Dengue tetravalent vaccine containing a common 30 nucleotide deletion in the 3′-UTR of dengue types 1,2,3, and 4, or antigenic chimeric dengue viruses 1,2,3, and 4
RE046631 · 2017-12-12
Assignee
Inventors
- Stephen S. Whitehead (Montgomery Village, MD)
- Brian R. Murphy (Bethesda, MD)
- Lewis Markoff (Bethesda, MD)
- Barry Falgout (Rockville, MD)
- Joseph E. Blaney (Gettysburg, PA)
- Kathryn A. Hanley (Las Cruces, NM)
- Ching-Juh Lai (Bethesda, MD)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The invention relates to a dengue virus tetravalent vaccine containing a common 30 nucleotide deletion (30) in the 3-untranslated region of the genome of dengue virus serotypes 1, 2, 3, and 4, or antigenic chimeric dengue viruses of serotypes 1, 2, 3, and 4.
Claims
1. A tetravalent immunogenic composition comprising a) a first attenuated virus that is immunogenic against dengue serotype 1, b) a second attenuated virus that is immunogenic against dengue serotype 2, c) a third attenuated virus that is immunogenic against dengue serotype 3, and d) a fourth attenuated virus that is immunogenic against dengue serotype 4, wherein each of a), b), c) and d) comprises a nucleic acid comprising i) a first nucleotide sequence encoding at least one structural protein from a first dengue virus, ii) a second nucleotide sequence encoding nonstructural proteins from the first dengue virus or a second dengue virus, and iii) a 3 untranslated region, wherein the 3 untranslated region contains a deletion of about 30 nucleotides corresponding to the TL2 stem-loop structure, and wherein both the 3 untranslated region and the second nucleotide sequence encoding nonstructural proteins are from either the first dengue virus or the second dengue virus; wherein the tetravalent immunogenic composition is not rDEN1/430, rDEN2/430, rDEN3/430, rDEN430.
2. The tetravalent immunogenic composition of claim 1, wherein the nucleic acid of at least one of a), b), c) or d) further comprises a mutation that confers a phenotype wherein the phenotype is temperature sensitivity in Vero cells or the human liver cell line HuH-7, host-cell restriction in mosquito cells or the human liver cell line HuH-7, host-cell adaptation for improved replication in Vero cells, or attenuation in mice or monkeys.
3. The composition of claim 1, wherein the first nucleotide sequence encoding at least one structural protein and the second nucleotide sequence encoding nonstructural proteins of at least one of a), b), c) and d) are from the first dengue virus.
4. The composition of claim 3, wherein the deletion of a) is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 1 genome corresponding to the TL2 stem-loop structure between about nucleotides 10562-10591.
5. The composition of claim 3, wherein the deletion of b) is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 2 genome corresponding to the TL2 stem-loop structure between about nucleotides 10541-10570.
6. The composition of claim 3, wherein the deletion of c) is by a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 3 genome corresponding to the TL2 stem-loop structure between about nucleotides 10535-10565.
7. The composition of claim 3, herein the deletion of d) is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 4 genome corresponding to the TL2 stem-loop structure between about nucleotides 10478-10507.
8. The composition of claim 1, wherein the first nucleotide sequence encoding at least one structural protein of at least one of a), b), c), and d) is from the first dengue virus and the second nucleotide sequence encoding nonstructural proteins of at least one of a), b), c), and d) is from the second dengue virus, wherein the serotype of the first dengue virus and the second dengue virus are different.
9. The composition of claim 8, wherein the serotype of the second dengue virus having the deletion is type 1.
10. The composition of claim 9, wherein the serotype of the first dengue virus of b) is type 2.
11. The composition of claim 9, wherein the serotype of the first dengue virus of c) is type 3.
12. The composition of claim 9, wherein the serotype of the first dengue virus of d) is type 4.
13. The composition of claim 9, wherein the first nucleotide sequence encodes at least two structural proteins of the first dengue virus.
14. The composition of claim 13, wherein the structural proteins are prM and E proteins.
15. The composition of claim 9, wherein the deletion of a) is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 1 genome corresponding to the TL2 stem-loop structure between about nucleotides 10562 and 10591.
16. The composition of claim 8, wherein the serotype of the second dengue virus having the deletion is type 2.
17. The composition of claim 16, wherein the serotype of the first dengue virus of a) is type 1.
18. The composition of claim 16, wherein the serotype of the first dengue virus of c) is type 3.
19. The composition of claim 16, wherein the serotype of the first dengue of d) virus is type 4.
20. The composition of claim 16, wherein the first nucleotide sequence encodes at least two structural proteins of the first dengue virus.
21. The composition of claim 20, wherein the structural proteins are prM and E proteins.
22. The composition of claim 16, wherein the deletion is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 2 genome corresponding to the TL2 stem-loop structure between about nucleotides 10541 and 10570.
23. The composition of claim 8, wherein the serotype of the second dengue virus having the deletion is type 3.
24. The composition of claim 23, wherein the serotype of the first dengue virus of a) is type 1.
25. The composition of claim 23, wherein the serotype of the first dengue virus of b) is type 2.
26. The composition of claim 23, wherein the serotype of the first dengue virus of d) is type 4.
27. The composition of claim 23, wherein the first nucleotide sequence encodes at least two structural proteins of the first dengue virus.
28. The composition of claim 27, wherein the structural proteins are prM and E proteins.
29. The composition of claim 23, wherein the deletion is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 3 genome corresponding to the TL2 stem-loop structure between about nucleotides 10535 and 10565.
30. The composition of claim 8, wherein the serotype of the second dengue virus having the deletion is type 4.
31. The composition of claim 30, wherein the serotype of the first dengue virus of a) is type 1.
32. The composition of claim 30, wherein the serotype of the first dengue virus of b) is type 2.
33. The composition of claim 30, wherein the serotype of the first dengue virus of c) is type 3.
34. The composition of claim 30, wherein the first nucleotide sequence encodes at least two structural proteins of the first dengue virus.
35. The composition of claim 34, wherein the structural proteins are prM and E proteins.
36. The composition of claim 30, wherein the deletion is a deletion of about 30 nucleotides from the 3 untranslated region of the dengue type 4 genome corresponding to the TL2 stem-loop structure between about nucleotides 10478 and 10507.
37. A method of inducing an immune response in a subject comprising administering an effective amount of the composition of claim 1 to the subject.
38. The method of claim 37 wherein the subject is a human.
39. A tetravalent vaccine comprising the composition of claim 1.
40. A method of preventing disease caused by dengue virus in a subject comprising administering an effective amount of the vaccine of claim 39 to the subject.
41. The method of claim 40 wherein the subject is a human.
