proteins
and/or for therapeutic purposes. In this context, typically after
preparing the inventive artificial nucleic acid as defined herein
or of the inventive composition or vaccine as defined herein, it is
typically applied or administered to a cell-free expression system,
a cell (e.g. an expression host cell or a somatic cell), a tissue
or an organism, e.g. in naked or complexed form or as a
(pharmaceutical) composition or vaccine as described herein,
preferably via transfection or by using any of the administration
modes as described herein. The method may be carried out in vitro,
in vivo or ex vivo. The method may furthermore be carried out in
the context of the treatment of a specific disease, particularly in
the treatment of infectious diseases, preferably Henipvirus and/or
a Nipah virus and/or a Hendra virus infection or a related disorder
as defined herein.
[0721] In this context, in vitro is defined herein as transfection
or transduction of the inventive artificial nucleic acid as defined
herein or of the inventive composition or vaccine as defined herein
into cells in culture outside of an organism; in vivo is defined
herein as transfection or transduction of the inventive artificial
nucleic acid or of the inventive composition or vaccine into cells
by application of the inventive mRNA or of the inventive
composition to the whole organism or individual and ex vivo is
defined herein as transfection or transduction of the inventive
artificial nucleic acid or of the inventive composition or vaccine
into cells outside of an organism or individual and subsequent
application of the transfected cells to the organism or
individual.
[0722] Likewise, according to another aspect, the present invention
also provides the use of the inventive artificial nucleic acid as
defined herein or of the inventive composition or vaccine as
defined herein, preferably for diagnostic or therapeutic purposes,
for expression of an encoded Henipvirus and/or a Nipah virus and/or
a Hendra virus antigenic peptide or protein, e.g. by applying or
administering the inventive artificial nucleic acid as defined
herein or of the inventive composition or vaccine as defined
herein, e.g. to a cell-free expression system, a cell (e.g. an
expression host cell or a somatic cell), a tissue or an organism.
The use may be applied for a (diagnostic) laboratory, for research,
for diagnostics, for commercial production of peptides or proteins
and/or for therapeutic purposes. In this context, typically after
preparing the inventive artificial nucleic acid as defined herein
or of the inventive composition or vaccine as defined herein, it is
typically applied or administered to a cell-free expression system,
a cell (e.g. an expression host cell or a somatic cell), a tissue
or an organism, preferably in naked form or complexed form, or as a
(pharmaceutical) composition or vaccine as described herein,
preferably via transfection or by using any of the administration
modes as described herein. The use may be carried out in vitro, in
vivo or ex vivo. The use may furthermore be carried out in the
context of the treatment of a specific disease, particularly in the
treatment of NIPAH virus infection or a related disorder.
[0723] In a particularly preferred embodiment, the invention
provides the artificial nucleic acid, the inventive composition or
the inventive vaccine for use as defined herein, preferably for use
as a medicament, for use in treatment or prophylaxis, preferably
treatment or prophylaxis of a Henipvirus and/or a Nipah virus
and/or a Hendra virus infection or a related disorder, or for use
as a vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0724] FIG. 1: shows that mRNA encoding Nipah virus F protein
(R6311) induces specific humoral immune responses after
immunization in mice. Further details are provided in Example
2.
[0725] FIG. 2: shows that mRNA encoding Henipavirus G protein is
expressed in cells after transfection. Further details are provided
in Example 3.
EXAMPLES
[0726] 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
[0727] 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.
[0728] RNA In Vitro Transcription on Linearized pDNA:
[0729] The DNA plasmids prepared according to paragraph 1 are
enzymatically linearized using EcoRl 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.RTM. (CureVac, Tubingen, Germany; WO2008077592)
and used for in vitro and in vivo experiments.
[0730] RNA In Vitro Transcription on PCR Amplified DNA
Templates:
[0731] 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.RTM.
(CureVac, Tubingen, 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-00009 TABLE 7 mRNA constructs used in the Example section:
SEQ ID NO: Name 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- 825 SEQ ID NO:
1397 Hendra-1994)-G(71-604) mRNA design 2; opt1
IgE-leader_Nipha(Bangladesh2004)-F 809 SEQ ID NO: 1381 mRNA design
2; opt1 SP-Influenza- 1043 SEQ ID NO: 1407
HA_Nipha(Bangladesh2004)-F mRNA design 2; opt1 SP-Osteonectin 1513
SEQ ID NO: 1543 BM40_Nipha(Bangladesh2004)-F mRNA design 2; opt1
SP-HsChemo- 1514 SEQ ID NO: 1544 tripsinogen_Nipha(Bangladesh2004)
mRNA design 2; opt1 SP-Nipha(Malaysia1999)-F(1- 1515 SEQ ID NO:
1545 26)_Nipha(Bangladesh2004)-F(27-546) mRNA design 2; opt1
Example 2: Vaccination of Mice with mRNA Encoding Nipah
[0732] 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.
[0733] Preparation of Protamine Complexed mRNA ("Vaccine
Composition 1"):
[0734] 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.
[0735] Immunization:
[0736] 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-00010 TABLE A Vaccination regimen (Example 2): Number of
mice Vaccine composition Dose Route/Volume 10 NIV F (Malaysia 1999)
R6311; 80 .mu.g i.d. 2 .times. 50 .mu.l Vaccine composition 1 10
100% RiLa Control i.d. 2 .times. 25 .mu.l
[0737] Detection of Specific Humoral Immune Responses:
[0738] Hela cells were transfected with 2 .mu.g of either R6311
vaccine composition using lipofectamine. The cells were harvested
20 h post transfection, and seeded at 1.times.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.
[0739] Results:
[0740] As shown in FIG. 1, the mRNA encoding Nipah virus F protein
(NIV F Malaysia 1999; R6311) is expressed in mice after i.d.
administration. Moreover, as specific anti-NIV F IgGs were detected
in sera of immunized mice, the results also show that the applied
mRNA vaccine is suitable to induce specific humoral immune
responses.
[0741] 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
[0742] 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.
[0743] For the analysis of Nipah virus protein and Hendra virus G
protein expression, HeLa cells were transfected with 2 .mu.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 .alpha..beta.-tubulin
was analyzed (.alpha..beta.-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 FIG. 2). The outline of the experiment is shown in Table
B. The result of the experiment is shown in FIG. 2.
TABLE-US-00011 TABLE B 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
[0744] Results:
[0745] As shown in FIG. 2, the mRNA encoding Henipavirus G protein
is expressed in HeLa cells as the immunostaining for cell lysates
of mRNA transfected cells was substantially increased compared to
the wfi control group. In particular, immunostaining at about 70
kDa (G monomer) and about 260 kDa (G multimer) were detected. The
results exemplify that the inventive mRNA encoding Henipavirus G
protein is translated in cells and that alternative mRNA constructs
according to the invention may also be translated in cells, which
is a prerequisite for an mRNA-based vaccine.
Example 4: Expression of Nipah Virus and Hendra Virus Proteins in
HeLa Cells and Analysis by FACS
[0746] 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 .mu.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 ant