42. The tetravalent immunogenic composition defined in claim 1, selected from the group consisting of: (1) rDEN130, rDEN230, rDEN330, rDEN430, (2) rDEN130, rDEN230, rDEN330, rDEN4/130, (3) rDEN130, rDEN230, rDEN330, rDEN4/230, (4) rDEN130, rDEN230, rDEN330, rDEN4/330, (5) rDEN130, rDEN230, rDEN3/130, rDEN430, (6) rDEN130, rDEN230, rDEN3/130, rDEN4/130, (7) rDEN130, rDEN230, rDEN3/130, rDEN4/230, (8) rDEN130, rDEN230, rDEN3/130, rDEN4/330, (9) rDEN130, rDEN230, rDEN3/230, rDEN430, (10) rDEN130, rDEN230, rDEN3/230, rDEN4/130, (11) rDEN130, rDEN230, rDEN3/230, rDEN4/230, (12) rDEN130, rDEN230, rDEN3/230, rDEN4/330, (13) rDEN130, rDEN230, rDEN3/430, rDEN430, (14) rDEN130, rDEN230, rDEN3/430, rDEN4/130, (15) rDEN130, rDEN230, rDEN3/430, rDEN4/230, (16) rDEN130, rDEN230, rDEN3/430, rDEN4/330, (17) rDEN130, rDEN2/130, rDEN330, rDEN430, (18) rDEN130, rDEN2/130, rDEN330, rDEN4/130, (19) rDEN130, rDEN2/130, rDEN330, rDEN4/230, (20) rDEN130, rDEN2/130, rDEN330, rDEN4/330, (21) rDEN130, rDEN2/130, rDEN3/130, rDEN430, (22) rDEN130, rDEN2/130, rDEN3/130, rDEN4/130, (23) rDEN130, rDEN2/130, rDEN3/130, rDEN4/230, (24) rDEN130, rDEN2/130, rDEN3/130, rDEN4/330, (25) rDEN130, rDEN2/130, rDEN3/230, rDEN430, (26) rDEN130, rDEN2/130, rDEN3/230, rDEN4/130, (27) rDEN130, rDEN2/130, rDEN3/230, rDEN4/230, (28) rDEN130, rDEN2/130, rDEN3/230, rDEN4/330, (29) rDEN130, rDEN2/130, rDEN3/430, rDEN430, (30) rDEN130, rDEN2/130, rDEN3/430, rDEN4/130, (31) rDEN130, rDEN2/130, rDEN3/430, rDEN4/230, (32) rDEN130, rDEN2/130, rDEN3/430, rDEN4/330, (33) rDEN130, rDEN2/330, rDEN330, rDEN430, (34) rDEN130, rDEN2/330, rDEN330, rDEN4/130, (35) rDEN130, rDEN2/330, rDEN330, rDEN4/230, (36) rDEN130, rDEN2/330, rDEN330, rDEN4/330, (37) rDEN130, rDEN2/330, rDEN3/130, rDEN430, (38) rDEN130, rDEN2/330, rDEN3/130, rDEN4/130, (39) rDEN130, rDEN2/330, rDEN3/130, rDEN4/230, (40) rDEN130, rDEN2/330, rDEN3/130, rDEN4/330, (41) rDEN130, rDEN2/330, rDEN3/230, rDEN430, (42) rDEN130, rDEN2/330, rDEN3/230, rDEN4/130, (43) rDEN130, rDEN2/330, rDEN3/230, rDEN4/230, (44) rDEN130, rDEN2/330, rDEN3/230, rDEN4/330, (45) rDEN130, rDEN2/330, rDEN3/430, rDEN430, (46) rDEN130, rDEN2/330, rDEN3/430, rDEN4/130, (47) rDEN130, rDEN2/330, rDEN3/430, rDEN4/230, (48) rDEN130, rDEN2/330, rDEN3/430, rDEN4/330, (49) rDEN130, rDEN2/430, rDEN330, rDEN430, (50) rDEN130, rDEN2/430, rDEN330, rDEN4/130, (51) rDEN130, rDEN2/430, rDEN330, rDEN4/230, (52) rDEN130, rDEN2/430, rDEN330, rDEN4/330, (53) rDEN130, rDEN2/430, rDEN3/130, rDEN430, (54) rDEN130, rDEN2/430, rDEN3/130, rDEN4/130, (55) rDEN130, rDEN2/430, rDEN3/130, rDEN4/230, (56) rDEN130, rDEN2/430, rDEN3/130, rDEN4/330, (57) rDEN130, rDEN2/430, rDEN3/230, rDEN430, (58) rDEN130, rDEN2/430, rDEN3/230, rDEN4/130, (59) rDEN130, rDEN2/430, rDEN3/230, rDEN4/230, (60) rDEN130, rDEN2/430, rDEN3/230, rDEN4/330, (61) rDEN130, rDEN2/430, rDEN3/430, rDEN430, (62) rDEN130, rDEN2/430, rDEN3/430, rDEN4/130, (63) rDEN130, rDEN2/430, rDEN3/430, rDEN4/230, (64) rDEN130, rDEN2/430, rDEN3/430, rDEN4/330, (65) rDEN1/230, rDEN230, rDEN330, rDEN430, (66) rDEN1/230, rDEN230, rDEN330, rDEN4/130, (67) rDEN1/230, rDEN230, rDEN330, rDEN4/230, (68) rDEN1/230, rDEN230, rDEN330, rDEN4/330, (69) rDEN1/230, rDEN230, rDEN3/130, rDEN430, (70) rDEN1/230, rDEN230, rDEN3/130, rDEN4/130, (71) rDEN1/230, rDEN230, rDEN3/130, rDEN4/230, (72) rDEN1/230, rDEN230, rDEN3/130, rDEN4/330, (73) rDEN1/230, rDEN230, rDEN3/230, rDEN430, (74) rDEN1/230, rDEN230, rDEN3/230, rDEN4/130, (75) rDEN1/230, rDEN230, rDEN3/230, rDEN4/230, (76) rDEN1/230, rDEN230, rDEN3/230, rDEN4/330, (77) rDEN1/230, rDEN230, rDEN3/430, rDEN430, (78) rDEN1/230, rDEN230, rDEN3/430, rDEN4/130, (79) rDEN1/230, rDEN230, rDEN3/430, rDEN4/230, (80) rDEN1/230, rDEN230, rDEN3/430, rDEN4/330, (81) rDEN1/230, rDEN2/130, rDEN330, rDEN430, (82) rDEN1/230, rDEN2/130, rDEN330, rDEN4/130, (83) rDEN1/230, rDEN2/130, rDEN330, rDEN4/230, (84) rDEN1/230, rDEN2/130, rDEN330, rDEN4/330, (85) rDEN1/230, rDEN2/130, rDEN3/130, rDEN430, (86) rDEN1/230, rDEN2/130, rDEN3/130, rDEN4/130, (87) rDEN1/230, rDEN2/130, rDEN3/130, rDEN4/230, (88) rDEN1/230, rDEN2/130, rDEN3/130, rDEN4/330, (89) rDEN1/230, rDEN2/130, rDEN3/230, rDEN430, (90) rDEN1/230, rDEN2/130, rDEN3/230, rDEN4/130, (91) rDEN1/230, rDEN2/130, rDEN3/230, rDEN4/230, (92) rDEN1/230, rDEN2/130, rDEN3/230, rDEN4/330, (93) rDEN1/230, rDEN2/130, rDEN3/430, rDEN430, (94) rDEN1/230, rDEN2/130, rDEN3/430, rDEN4/130, (95) rDEN1/230, rDEN2/130, rDEN3/430, rDEN4/230, (96) rDEN1/230, rDEN2/130, rDEN3/430, rDEN4/330, (97) rDEN1/230, rDEN2/330, rDEN330, rDEN430, (98) rDEN1/230, rDEN2/330, rDEN330, rDEN4/130, (99) rDEN1/230, rDEN2/330, rDEN330, rDEN4/230, (100) rDEN1/230, rDEN2/330, rDEN330, rDEN4/330, (101) rDEN1/230, rDEN2/330, rDEN3/130, rDEN430, (102) rDEN1/230, rDEN2/330, rDEN3/130, rDEN4/130, (103) rDEN1/230, rDEN2/330, rDEN3/130, rDEN4/230, (104) rDEN1/230, rDEN2/330, rDEN3/130, rDEN4/330, (105) rDEN1/230, rDEN2/330, rDEN3/230, rDEN430, (106) rDEN1/230, rDEN2/330, rDEN3/230, rDEN4/130, (107) rDEN1/230, rDEN2/330, rDEN3/230, rDEN4/230, (108) rDEN1/230, rDEN2/330, rDEN3/230, rDEN4/330, (109) rDEN1/230, rDEN2/330, rDEN3/430, rDEN430, (110) rDEN1/230, rDEN2/330, rDEN3/430, rDEN4/130, (111) rDEN1/230, rDEN2/330, rDEN3/430, rDEN4/230, (112) rDEN1/230, rDEN2/330, rDEN3/430, rDEN4/330, (113) rDEN1/230, rDEN2/430, rDEN330, rDEN430, (114) rDEN1/230, rDEN2/430, rDEN330, rDEN4/130, (115) rDEN1/230, rDEN2/430, rDEN330, rDEN4/230, (116) rDEN1/230, rDEN2/430, rDEN330, rDEN4/330, (117) rDEN1/230, rDEN2/430, rDEN3/130, rDEN430, (118) rDEN1/230, rDEN2/430, rDEN3/130, rDEN4/130, (119) rDEN1/230, rDEN2/430, rDEN3/130, rDEN4/230, (120) rDEN1/230, rDEN2/430, rDEN3/130, rDEN4/330, (121) rDEN1/230, rDEN2/430, rDEN3/230, rDEN430, (122) rDEN1/230, rDEN2/430, rDEN3/230, rDEN4/130, (123) rDEN1/230, rDEN2/430, rDEN3/230, rDEN4/230, (124) rDEN1/230, rDEN2/430, rDEN3/230, rDEN4/330, (125) rDEN1/230, rDEN2/430, rDEN3/430, rDEN430, (126) rDEN1/230, rDEN2/430, rDEN3/430, rDEN4/130, (127) rDEN1/230, rDEN2/430, rDEN3/430, rDEN4/230, (128) rDEN1/230, rDEN2/430, rDEN3/430, rDEN4/330, (129) rDEN1/330, rDEN230, rDEN330, rDEN430, (130) rDEN1/330, rDEN230, rDEN330, rDEN4/130, (131) rDEN1/330, rDEN230, rDEN330, rDEN4/230, (132) rDEN1/330, rDEN230, rDEN330, rDEN4/330, (133) rDEN1/330, rDEN230, rDEN3/130, rDEN430, (134) rDEN1/330, rDEN230, rDEN3/130, rDEN4/130, (135) rDEN1/330, rDEN230, rDEN3/130, rDEN4/230, (136) rDEN1/330, rDEN230, rDEN3/130, rDEN4/330, (137) rDEN1/330, rDEN230, rDEN3/230, rDEN430, (138) rDEN1/330, rDEN230, rDEN3/230, rDEN4/130, (139) rDEN1/330, rDEN230, rDEN3/230, rDEN4/230, (140) rDEN1/330, rDEN230, rDEN3/230, rDEN4/330, (141) rDEN1/330, rDEN230, rDEN3/430, rDEN430, (142) rDEN1/330, rDEN230, rDEN3/430, rDEN4/130, (143) rDEN1/330, rDEN230, rDEN3/430, rDEN4/230, (144) rDEN1/330, rDEN230, rDEN3/430, rDEN4/330, (145) rDEN1/330, rDEN2/130, rDEN330, rDEN430, (146) rDEN1/330, rDEN2/130, rDEN330, rDEN4/130, (147) rDEN1/330, rDEN2/130, rDEN330, rDEN4/230, (148) rDEN1/330, rDEN2/130, rDEN330, rDEN4/330, (149) rDEN1/330, rDEN2/130, rDEN3/130, rDEN430, (150) rDEN1/330, rDEN2/130, rDEN3/130, rDEN4/130, (151) rDEN1/330, rDEN2/130, rDEN3/130, rDEN4/230, (152) rDEN1/330, rDEN2/130, rDEN3/130, rDEN4/330, (153) rDEN1/330, rDEN2/130, rDEN3/230, rDEN430, (154) rDEN1/330, rDEN2/130, rDEN3/230, rDEN4/130, (155) rDEN1/330, rDEN2/130, rDEN3/230, rDEN4/230, (156) rDEN1/330, rDEN2/130, rDEN3/230, rDEN4/330, (157) rDEN1/330, rDEN2/130, rDEN3/430, rDEN430, (158) rDEN1/330, rDEN2/130, rDEN3/430, rDEN4/130, (159) rDEN1/330, rDEN2/130, rDEN3/430, rDEN4/230, (160) rDEN1/330, rDEN2/130, rDEN3/430, rDEN4/330, (161) rDEN1/330, rDEN2/330, rDEN330, rDEN430, (162) rDEN1/330, rDEN2/330, rDEN330, rDEN4/130, (163) rDEN1/330, rDEN2/330, rDEN330, rDEN4/230, (164) rDEN1/330, rDEN2/330, rDEN330, rDEN4/330, (165) rDEN1/330, rDEN2/330, rDEN3/130, rDEN430, (166) rDEN1/330, rDEN2/330, rDEN3/130, rDEN4/130, (167) rDEN1/330, rDEN2/330, rDEN3/130, rDEN4/230, (168) rDEN1/330, rDEN2/330, rDEN3/130, rDEN4/330, (169) rDEN1/330, rDEN2/330, rDEN3/230, rDEN430, (170) rDEN1/330, rDEN2/330, rDEN3/230, rDEN4/130, (171) rDEN1/330, rDEN2/330, rDEN3/230, rDEN4/230, (172) rDEN1/330, rDEN2/330, rDEN3/230, rDEN4/330, (173) rDEN1/330, rDEN2/330, rDEN3/430, rDEN430, (174) rDEN1/330, rDEN2/330, rDEN3/430, rDEN4/130, (175) rDEN1/330, rDEN2/330, rDEN3/430, rDEN4/230, (176) rDEN1/330, rDEN2/330, rDEN3/430, rDEN4/330, (177) rDEN1/330, rDEN2/430, rDEN330, rDEN430, (178) rDEN1/330, rDEN2/430, rDEN330, rDEN4/130, (179) rDEN1/330, rDEN2/430, rDEN330, rDEN4/230, (180) rDEN1/330, rDEN2/430, rDEN330, rDEN4/330, (181) rDEN1/330, rDEN2/430, rDEN3/130, rDEN430, (182) rDEN1/330, rDEN2/430, rDEN3/130, rDEN4/130, (183) rDEN1/330, rDEN2/430, rDEN3/130, rDEN4/230, (184) rDEN1/330, rDEN2/430, rDEN3/130, rDEN4/330, (185) rDEN1/330, rDEN2/430, rDEN3/230, rDEN430, (186) rDEN1/330, rDEN2/430, rDEN3/230, rDEN4/130, (187) rDEN1/330, rDEN2/430, rDEN3/230, rDEN4/230, (188) rDEN1/330, rDEN2/430, rDEN3/230, rDEN4/330, (189) rDEN1/330, rDEN2/430, rDEN3/430, rDEN430, (190) rDEN1/330, rDEN2/430, rDEN3/430, rDEN4/130, (191) rDEN1/330, rDEN2/430, rDEN3/430, rDEN4/230, (192) rDEN1/330, rDEN2/430, rDEN3/430, rDEN4/330, (193) rDEN1/430, rDEN230, rDEN330, rDEN430, (194) rDEN1/430, rDEN230, rDEN330, rDEN4/130, (195) rDEN1/430, rDEN230, rDEN330, rDEN4/230, (196) rDEN1/430, rDEN230, rDEN330, rDEN4/330, (197) rDEN1/430, rDEN230, rDEN3/130, rDEN430, (198) rDEN1/430, rDEN230, rDEN3/130, rDEN4/130, (199) rDEN1/430, rDEN230, rDEN3/130, rDEN4/230, (200) rDEN1/430, rDEN230, rDEN3/130, rDEN4/330, (201) rDEN1/430, rDEN230, rDEN3/230, rDEN430, (202) rDEN1/430, rDEN230, rDEN3/230, rDEN4/130, (203) rDEN1/430, rDEN230, rDEN3/230, rDEN4/230, (204) rDEN1/430, rDEN230, rDEN3/230, rDEN4/330, (205) rDEN1/430, rDEN230, rDEN3/430, rDEN430, (206) rDEN1/430, rDEN230, rDEN3/430, rDEN4/130, (207) rDEN1/430, rDEN230, rDEN3/430, rDEN4/230, (208) rDEN1/430, rDEN230, rDEN3/430, rDEN4/330, (209) rDEN1/430, rDEN2/130, rDEN330, rDEN430, (210) rDEN1/430, rDEN2/130, rDEN330, rDEN4/130, (211) rDEN1/430, rDEN2/130, rDEN330, rDEN4/230, (212) rDEN1/430, rDEN2/130, rDEN330, rDEN4/330, (213) rDEN1/430, rDEN2/130, rDEN3/130, rDEN430, (214) rDEN1/430, rDEN2/130, rDEN3/130, rDEN4/130, (215) rDEN1/430, rDEN2/130, rDEN3/130, rDEN4/230, (216) rDEN1/430, rDEN2/130, rDEN3/130, rDEN4/330, (217) rDEN1/430, rDEN2/130, rDEN3/230, rDEN430, (218) rDEN1/430, rDEN2/130, rDEN3/230, rDEN4/130, (219) rDEN1/430, rDEN2/130, rDEN3/230, rDEN4/230, (220) rDEN1/430, rDEN2/130, rDEN3/230, rDEN4/330, (221) rDEN1/430, rDEN2/130, rDEN3/430, rDEN430, (222) rDEN1/430, rDEN2/130, rDEN3/430, rDEN4/130, (223) rDEN1/430, rDEN2/130, rDEN3/430, rDEN4/230, (224) rDEN1/430, rDEN2/130, rDEN3/430, rDEN4/330, (225) rDEN1/430, rDEN2/330, rDEN330, rDEN430, (226) rDEN1/430, rDEN2/330, rDEN330, rDEN4/130, (227) rDEN1/430, rDEN2/330, rDEN330, rDEN4/230, (228) rDEN1/430, rDEN2/330, rDEN330, rDEN4/330, (229) rDEN1/430, rDEN2/330, rDEN3/130, rDEN430, (230) rDEN1/430, rDEN2/330, rDEN3/130, rDEN4/130, (231) rDEN1/430, rDEN2/330, rDEN3/130, rDEN4/230, (232) rDEN1/430, rDEN2/330, rDEN3/130, rDEN4/330, (233) rDEN1/430, rDEN2/330, rDEN3/230, rDEN430, (234) rDEN1/430, rDEN2/330, rDEN3/230, rDEN4/130, (235) rDEN1/430, rDEN2/330, rDEN3/230, rDEN4/230, (236) rDEN1/430, rDEN2/330, rDEN3/230, rDEN4/330, (237) rDEN1/430, rDEN2/330, rDEN3/430, rDEN430, (238) rDEN1/430, rDEN2/330, rDEN3/430, rDEN4/130, (239) rDEN1/430, rDEN2/330, rDEN3/430, rDEN4/230, (240) rDEN1/430, rDEN2/330, rDEN3/430, rDEN4/330, (241) rDEN1/430, rDEN2/430, rDEN330, rDEN430, (242) rDEN1/430, rDEN2/430, rDEN330, rDEN4/130, (243) rDEN1/430, rDEN2/430, rDEN330, rDEN4/230, (244) rDEN1/430, rDEN2/430, rDEN330, rDEN4/330, (245) rDEN1/430, rDEN2/430, rDEN3/130, rDEN430, (246) rDEN1/430, rDEN2/430, rDEN3/130, rDEN4/130, (247) rDEN1/430, rDEN2/430, rDEN3/130, rDEN4/230, (248) rDEN1/430, rDEN2/430, rDEN3/130, rDEN4/330, (249) rDEN1/430, rDEN2/430, rDEN3/230, rDEN430, (250) rDEN1/430, rDEN2/430, rDEN3/230, rDEN4/130, (251) rDEN1/430, rDEN2/430, rDEN3/230, rDEN4/230, (252) rDEN1/430, rDEN2/430, rDEN3/230, rDEN4/330, (254) rDEN1/430, rDEN2/430, rDEN3/430, rDEN4/130, (255) rDEN1/430, rDEN2/430, rDEN3/430, rDEN4/230, and (256) rDEN1/430, rDEN2/430, rDEN3/430, rDEN4/330.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(13) TABLE-US-00001 Brief Description of the Sequences GenBank Accession Serotype No. or description DEN1 U88535 DEN2 Tonga/74 DEN3 Sleman/78 DEN4 AF326825
(14) TABLE-US-00002 BriefDescriptionoftheSEQIDNOs Figure, Table,or SEQ Identification Appendix IDNO. TL2regionofDEN1 FIG.2C 1 TL2regionofDEN2 FIG.2C 2 TL2regionofDEN3 FIG.2C 3 TL2regionofDEN4 FIG.2C 4 TL2regionofDEN130 FIG.2B 5 TL2regionofDEN230 FIG.2B 6 TL2regionofDEN330 FIG.2B 7 TL2regionofDEN430 FIG.2B 8 TL2regionofp2 FIG.6B 9 TL2regionofp230 FIG.6B 10 TL2regionofp3 FIG.8B 11 TL2regionofp330 FIG.8B 12 Spellinkerinp3 FIG.8A 13 rDEN2/4junction1 FIG.9B 14-nt,15-aa rDEN2/4junction2 FIG.9B 16-nt,17-aa rDEN2/4junction3 FIG.9B 18-nt,19-aa rDEN3/4junction1 FIG.11B 20-nt,21-aa rDEN3/4junction2 FIG.11B 22-nt,23-aa rDEN3/4junction3 FIG.11B 24-nt,25-aa rDEN1/4junction1 FIG.12B 26-nt,27-aa rDEN1/4junction2 FIG.12B 28-nt,29-aa rDEN1/4junction3 FIG.12B 30-nt,31-aa D4selectedNS4Bregion Table15 32-nt,33-aa D1selectedNS4Bregion Table15 34-nt,35-aa D2selectedNS4Bregion Table15 36-nt,37-aa D3selectedNS4Bregion Table15 38-nt,39-aa CCACGGGCGCCGT Table26 40 AAGGCCTGGA Table26 41 TATCCCCGGGAC Table26 42 AGAGCTCTCTC Table26 43 GAATCTCCACCCGGA Table26 44 CTGTCGAATC Table26 45 DEN2(Tonga/74) Appendix1 46-nt,47-aa cDNAplasmidp2 DEN3(Sleman/78) Appendix2 48-nt,49-aa cDNAplasmidp3 DEN1(PuertoRico/94)CME Appendix3 50-nt,51-aa chimericregion DEN1(PuertoRico/94)ME Appendix4 52-nt,53-aa chimericregion
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Introduction
(15) A molecular approach is herewith used to develop a genetically stable live attenuated tetravalent dengue virus vaccine. Each component of the tetravalent vaccine, namely, DEN1, DEN2, DEN3, and DEN4, must be attenuated, genetically stable, and immunogenic. A tetravalent vaccine is needed to ensure simultaneous protection against each of the four dengue viruses, thereby precluding the possibility of developing the more serious illnesses dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which occur in humans during secondary infection with a heterotypic wild-type dengue virus. Since dengue viruses can undergo genetic recombination in nature (Worobey, M. et al. 1999 PNAS USA 96:7352-7), the tetravalent vaccine should be genetically incapable of undergoing a recombination event between its four virus components that could lead to the generation of viruses lacking attenuating mutations. Previous approaches to develop a tetravalent dengue virus vaccine have been based on independently deriving each of the four virus components through separate mutagenic procedures, such as passage in tissue culture cells derived from a heterologous host. This strategy has yielded attenuated vaccine candidates (Bhamarapravati, N. and Sutee, Y. 2000 Vaccine 18:44-7). However, it is possible that gene exchanges among the four components of these independently derived tetravalent vaccines could occur in vaccinees, possibly creating a virulent recombinant virus. Virulent polioviruses derived from recombination have been generated in vaccinees following administration of a trivalent poliovirus vaccine (Guillot, S. et al. 2000 J Virol 74:8434-43).
(16) The present invention describes: (1) improvements to the previously described rDEN430 vaccine candidate, 2) attenuated rDEN130, rDEN230, and rDEN330 recombinant viruses containing a 30 nucleotide deletion (30) in a section of the 3 untranslated region (UTR) that is homologous to that in the rDEN430 recombinant virus, (3) a method to generate a tetravalent dengue virus vaccine composed of rDEN130, rDEN230, rDEN330, and rDEN430, 4) attenuated antigenic chimeric viruses, rDEN1/430, rDEN2/430, and rDEN3/430, for which the CME, ME, or E gene regions of rDEN430 have been replaced with those derived from DEN1, DEN2, or DEN3; alternatively rDEN1/330, rDEN2/330, and rDEN4/330 for which CME, ME, or E gene regions of rDEN330 have been replaced with those derived from DEN1, 2, or 4; alternatively rDEN1/230, rDEN3/230, and rDEN4/230 for which CME, ME, or E gene regions of rDEN230 have been replaced with those derived from DEN1, 3, or, 4; and alternatively rDEN2/130, rDEN3/130, and rDEN4/130 for which CME, ME, or E gene regions of rDEN130 have been replaced with those derived from DEN2, 3, or 4, and 5) a method to generate a tetravalent dengue virus vaccine composed of rDEN1/430, rDEN2/430, rDEN3/430, and rDEN430, alternatively rDEN1/330, rDEN2/330, rDEN4/330, and rDEN330, alternatively rDEN1/230, rDEN3/230, rDEN4/230, and rDEN230, and alternatively rDEN2/130, rDEN3/130, rDEN4/130, and rDEN130. These tetravalent vaccines are unique since they contain a common shared attenuating mutation which eliminates the possibility of generating a virulent wild-type virus in a vaccinee since each component of the vaccine possesses the same 30 attenuating deletion mutation. In addition, the rDEN130, rDEN230, rDEN330, rDEN430 tetravalent vaccine is the first to combine the stability of the 30 mutation with broad antigenicity. Since the 30 deletion mutation is in the 3 UTR of each virus, all of the proteins of the four component viruses are available to induce a protective immune response. Thus, the method provides a mechanism of attenuation that maintains each of the proteins of DEN1, DEN2, DEN3, and DEN4 viruses in a state that preserves the full capability of each of the proteins of the four viruses to induce humoral and cellular immune responses against all of the structural and non-structural proteins present in each dengue virus serotype.
(17) As previously described, the DEN4 recombinant virus, rDEN430 (previously referred to as 2A30), was engineered to contain a 30 nucleotide deletion in the 3UTR of the viral genome (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13; Men, R. et al. 1996 J Virol 70:3930-7). Evaluation in rhesus monkeys showed the virus to be significantly attenuated relative to wild-type parental virus, yet highly immunogenic and completely protective. Also, a phase I clinical trial with adult human volunteers showed the rDEN430 recombinant virus to be safe and satisfactorily immunogenic (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). To develop a tetravalent vaccine bearing a shared attenuating mutation in a untranslated region, we selected the 30 mutation to attenuate wild-type dengue viruses of serotypes 1, 2, and 3 since it attenuated wild-type DEN4 virus for rhesus monkeys and was safe in humans (
(18) The 30 mutation was first described and characterized in the DEN4 virus (Men, R. et al. 1996 J Virol 70:3930-7). In DEN4, the mutation consists of the removal of 30 contiguous nucleotides comprising nucleotides 10478-10507 of the 3 UTR (
Immunogenic Dengue Chimeras and Methods for their Preparation
(19) Immunogenic dengue chimeras and methods for preparing the dengue chimeras are provided herein. The immunogenic dengue chimeras are useful, alone or in combination, in a pharmaceutically acceptable carrier as immunogenic compositions to minimize, inhibit, or immunize individuals and animals against infection by dengue virus.
(20) Chimeras of the present invention comprise nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences selected from the backbone of a dengue virus of a different serotype. These chimeras can be used to induce an immunogenic response against dengue virus.
(21) In another embodiment, the preferred chimera is a nucleic acid chimera comprising a first nucleotide sequence encoding at least one structural protein from a dengue virus of a first serotype, and a second nucleotide sequence encoding non-structural proteins from a dengue virus of a second serotype different from the first. In another embodiment the dengue virus of the second serotype is DEN4. In another embodiment, the structural protein can be the C protein of a dengue virus of the first serotype, the prM protein of a dengue virus of the first serotype, the E protein of a dengue virus of the first serotype, or any combination thereof.
(22) The term residue is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
(23) Furthermore, one of skill in the art will recognize that individual substitutions, deletions or additions in the amino acid sequence, or in the nucleotide sequence encoding for the amino acids, which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified variations, wherein the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(24) As used herein, the terms virus chimera, chimeric virus, dengue chimera and chimeric dengue virus means an infectious construct of the invention comprising nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences derived from the backbone of a dengue virus of a different serotype.
(25) As used herein, infectious construct indicates a virus, a viral construct, a viral chimera, a nucleic acid derived from a virus or any portion thereof, which may be used to infect a cell.
(26) As used herein, nucleic acid chimera means a construct of the invention comprising nucleic acid comprising nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences derived from the backbone of a dengue virus of a different serotype. Correspondingly, any chimeric virus or virus chimera of the invention is to be recognized as an example of a nucleic acid chimera.
(27) The structural and nonstructural proteins of the invention are to be understood to include any protein comprising or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, three or more amino acid residues thereof.
(28) Dengue Chimeras
(29) Dengue virus is a mosquito-borne flavivirus pathogen. The dengue virus genome contains a 5 untranslated region (5 UTR), followed by a capsid protein (C) encoding region, followed by a premembrane/membrane protein (prM) encoding region, followed by an envelope protein (E) encoding region, followed by the region encoding the nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finally a 3 untranslated region (3 UTR). The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1-NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.
(30) The dengue chimeras of the invention are constructs formed by fusing structural protein genes from a dengue virus of one serotype, e.g. DEN1, DEN2, DEN3, or DEN4, with non-structural protein genes from a dengue virus of a different serotype, e.g., DEN1, DEN2, DEN3, or DEN4.
(31) The attenuated, immunogenic dengue chimeras provided herein contain one or more of the structural protein genes, or antigenic portions thereof, of the dengue virus of one serotype against which immunogenicity is to be conferred, and the nonstructural protein genes of a dengue virus of a different serotype.
(32) The chimera of the invention contains a dengue virus genome of one serotype as the backbone, in which the structural protein gene(s) encoding C, prM, or E protein(s) of the dengue genome, or combinations thereof, are replaced with the corresponding structural protein gene(s) from a dengue virus of a different serotype that is to be protected against. The resulting viral chimera has the properties, by virtue of being chimerized with a dengue virus of another serotype, of attenuation and is therefore reduced in virulence, but expresses antigenic epitopes of the structural gene products and is therefore immunogenic.
(33) The genome of any dengue virus can be used as the backbone in the attenuated chimeras described herein. The backbone can contain mutations that contribute to the attenuation phenotype of the dengue virus or that facilitate replication in the cell substrate used for manufacture, e.g., Vero cells. The mutations can be in the nucleotide sequence encoding non-structural proteins, the 5 untranslated region or the 3 untranslated region. The backbone can also contain further mutations to maintain the stability of the attenuation phenotype and to reduce the possibility that the attenuated virus or chimera might revert back to the virulent wild-type virus. For example, a first mutation in the 3 untranslated region and a second mutation in the 5 untranslated region will provide additional attenuation phenotype stability, if desired. In particular, a mutation that is a deletion of 30 nts from the 3 untranslated region of the DEN4 genome between nts 10478-10507 results in attenuation of the DEN4 virus (Men et al. 1996 J. Virology 70:3930-3933; Durbin et al. 2001 Am J Trop Med 65:405-413, 2001). Therefore, the genome of any dengue type 4 virus containing such a mutation at this locus can be used as the backbone in the attenuated chimeras described herein. Furthermore, other dengue virus genomes containing an analogous deletion mutation in the 3 untranslated region of the genomes of other dengue virus serotypes may also be used as the backbone structure of this invention.
(34) Such mutations may be achieved by site-directed mutagenesis using techniques known to those skilled in the art. It will be understood by those skilled in the art that the virulence screening assays, as described herein and as are well known in the art, can be used to distinguish between virulent and attenuated backbone structures.
(35) Construction of Dengue Chimeras
(36) The dengue virus chimeras described herein can be produced by substituting at least one of the structural protein genes of the dengue virus of one serotype against which immunity is desired into a dengue virus genome backbone of a different serotype, using recombinant engineering techniques well known to those skilled in the art, namely, removing a designated dengue virus gene of one serotype and replacing it with the desired corresponding gene of dengue virus of a different serotype. Alternatively, using the sequences provided in GenBank, the nucleic acid molecules encoding the dengue proteins may be synthesized using known nucleic acid synthesis techniques and inserted into an appropriate vector. Attenuated, immunogenic virus is therefore produced using recombinant engineering techniques known to those skilled in the art.
(37) As mentioned above, the gene to be inserted into the backbone encodes a dengue structural protein of one serotype. Preferably the dengue gene of a different serotype to be inserted is a gene encoding a C protein, a prM protein and/or an E protein. The sequence inserted into the dengue virus backbone can encode both the prM and E structural proteins of the other serotype. The sequence inserted into the dengue virus backbone can encode the C, prM and E structural proteins of the other serotype. The dengue virus backbone is the DEN1, DEN2, DEN3, or DEN4 virus genome, or an attenuated dengue virus genome of any of these serotypes, and includes the substituted gene(s) that encode the C, prM and/or E structural protein(s) of a dengue virus of a different serotype, or the substituted gene(s) that encode the prM and/or E structural protein(s) of a dengue virus of a different serotype.
(38) Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of dengue virus of any of the serotypes can be evaluated for usefulness as vaccines by screening them for phenotypic markers of attenuation that indicate reduction in virulence with retention of immunogenicity. Antigenicity and immunogenicity can be evaluated using in vitro or in vivo reactivity with dengue antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.
(39) Dengue Vaccines
(40) The preferred chimeric viruses and nucleic acid chimeras provide live, attenuated viruses useful as immunogens or vaccines. In a preferred embodiment, the chimeras exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.
(41) The chimeric viruses or nucleic acid chimeras of this invention can comprise the structural genes of a dengue virus of one serotype in a wild-type or an attenuated dengue virus backbone of a different serotype. For example, the chimera may express the structural protein genes of a dengue virus of one serotype in either of a dengue virus or an attenuated dengue virus background of a different serotype.
(42) The strategy described herein of using a genetic background that contains nonstructural regions of a dengue virus genome of one serotype, and, by chimerization, the properties of attenuation, to express the structural protein genes of a dengue virus of a different serotype has lead to the development of live, attenuated dengue vaccine candidates that express structural protein genes of desired immunogenicity. Thus, vaccine candidates for control of dengue pathogens can be designed.
(43) Viruses used in the chimeras described herein are typically grown using techniques known in the art. Virus plaque or focus forming unit (FFU) titrations are then performed and plaques or FFU are counted in order to assess the viability, titer and phenotypic characteristics of the virus grown in cell culture. Wild type viruses are mutagenized to derive attenuated candidate starting materials.
(44) Chimeric infectious clones are constructed from various dengue serotypes. The cloning of virus-specific cDNA fragments can also be accomplished, if desired. The cDNA fragments containing the structural protein or nonstructural protein genes are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from dengue RNA with various primers. Amplified fragments are cloned into the cleavage sites of other intermediate clones. Intermediate, chimeric dengue clones are then sequenced to verify the sequence of the inserted dengue-specific cDNA.
(45) Full genome-length chimeric plasmids constructed by inserting the structural or nonstructural protein gene region of dengue viruses into vectors are obtainable using recombinant techniques well known to those skilled in the art.
(46) Methods of Administration
(47) The viral chimeras described herein are individually or jointly combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunogen or vaccine to humans or animals. The terms pharmaceutically acceptable carrier or pharmaceutically acceptable vehicle are used herein to mean any composition or compound including, but not limited to, water or saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner.
(48) The immunogenic or vaccine formulations may be conveniently presented in viral plaque forming unit (PFU) unit or focus forming unit (FFU) dosage form and prepared by using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.
(49) Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art.
(50) The immunogenic or vaccine composition may be administered through different routes, such as oral or parenteral, including, but not limited to, buccal and sublingual, rectal, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The composition may be administered in different forms, including, but not limited to, solutions, emulsions and suspensions, microspheres, particles, microparticles, nano-particles and liposomes. It is expected that from about 1 to about 5 doses may be required per immunization schedule. Initial doses may range from about 100 to about 100,000 PFU or FFU, with a preferred dosage range of about 500 to about 20,000 PFU or FFU, a more preferred dosage range of from about 1000 to about 12,000 PFU or FFU and a most preferred dosage range of about 1000 to about 4000 PFU or FFU. Booster injections may range in dosage from about 100 to about 20,000 PFU or FFU, with a preferred dosage range of about 500 to about 15,000, a more preferred dosage range of about 500 to about 10,000 PFU or FFU, and a most preferred dosage range of about 1000 to about 5000 PFU or FFU. For example, the volume of administration will vary depending on the route of administration. Intramuscular injections may range in volume from about 0.1 ml to 1.0 ml.
(51) The composition may be stored at temperatures of from about 100 C. to about 4 C. The composition may also be stored in a lyophilized state at different temperatures including room temperature. The composition may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to, filtration. The composition may also be combined with bacteriostatic agents to inhibit bacterial growth.
(52) Administration Schedule
(53) The immunogenic or vaccine composition described herein may be administered to humans, especially individuals travelling to regions where dengue virus infection is present, and also to inhabitants of those regions. The optimal time for administration of the composition is about one to three months before the initial exposure to the dengue virus. However, the composition may also be administered after initial infection to ameliorate disease progression, or after initial infection to treat the disease.
(54) Adjuvants
(55) A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the chimeric virus in the immunogen or vaccine composition of this invention. Such adjuvants include, but are not limited to, the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, Freund's complete adjuvant (for animals), Freund's incomplete adjuvant; sorbitan monooleate, squalene, CRL-8300 adjuvant, alum, QS 21, muramyl dipeptide, CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs, trehalose, bacterial extracts, including mycobacterial extracts, detoxified endotoxins, membrane lipids, or combinations thereof.
(56) Nucleic Acid Sequences
(57) Nucleic acid sequences of dengue virus of one serotype and dengue virus of a different serotype are useful for designing nucleic acid probes and primers for the detection of dengue virus chimeras in a sample or specimen with high sensitivity and specificity. Probes or primers corresponding to dengue virus can be used to detect the presence of a vaccine virus. The nucleic acid and corresponding amino acid sequences are useful as laboratory tools to study the organisms and diseases and to develop therapies and treatments for the diseases.
(58) Nucleic acid probes and primers selectively hybridize with nucleic acid molecules encoding dengue virus or complementary sequences thereof. By selective or selectively is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the dengue virus sequence. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in the sample. The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term selectively hybridizes excludes the occasional randomly hybridizing nucleic acids, and thus has the same meaning as specifically hybridizing. The selectively hybridizing nucleic acid probes and primers of this invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes, preferably 85% or more.
(59) The present invention also contemplates sequences, probes and primers that selectively hybridize to the encoding nucleic acid or the complementary, or opposite, strand of the nucleic acid. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-species hybridization capability is maintained. By probe or primer is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes or primers can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides. Isolated nucleic acids are provided herein that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least five nucleotides complementary to the sequence of interest as described in Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
(60) If used as primers, the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of detecting the presence of dengue virus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.
(61) The nucleic acid sequences encoding dengue virus can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant dengue virus peptide and/or polypeptides.
(62) The nucleic acid sequences of the invention include a diagnostic probe that serves to report the detection of a cDNA amplicon amplified from the viral genomic RNA template by using a reverse-transcription/polymerase chain reaction (RT/PCR), as well as forward and reverse amplimers that are designed to amplify the cDNA amplicon. In certain instances, one of the amplimers is designed to contain a vaccine virus-specific mutation at the 3-terminal end of the amplimer, which effectively makes the test even more specific for the vaccine strain because extension of the primer at the target site, and consequently amplification, will occur only if the viral RNA template contains that specific mutation.
(63) Automated PCR-based nucleic acid sequence detection systems have been recently developed. TaqMan assay (Applied Biosystems) is widely used. A more recently developed strategy for diagnostic genetic testing makes use of molecular beacons (Tyagi and Kramer, 1996 Nature Biotechnology 14:303-308). Molecular beacon assays employ quencher and reporter dyes that differ from those used in the TaqMan assay. These and other detection systems may used by one skilled in is the art.
EXAMPLE 1
Improvement of Dengue Virus Vaccine Candidate rDEN430
(64) The safety of recombinant live-attenuated dengue-4 vaccine candidate rDEN430 was evaluated in twenty human volunteers who received a dose of 5.0 log.sub.10plaque forming units (PFU) (Durbin A. P. et al. 2001 Am J Trop Med Hyg 65:405-413). The vaccine candidate was found to be safe, well-tolerated and immunogenic in all of the vaccinees. However, five of the vaccinees experienced a transient elevation in alanine aminotransferase levels, three experienced neutropenia and ten vaccinees developed an asymptomatic macular rash, suggesting that it may be necessary to further attenuate this vaccine candidate.
(65) Currently, a randomized, double-blind, placebo-controlled, dose de-escalation study is being conducted to determine the human infectious dose 50 (HID.sub.50) of rDEN430. Each dose cohort consists of approximately twenty vaccinees and four placebo recipients. To date, complete data for doses of 3.0 log.sub.10PFU and 2.0 log.sub.10PFU has been collected. rDEN430 infected 100% of vaccinees when 3.0 log.sub.10PFU was administered and 95% of vaccinees when 2.0 log.sub.10PFU was administered (Table 1). The vaccine candidate caused no symptomatic illness at either dose (Table 1). One vaccinee who received 3.0 log.sub.10PFU experienced a transient elevation in alanine aminotransferase levels and approximately one fourth of the vaccinees experienced neutropenia at both doses (Table 1). Neutropenia was transient and mild. More than half of the vaccinees developed a macular rash at both doses; the occurrence of rash was not correlated with vaccination dose or with viremia (Table 1 and Table 2). Neither peak titer nor onset of viremia differed between the 3.0 log.sub.10PFU and 2.0 log.sub.10PFU doses, though both measures of viremia were significantly lower for these doses than for a dose of 5.0 log.sub.10PFU (Table 3). The vaccine candidate was immunogenic in 95% of vaccinees at both doses and neutralizing antibody did not decline between days 28 and 42 post-vaccination (Table 4). Although the HID.sub.50 has not been determined yet, it is clearly less than 2.0 log.sub.10PFU. Interestingly, decreases in the dose of vaccine have had no consistent effect on immunogenicity, viremia, benign neutropenia or the occurrence of rash. Thus will not necessarily be possible to further attenuate rDEN430 by decreasing the dose of virus administered, and other approaches must be developed.
(66) TABLE-US-00003 TABLE 1 rDEN430 clinical summary No. No. Mean No. volunteers with: No. of in- with peak Neutro- subjects Dose.sup.a fected viremia titer.sup.b Fever Rash penia.sup.c ALT 20 5.0 20 14 1.2 (0.2) 1.sup.d 10 3 5 20 3.0 20 7 0.4 (0.0) 0 11 5 1.sup.e 20 2.0 19 11 0.6 (0.1) 1.sup.d 16 4 0 8 0 0 0 0 0 0 0 0 .sup.aLog.sub.10 pfu .sup.bLog.sub.10 pfu/mL .sup.cNeutropenia defined as ANC < 1500/dl .sup.dT Max in volunteer = 100.4 F. .sup.eALT day 0 = 78, ALT max = 91 (day 14)
(67) TABLE-US-00004 TABLE 2 Pattern of rash in vaccinees No. No. Mean with with Viremia Viremia Mean day of duration Dose.sup.a viremia rash & rash no rash onset SD (days SD) 5 14/20 10/20 9/20 5/20 .sub.8.1 1.3 [A].sup.a 3.6 2.0 [A] 3 7/20 11/20 6/20 1/20 12.2 1.4 [B] 5.0 2.1 [A] 2 11/20 16/20 9/20 2/20 11.2 1.4 [B] 6.9 1.7 [B] .sup.alog.sub.10 pfu .sup.bMeans in each column with different letters are significantly different ( = 0.05)
(68) TABLE-US-00005 TABLE 3 rDEN430 viremia summary Mean onset of Mean duration # with Mean peak titer viremia of viremia Dose.sup.a viremia (log.sub.10 pfu/mL) (day SD) (day SD) 5 14 1.2 0.2 [A] 5.8 2.4 [A].sup.b 4.4 2.4 [A] 3 7 0.4 0.0 [B] 9.1 2.5 [B] 1.6 1.0 [B] 2 11 0.6 0.1 [B] 8.7 2.4 [B] 2.6 2.0 [A] .sup.alog.sub.10 pfu .sup.bMeans in each column with different letters are significantly different ( = 0.05)
(69) TABLE-US-00006 TABLE 4 Immunogenicity of rDEN430 Geometric mean serum neutralizing % No. of Dose No. antibody titer (range) serocon- subjects (log.sub.10) infected Day 28 Day 42 version 20 5.0 20 567 (72-2455) 399 (45-1230) 100 20 3.0 20 156 (5-2365) 158 (25-1222) 95 20 2.0 19 163 (5-943) 165 (5-764) 95 8 0 0 0 0 0
(70) Two approaches have been taken to further attenuate rDEN430. This first is the generation and characterization of attenuating point mutations in rDEN4 using 5 fluorouracil mutagenesis (Blaney, J. E. Jr. et al. 2002 Virology 300: 125-139; Blaney, J. E. Jr. et al. 2001 J. Virol. 75: 9731-9740). This approach has identified a panel of point mutations that confer a range of temperature sensitivity (ts) and small plaque (sp) phenotypes in Vero and HuH-7 cells and attenuation (att) phenotypes in suckling mouse brain and SCID mice engrafted with HuH-7 cells (SCID-HuH-7 mice). In this example, a subset of these mutations has been introduced to rDEN430 and the phenotypes of the resulting viruses evaluated.
(71) A second approach was to create a series of paired charge-to-alanine mutations in contiguous pairs of charged amino acid residues in the rDEN4 NS5 gene. As demonstrated previously, mutation of 32 individual contiguous pairs of charged amino acid residues in rDEN4 NS5 conferred a range of ts phenotypes in Vero and HuH-7 cells and a range of att phenotypes in suckling mouse brain (Hanley, K. H. et al. 2002 J. Virol. 76 525-531). As demonstrated below, these mutations also confer an att phenotype in SCID-HuH-7 mice. These mutations have been introduced, either as single pairs or sets of two pairs, into rDEN430 to determine whether they are compatible with the 30 mutation and whether they enhance the att phenotypes of rDEN430.
(72) A panel of rDEN4 viruses bearing individual point mutations have been characterized which possess temperature sensitive and/or small plaque phenotypes in tissue culture and varying levels of attenuated replication in suckling mouse brain when compared to wild type rDEN4 virus (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol. 75:9731-9740). Three mutations have been selected to combine with the 30 deletion mutation to evaluate their ability to further restrict replication of rDEN430 in rhesus monkeys. First, the missense mutation in NS3 at nucleotide 4995 (Ser>Pro) which confers temperature sensitivity in Vero and HuH-7 cells and restricted replication in suckling mouse brain was previously combined with the 30 mutation (Blaney, J. E. et al. 2001 J Virol. 75:9731-9740). The resulting virus, rDEN430-4995, was found to be more restricted (1,000-fold) in mouse brain replication than rDEN430 virus (<5-fold) when compared to wild type rDEN4 virus. Second, a missense mutation at nucleotide 8092 (Glu>Gly) which also confers temperature sensitivity in Vero and HuH-7 cells and 10,000-fold restricted replication in suckling mouse brain was combined with the 30 mutation here. Third, a substitution in the 3 UTR at nucleotide 10634 which confers temperature sensitivity in Vero and HuH-7 cells, small plaque size in HuH-7 cells, and approximately 1,000-fold restricted replication in suckling mouse brain and SCID mice transplanted with HuH-7 cells was combined with the 30 mutation here (Blaney, J. E. et al. 2002 Virology 300:125-139).
(73) For the present investigation, subcloned fragments of p4 (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13) containing the above mutations were introduced into the p430 cDNA clone. For transcription and recovery of virus, cDNA was linearized with Acc65I (isoschizomer of KpnI which cleaves leaving only a single 3 nucleotide) and used as template for transcription by SP6 RNA polymerase as previously described (Blaney, J. E. et al. 2002 Virology 300:125-139). C6/36 mosquito cells were transfected using liposome-mediated transfection and cell culture supernatants were harvested between days five and seven. Recovered virus was terminally diluted twice in Vero cells and passaged two (rDEN430-4995) or three (rDEN430-8092 and rDEN430-10634) times in Vero cells.
(74) The complete genomic sequences of rDEN430-4995, rDEN430-8092, and rDEN430-10634 viruses were determined as previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). As expected, each rDEN430 virus derivative contained the 30 mutation. Unexpectedly, in rDEN430-4995 virus, the nucleotide changes in the codon containing nucleotide 4995, resulted in a Ser>Leu amino acid change rather than a Ser>Pro change since the p430-4995 cDNA was designed to introduce the Ser>Pro change (Table 5). The p430-4995 cDNA clone was indeed found to encode a Ser>Pro change at nucleotide 4995, so it is unclear how the virus population acquired the Ser>Leu mutation. Nevertheless, this virus was evaluated to assess the phenotype specified by this missense mutation. rDEN430-4995 virus was also found to contain an incidental mutation at nucleotides 4725-6 which resulted in a single amino acid change (Ser>Asp). The rDEN430-8092 and rDEN430-10634 viruses contained the appropriate nucleotide substitutions as well as additional incidental mutations in E, NS4B and NS4B, respectively (Table 5).
(75) TABLE-US-00007 TABLE 5 Missense and UTR mutations present in rDEN430 virus derivatives bearing introduced point mutations. Amino Amino Nucleotide Nucleotide acid acid Virus Gene position substitution position.sup.a change rDEN430- NS3 4725 U > G 1542 Ser > Asp 4995 NS3 4726 C > A 1542 Ser > Asp NS3 4995.sup.b U > C 1632 Ser > Leu rDEN430- E 1612 A > C 504 Asp > Ala 8092 NS4B 7131 A > G 2344 Thr > Ala NS5 8092.sup.b A > G 2664 Glu > Gly rDEN430- NS4B 6969 A > U 2290 Met > Leu 10634 NS4B 7182 G > C 2361 Gly > Arg 3 UTR 10634.sup.b U > C none none .sup.aAmino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nucleotides 102-104) as position 1. .sup.bMutation restricts replication in mouse models of DEN4 infection which were introduced by Kunkel mutagenesis.
(76) Replication of the three modified rDEN430 viruses were compared to rDEN430 and wild type rDEN4 virus in the suckling mouse brain model and SCID mice transplanted with HuH-7 cells (SCID-HuH-7 mice). Experiments were conducted as previously described (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol. 75:9731-9740). Briefly, for infection of suckling mouse brain, groups of six seven-day-old mice were inoculated intracerebrally with 4.0 log.sub.10PFU of virus and the brain of each mouse was removed five days later. Clarified supernatants of 10% brain suspensions were then frozen at 70 C., and the virus titer was determined by plaque assay in Vero cells. For analysis of DEN4 virus replication in SCID-HuH-7 mice, four to six week-old SCID mice were injected intraperitoneally with 10.sup.7 HuH-7 cells. Five to six weeks after transplantation, mice were infected by direct inoculation into the tumor with 4.0 log.sub.10PFU of virus, and serum for virus titration was obtained by tail-nicking on day 7. The virus titer was determined by plaque assay in Vero cells.
(77) Wild type rDEN4 virus replicated to 6.0 log.sub.10PFU/g in suckling mouse brain, and rDEN430 was restricted in replication by 0.7 log.sub.10PFU/g, which is similar to previous observations (Table 6) (Blaney, J. E. et al. 2001 J Virol. 75:9731-9740). rDEN430-4995, rDEN430-8092, and rDEN430-10634 viruses were found to have restricted replication in suckling mouse brain when compared to rDEN4 virus of 3.3, 2.8, and 2.4 log.sub.10PFU/g, respectively. These results indicate that the additional attenuating mutations serve to further restrict replication of the rDEN430 virus in mouse brain ranging from 50-fold (rDEN430-10634) to 400-fold (rDEN430-4995). In SCID-HuH-7 mice, virus titer of rDEN430 virus was 0.4 log.sub.10PFU/ml lower than rDEN4 virus, which is also similar to previous studies (Blaney, J. E. et al. 2002 Virology 300:125-139). Each modified rDEN430 virus was found to be further restricted in replication in SCID-HuH-7 mice (Table 6). rDEN430-4995, rDEN430-8092, and rDEN430-10634 viruses had restricted replication in SCID-HuH-7 mice when compared to rDEN4 virus of 2.9, 1.1, and 2.3 log.sub.10PFU/g below the level of wild type rDEN4 virus, respectively. Two important observations were made: (1) The 4995, 8092 and 10634 mutations were compatible for viability with the 30 mutation, and (2) These three modified rDEN430 viruses had between a 10 and 1,000-fold reduction in replication in comparison to rDEN4 wild-type virus, which allows viruses with a wide range of attenuation in this model to be further evaluated in monkeys or humans.
(78) TABLE-US-00008 TABLE 6 Addition of point mutations in NS3, NS5, or the 3 UTR to rDEN430 virus further attenuates the virus for suckling mouse brain and SCID-HuH-7 mice. Replication in suckling Replication in mouse brain.sup.a SCID-HuH-7 mice.sup.c Virus Mean Virus Mean titer SE log.sub.10-unit titer SE log.sub.10-unit No. of log.sub.10 PFU/g reduction No. of log.sub.10 PFU/ml reduction Virus mice brain from wt.sup.b mice serum from wt.sup.b rDEN4 12 6.0 0.1 13 6.4 0.2 rDEN430 12 5.3 0.1 0.7 20 6.0 0.2 0.4 rDEN430-4995 6 2.7 0.4 3.3 5 3.5 0.3 2.9 rDEN430-8092 6 3.2 0.2 2.8 7 5.0 0.4 1.1 rDEN430-10634 12 3.6 0.1 2.4 5 4.4 0.3 2.3 .sup.aGroups of 6 suckling mice were inoculated i.c. with 10.sup.4 PFU of virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. .sup.bComparison of mean virus titers of mice inoculated with mutant virus and concurrent rDEN4 wt control. .sup.cGroups of HuH-7-SCID mice were inoculated directly into the tumor with 10.sup.4 PFU virus. Serum was collected on day 6 and 7 and titered in Vero cells.
(79) Based on the findings in the two mouse models of DEN4 virus infection, each of the rDEN430-4995, rDEN430-8092, and rDEN430-10634 viruses was evaluated in the rhesus macaque model of DEN4 infection which has been previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). Briefly, groups of four (rDEN430-4995, rDEN430-8092, and rDEN430-10634) or two (rDEN4, rDEN430, mock) monkeys were inoculated with 5.0 log.sub.10PFU virus subcutaneously. Monkeys were observed daily and serum was collected on days 0 to 6, 8, 10, and 12, and virus titers were determined by plaque assay in Vero cells for measurement of viremia. On day 28, serum was drawn and the level of neutralizing antibodies was tested by plaque reduction assay in Vero cells as previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413).
(80) Viremia was detected beginning on day 1 post-infection and ended by day 4 in all monkeys (Table 7,
(81) TABLE-US-00009 TABLE 7 Addition of point mutations to rDEN430 further attenuates the virus for rhesus monkeys. Geometric mean No. of Mean no. serum neutralizing monkeys of viremic Mean peak antibody titer No. of with days per virus titer (reciprocal dilution) Virus.sup.a monkeys viremia monkey.sup.b (log.sub.10 PFU/ml SE) Day 0 Day 28 mock 2 0 0 <0.7 <10 <10 rDEN4 2 2 3.0 2.2 0.6 <10 398 rDEN430 2 2 2.0 1.1 0.4 <10 181 rDEN430-4995 4 2 0.8 0.9 0.2 <10 78 rDEN430-8092 4 2 0.5 0.7 0.1 <10 61 rDEN430-10634 4 4 1.3 1.3 0.2 <10 107 .sup.aGroups of rhesus monkeys were inoculated subcutaneously with 10.sup.5 PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 12, and 28. Virus titer was determined by plaque assay in Vero cells. .sup.bViremia was not detected in any monkey after day 4.
(82) Serum collected on day 0 and 28 was tested for the level of neutralizing antibodies against rDEN4. No detectable neutralizing antibodies were found against DEN4 on day 0, as expected, since the monkeys were pre-screened to be negative for neutralizing antibodies against flaviviruses (Table 7). On day 28, monkeys infected with rDEN4 had a mean serum neutralizing antibody titer (reciprocal dilution) of 398 which was approximately two-fold higher than monkeys infected with rDEN430 virus (1:181). This result and the two-fold higher level of viremia in rDEN4 virus-infected monkeys are similar to results obtained previously (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). Monkeys infected with rDEN430-4995 (1:78), rDEN430-8092 (1:61), and rDEN430-10634 (1:107) viruses each had a reduced mean serum neutralizing antibody titer compared to monkeys infected with rDEN430 virus. The four monkeys which had no detectable viremia did have serum neutralizing antibody titers indicating that they were indeed infected. Despite the slight increase in mean peak virus titer of rDEN430-10634 virus compared with rDEN430 virus, rDEN430-10634 virus had a lower mean serum neutralizing antibody titer compared to monkeys infected with rDEN430 virus. This and the lower mean number of viremic days per monkey suggests that the 10634 mutation can attenuate the replication of rDEN430 virus in monkeys.
(83) On day 28 after inoculation, all monkeys were challenged with 5.0 log.sub.10PFU wild type rDEN4 virus subcutaneously. Monkeys were observed daily and serum was collected on days 28 to 34, 36, and 38, and virus titers were determined by plaque assay in Vero cells for measurement of viremia after challenge. Twenty eight days after rDEN4 virus challenge, serum was drawn and the level of neutralizing antibodies was tested by plaque reduction assay in Vero cells. Mock-inoculated monkeys had a mean peak virus titer of 2.3 log.sub.10PFU/ml after challenge with a mean number of viremic days of 3.5 (Table 8). However, monkeys inoculated with rDEN4, rDEN430, or each of the modified rDEN430 viruses had no detectable viremia, indicating that despite the decreased replication and immunogenicity of rDEN430-4995, rDEN430-8092, and rDEN430-10634 viruses, each was sufficiently immunogenic to induce protection against wild type rDEN4. Increases in mean neutralizing antibody titer were minimal (<3-fold) following challenge in all inoculation groups except mock-infected providing further evidence that the monkeys were protected from the challenge.
(84) TABLE-US-00010 TABLE 8 rDEN430 containing additional point mutations protects rhesus monkeys from wt DEN4 virus challenge Mean no. of Geometric mean serum viremic days per neutralizing antibody monkey after Mean peak titer (reciprocal No. of rDEN4 virus titer dilution) Virus.sup.a monkeys challenge (log.sub.10 PFU/ml SE) Day 28 Day 56 Mock 2 3.5 2.3 0.1 <10 358 rDEN4 2 0.0 <0.7 398 753 rDEN430 2 0.0 <0.7 181 202 rDEN430-4995 4 0.0 <0.7 78 170 rDEN430-8092 4 0.0 <0.7 61 131 rDEN430-10634 4 0.0 <0.7 107 177 .sup.a28 days after primary inoculation with the indicated viruses, rhesus monkeys were challenged subcutaneously with 10.sup.5 PFU rDEN4 virus in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Virus titer was determined by plaque assay in Vero cells.
(85) Taken together, these results indicate that the three point mutations, 4995, 8092, and 10634) described above do further attenuate the rDEN430 vaccine candidate in suckling mouse brain, SCID-HuH-7 mice, and rhesus monkeys. Because of additional incidental mutations (Table 4) present in each modified rDEN430 virus, the phenotypes cannot be directly attributed to the individual 4995, 8092, and 10634 point mutations. However, the presence of similar mouse-attenuation phenotypes in other rDEN4 viruses bearing one of these three mutations supports the contention that the 4995, 8092, and 10634 point mutations are responsible for the att phenotypes of the modified rDEN430 viruses. Since rDEN430-4995, rDEN430-8092, and rDEN430-10634 virus demonstrated decreased replication in rhesus monkeys while retaining sufficient immunogenicity to confer protective immunity, these viruses are contemplated as dengue vaccines for humans.
(86) DEN4 viruses carrying both 30 and charge-to-alanine mutations were next generated. A subset of seven groups of charge-to-alanine mutations described above were identified that conferred between a 10-fold and 1,000-fold decrease in replication in SCID-HuH-7 mice and whose unmutated sequence was well-conserved across the four dengue serotypes. These mutations were introduced as single pairs and as two sets of pairs to rDEN430 using conventional cloning techniques. Transcription and recovery of virus and terminal dilution of viruses were conducted as described above. Assay of the level of temperature sensitivity of the charge-cluster-to-alanine mutant viruses in Vero and HuH-7 cells, level of replication in the brain of suckling mice and level of replication in SCID-HuH-7 mice was conducted as described above.
(87) Introduction of one pair of charge-to-alanine mutations to rDEN4 produced recoverable virus in all cases (Table 9). Introduction of two pairs of charge-to-alanine mutations produced recoverable virus in two out of three cases (rDEN430-436-437-808-809 was not recoverable).
(88) rDEN430 is not ts in Vero or HuH-7 cells. In contrast, seven of the seven sets of charge-to-alanine mutations used in this example conferred a ts phenotype in HuH-7 cells and five also conferred a ts phenotype in Vero cells. All six viruses carrying both 30 and charge-to-alanine mutations showed a ts phenotype in both Vero and HuH-7 cells (Table 9). rDEN430 is not attenuated in suckling mouse brain, whereas five of the seven sets of charge-to-alanine mutations conferred an att phenotype in suckling mouse brain (Table 10). Four of the viruses carrying both 30 and charge-to-alanine mutations were attenuated in suckling mouse brain (Table 10). In one case (rDEN430-23-24-396-397) combination of two mutations that did not attenuate alone resulted in an attenuated virus. Generally, viruses carrying both 30 and charge-to-alanine mutations showed levels of replication in the suckling mouse brain more similar to their charge-to-alanine mutant parent virus than to rDEN430.
(89) rDEN430 is attenuated in SCID-HuH-7 mice, as are six of the seven charge-to-alanine mutant viruses used in this example. Viruses carrying both 30 and charge-to-alanine mutations tended to show similar or slightly lower levels of replication in SCID-HuH-7 mice compared to their charge-to-alanine mutant parent virus (Table 10). In three cases, viruses carrying both 30 and charge-to-alanine mutations showed at least a fivefold greater reduction in SCID-HuH-7 mice than rDEN430.
(90) The complete genomic sequence of five viruses (rDEN4-200-201, rDEN430-200-201, rDEN4-436-437 [clone 1], rDEN430-436-437, and rDEN4-23-24-200-201) that replicated to >10.sup.5 PFU/ml in Vero cells at 35 C. and that showed a hundredfold or greater reduction in replication in SCID-HuH-7 mice was determined (Table 11). Each of the five contained one or more incidental mutations. In one virus, rDEN430-436-437, the one additional mutation has been previously associated with Vero cell adaptation (Blaney, J. E. Jr. et al. 2002 Virology 300:125-139). Each of the remaining viruses contained at least one incidental mutation whose phenotypic effect is unknown. Consequently, the phenotypes described cannot be directly attributed to the charge-to-alanine mutations. However, the fact that rDEN4 and rDEN430 viruses carrying the same charge-to-alanine mutations shared similar phenotypes provides strong support for the ability of charge-to-alanine mutations to enhance the attenuation of rDEN430. Because rDEN4-436-437 [clone 1] contained 4 incidental mutations, a second clone of this virus was prepared. rDEN4-436-437 [clone2] contained only one incidental mutation (Table 11), and showed the same phenotypes as rDEN4-436-437 in cell culture and SCID-HuH-7 mice. rDEN4-436-437 [clone 2] was used in the rhesus monkey study described below.
(91) TABLE-US-00011 TABLE 9 Addition of charge-to-alanine mutations to rDEN430 confers a ts phenotype in both Vero and HuH-7 cells. Mean virus titer (log.sub.10 PFU/ml) at indicated temperature ( C.).sup.a AA No. nt Vero HuH-7 Virus changed.sup.b changed 35 37 38 39 .sup.c 35 37 38 39 rDEN4 none 0 7.4 7.1 7.7 7.2 0.2 7.7 7.5 7.5 7.4 0.3 rDEN430 none 30 6.6 6.6 6.5 6.5 0.1 7.4 6.9 7.0 6.4 1.0 rDEN4-23-24 KE 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 rDEN430-23-24 6.1 5.5 4.9 <1.7 4.4 6.5 5.9 4.7 <1.7 >4.2 rDEN4-200-201 KH 4 5.3 4.8 4.8 4.3 1.0 5.7 5.4 2.7 <1.7 >4.0 rDEN430-200-201 6.0 5.3 5.6 <1.7 >4.3 5.8 5.0 5.9 <1.7 >4.1 rDEN4-436-437 DK 4 5.2 4.2 3.4 1.9 3.3 5.9 4.9 3.2 <1.7 >4.2 rDEN430-436-437 6.3 5.7 5.5 <1.7 >4.6 6.5 5.7 5.1 <1.7 >4.8 [clone1] rDEN4-808-809 ED 3 4.6 4.1 <1.7 <1.7 >2.9 5.2 <1.7 <1.7 <1.7 >3.5 rDEN430-808-809 5.6 4.9 4.9 <1.7 >3.9 5.9 4.8 5.1 <1.7 >4.2 rDEN4-23-24-200-201 KE, KH 7 6.0 5.2 4.2 <1.7 >4.3 6.9 6.3 <1.7 <1.7 >5.2 rDEN430-23-24-200-201 4.5 4.2 4.8 <1.7 >2.8 4.9 4.5 2.9 <1.7 >3.2 rDEN4-23-24-396-397 KE, RE 7 6.5 5.8 5.5 <1.7 >4.8 7.1 5.9 5.4 <1.7 >5.4 rDEN430-23-24-396-397 6.1 5.2 4.8 <1.7 >4.4 6.9 5.4 4.9 <1.7 >5.2 rDEN-436-437-808-809 DK, ED 7 4.9 4.9 5.1 <1.7 >3.2 5.5 3.2 <1.7 <1.7 >3.8 .sup.aUnderlined values indicate a 2.5 or 3.5 log.sub.10 PFU/ml reduction in titer in Vero or HuH-7 cells, respectively, at the indicated temperature when compared to the permissive temperature (35 C.). .sup.bAmino acid pair(s) changed to pair of Ala residues. .sup.cReduction in titer (log.sub.10 pfu/ml) compared to the permissive temperature (35 C.).
(92) TABLE-US-00012 TABLE 10 Addition of charge-to-alanine mutations attenuates rDEN430 in suckling mouse brain and enhances attenuation in SCID-HuH-7 mice. Replication in suckling mice.sup.a Replication in SCID-HuH-7 mice.sup.b Mean log Mean log Mean virus titer SE reduction from Mean virus titer SE reduction from Virus n (log.sub.10 PFU/g brain) wt.sup.b n (log.sub.10 PFU/ml serum) wt.sup.d rDEN4 18 6.2 0.4 33 5.4 0.3 rDEN430 12 5.9 0.8 0.2 8 3.4 0.3 2.3 rDEN4-23-24 18 4.7 0.1 1.6 19 4.7 0.5 1.3 rDEN430-23-24 6 5.6 0.3 0.7 7 4.6 0.4 1.5 rDEN4-200-201 12 5.5 0.5 0.6 12 3.7 0.2 2.6 rDEN430-200-201 6 5.5 0.6 0.1 4 3.3 0.6 1.8 rDEN4-436-437 18 2.7 0.4 3.5 10 2.9 0.7 2.5 rDEN430-436-437 [clone 1] 6 2.9 0.3 3.4 4 2.3 0.4 2.8 rDEN4-808-809 6 1.8 0.1 3.1 8 3.2 0.4 3.0 rDEN430-808-809 12 3.9 0.7 2.1 4 3.7 0.6 2.4 rDEN4-23-24-200-201 12 5.3 0.5 0.7 13 3.4 0.1 2.9 rDEN430-23-24-200-201 6 3.0 0.2 2.6 5 1.8 0.1 3.3 rDEN4-23-24-396-397 12 4.6 0.9 1.5 8 3.6 0.3 2.3 rDEN430-23-24-396-397 6 3.0 0.2 2.6 5 2.2 0.3 2.9 rDEN-436-437-808-809 6 <1.7 0.0 3.6 8 2.1 0.3 2.4 .sup.aGroups of six suckling mice were inoculated i.c. with 10.sup.4 PFU virus in a 30 l inoculum. The brain was removed 5 days later, homogenized, and virus was quantitated by titration in Vero cells. .sup.bDetermined by comparing the mean viral titers in mice inoculated with sample virus and concurrent wt controls (n = 6). The attenuation (att) phenotype is defined as a reduction of 1.5 log.sub.10 PFU/g compared to wt virus; reductions of 1.5 are listed in boldface. .sup.cGroups of SCID-HuH-7 mice were inoculated directly into the tumor with 10.sup.4 PFU virus. .sup.dDetermined by comparing mean viral titers in mice inoculated with sample virus and concurrent wt controls.
The attenuation phenotype is defined as a reduction of 1.5 log.sub.10PFU/g compared to wt virus; reductions of 1.5 are listed in boldface.
(93) TABLE-US-00013 TABLE 11 Missense and UTR mutations present in rDEN4 virus derivatives bearing charge-to-alanine and the 30 mutation. Nucleotide Nucleotide Amino acid Virus Gene.sup.a,b position substitution position.sup.c Amino acid change.sup.b rDEN4-200-201 prM 626 A > T 61 Glu > Asp NS4A 6659 C > T 93 Leu > Phe NS5 8160-8165 AAACA > GCAGC 200-201 LysHis > AlaAla rDEN430-200-201 NS3 4830 G > A 102 Gly > Arg NS5 8106 G > A 181 Val > Ile NS5 8160-8165 AAACA > GCAGC 200-201 LysHis > AlaAla 3 UTR 10478-10507 30 deletion None None rDEN4-436-437 [clone 1] E 2331 T > G 464 Trp > Gly NS1 2845 C > T 140 Ser > Phe NS3* 4891 T > C 122 Ile > Thr NS5 8869-8873 GACAA > GCAGC 436-437 AspLys > AlaAla NS5 9659 A > G 699 Lys > Arg rDEN4-436-437 [clone 2] NS4B 7153 T > C 108 Val > Ala NS5 8869-8873 GACAA > GCAGC 436-437 AspLys > AlaAla rDEN430-436-437 NS4B* 7163 A > C 111 Leu > Phe NS5 8869-73 GACAA > GCAGC 436-437 AspLys > AlaAla 3 UTR 10478-10507 30 deletion None None rDEN4-23-24-200-201 NS3 6751 A > C 124 Lys > Thr NS5 7629-7633 AAAGA > GCAGC 23-24 LysGlu > AlaAla NS5 8160-8165 AAACA > GCAGC 200-201 LysHis > AlaAla .sup.aAsterisk indicates previously identified Vero cell adaptation mutation. .sup.bBold values indicate mutations designed to occur in the designated virus. .sup.cAmino acid position in the protein product of the designated DEN4 gene; numbering starts with the amino terminus of the protein.
(94) Based on the attenuation in the SCID-HuH7 mouse model, four of the charge-to-alanine mutant viruses (rDEN4-200-201, rDEN430-200-201, rDEN4-436-437 [clone 2], rDEN430-436-437) were evaluated in rhesus macaques as described above. As with the study of viruses carrying attenuating point mutations, viremia was detected on day 1 post-infection and ended by day 4 in all monkeys (
(95) As expected, none of the monkeys in this study showed detectable levels of neutralizing antibody on day 0. On day 28, every monkey infected with a virus showed a detectable levels of neutralizing antibody, indicating that all of the monkeys, even those that showed no detectable viremia, had indeed been infected. As in the study of attenuating point mutations, monkeys infected with rDEN4 had a mean serum neutralizing antibody titer (reciprocal dilution) which was approximately twice that of monkeys that had been infected with rDEN430. Monkeys infected with rDEN4-200-201 and rDEN4-436-437 [clone 2] had similar mean neutralizing antibody titers to rDEN4, and those infected with rDEN430-200-201 and rDEN430-436-437 had similar mean neutralizing antibody titers to rDEN4. In each case the addition of the 30 mutation to a virus resulted in a two-fold decrease in neutralizing antibody. Thus, although the addition of charge-to-alanine mutations to rDEN430 decreased mean peak viremia below that of rDEN430 alone, it did not affect levels of neutralizing antibody.
(96) TABLE-US-00014 TABLE 12 Addition of paired charge-to-alanine mutations to rDEN430 further attenuates the virus for rhesus monkeys. Geometric mean serum neutralizing No. of Mean no. antibody titer monkeys of viremic Mean peak (reciprocal No. of with days per virus titer dilution) Virus.sup.a monkeys viremia monkey.sup.b (log.sub.10 PFU/ml SE) Day 0 Day 28 mock 2 0 0 <0.7 <5 <5 rDEN4 2 2 2.5 2.6 0.3 <5 276 rDEN430 2 2 2.0 2.1 0.1 <5 131 rDEN4-200, 201 4 4 2.3 1.8 0.3 <5 212 rDEN430-200, 201 4 3 1.5 1.3 0.2 <5 139 rDEN4-436, 437 [cl 2) 4 4 3.3 1.8 0.2 <5 273 rDEN430-436, 437 4 3 1.3 1.0 0.0 <5 143 .sup.aGroups of rhesus monkeys were inoculated subcutaneously with 10.sup.5 PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10 and 28. Virus titer was determined by plaque assay in Vero cells. .sup.bViremia was not detected in any monkey after day 4.
(97) After challenge with rDEN4 on day 28, mock-infected monkeys had a mean peak virus titer of 1.5 log.sub.10PFU/ml and a mean number of viremic days of 3.0 (Table 13). However, none of the monkeys previously inoculated with rDEN4, rDEN430 or the charge-to-alanine mutant viruses showed detectable viremia. Additionally, none of the monkeys showed a greater than four-fold increase in serum neutralizing antibody titer. Together these data indicate that infection with any of the viruses, including those carrying both 30 and the charge-to-alanine mutations, protected rhesus macaques from challenge with rDEN4.
(98) TABLE-US-00015 TABLE 13 rDEN430 containing charge-to-alanine mutations protects rhesus monkeys from wt DEN4 virus challenge Mean no. of Geometric mean serum viremic days neutralizing antibody per monkey Mean peak titer (reciprocal No. of after rDEN4 virus titer dilution) Virus.sup.a monkeys challenge (log.sub.10 PFU/ml SE) Day 28 Day 56 mock 2 3.0 1.5 0.7 <5 284 rDEN4 2 0.0 <0.7 276 316 rDEN430 2 0.0 <0.7 131 96 rDEN4-200, 201 4 0.0 <0.7 212 356 rDEN430-200, 201 4 0.0 <0.7 139 132 rDEN4-436, 4 0.0 <0.7 273 401 437 [cl 2] rDEN430-436, 437 4 0.0 <0.7 143 182 .sup.a28 days after primary inoculation with the indicated viruses, rhesus monkeys were challenged subcutaneously with 10.sup.5 PFU rDEN4 virus in a 1 ml dose. Serum was collected on days 28 to 34, 36, 10, and 56. Virus titer was determined by plaque assay in Vero cells.
(99) Addition of charge-to-alanine mutations to rDEN430 can confer a range of is phenotypes in both Vero and HuH-7 cells and att phenotypes in suckling mouse brain and can either enhance or leave unchanged attenuation in SCID-HuH-7 mice. Most importantly, addition of these mutations can decrease the viremia produced by rDEN430 in rhesus macaques without decreasing neutralizing antibody titer or protective efficacy. Thus addition of such mutations to rDEN430 is contemplated as enhancing attenuation in humans. Also, mutations are contemplated as being added that do not change the overall level of attenuation, but stabilize the attenuation phenotype because they themselves are independently attenuating even in the absence of the 30 mutation. Charge-to-alanine mutations are particularly useful because they occur outside of the structural gene regions, and so can be used to attenuate structural gene chimeric viruses. Moreover, they involve at least three nucleotide changes, making them unlikely to revert to wild type sequence.
(100) A series of point mutations that enhance the replication of rDEN4 in Vero cells tissue culture have been identified; these are primarily located in the NS4B gene (Blaney, J. E. et. al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol 75:9731-9740). Vero cell adaptation mutations confer two desirable features upon a vaccine candidate. First, they enhance virus yield in Vero cells, the intended substrate for vaccine production, and thus render vaccine production more cost-effective. Second, although each of these Vero adaptation mutations are point mutations, they are likely to be extremely stable during vaccine manufacture, because they give a selective advantage in Vero cells. At least one Vero cell adaptation mutation, at position 7129, was also shown to decrease mosquito infectivity of rDEN4; poor mosquito infectivity is another desirable characteristic of a dengue vaccine candidate. To investigate the generality of this finding, we tested the effect of the remaining Vero cell adaptation mutations on the ability of rDEN4 to infect Aedes aegypti mosquitoes fed on an infectious bloodmeal. Table 14 shows the infectivity of each virus carrying a single Vero cell adaptation mutation at high titer. Of these, only one mutation, at position 7182, was associated with a large decrease in mosquito infectivity. Thus 7182 may be a particularly valuable mutation to include in an rDEN4 vaccine candidate, as it has opposite effects on replication in Vero cells and in mosquitoes.
(101) TABLE-US-00016 TABLE 14 Effect of Vero cell adaptation mutations on rDEN4 mosquito infectivity Aedes aegypti (oral infection) Dose.sup.a % infected.sup.b Virus (log.sub.10 pfu) No. tested Midgut Head rDEN4 4.3 27 70 25 rDEN4-4891 4.4 23 74 13 rDEN4-4995 4.8 20 80 50 rDEN4-7153 4.8 20 80 30 rDEN4-7546 4.6 20 55 10 rDEN4-7162 5.0 20 55 25 rDEN4-7163 4.9 15 73 72 rDEN4-7182 5.0 20 20 0 rDEN4-7630 4.3 10 70 10 .sup.aVirus titer ingested, assuming a 2 il bloodmeal. .sup.bPercentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection.
EXAMPLE 2
Generation and Characterization of a Recombinant DEN1 Virus Containing the 30 Mutation
(102) We first sought to determine if the 30 mutation was able to satisfactorily attenuate a wild-type DEN virus other than the DEN4 serotype. To do this, the 30 mutation was introduced into the cDNA for DEN1 (Western Pacific). The pRS424DEN1WP cDNA clone (Puri, B. et al. 2000 Virus Genes 20:57-63) was digested with BamHI and used as template in a PCR using Pfu polymerase with forward primer 30 (DEN1 nt 10515-10561 and 10592-10607) and the M13 reverse sequencing primer (101 nt beyond the 3 end of DEN1 genome sequence). The resulting PCR product was 292 bp and contained the 30 mutation. The pRS424DEN1WP cDNA was partially digested with Apa I, then digested to completion with Sac II and the vector was gel isolated, mixed with PCR product, and used to transform yeast strain YPH857 to yield growth on plates lacking tryptophan (Polo, S. et al. 1997 J Virol 71:5366-74). Positive yeast colonies were confirmed by PCR and restriction enzyme analysis. DNA isolated from two independent yeast colonies was used to transform E. coli strain STBL2. Plasmid DNA suitable for generating RNA transcripts was prepared and the presence of the 30 mutation was verified by sequence analysis.
(103) For transcription and generation of virus, cDNA (designated pRS424DEN130) that was linearized with Sac II was used as template in a transcription reaction using SP6 RNA polymerase as described (Polo, S. et al. 1997 J Virol 71:5366-74). Transcription reactions were electroporated into LLC-MK2 cells and infection was confirmed by observation of CPE and immunofluorescence and harvested on day 14. Virus stocks were amplified on C6/36 mosquito cells and titered on LLC-MK2 cells. The genome of the resulting virus, rDEN130, was sequenced to confirm the presence of the 30 mutation. The 30 mutation removes nucleotides 10562-10591 of DEN1 (
(104) TABLE-US-00017 TABLE 15 Missense mutations present among the recombinant DEN1 viruses and correlation of NS4B region mutations with those found in DEN4 Trans- Nucleo- Nucleo- Amino Amino fection tide tide acid acid Virus cell type Gene position change position change wt rDEN1 LLC-MK2 prM 816 C > U 241 Ala > Val NS4B 7165.sup.a U > G 2357 Phe > Leu NS4B 7173.sup.b U > C 2360 Val > Ala rDEN130 LLC-MK2 E 1748 A > U 552 Thr > Ser rDEN130 Vero E 1545 A > G 484 Lys > Arg .sup.aSame nucleotide as 7154 in rDEN4. .sup.bSame nucleotide as 7162 in rDEN4
(105) * Nucleotide and amino acid comparison of selected NS4B region:
(106) TABLE-US-00018 777777 DEN4 111111 base 345678 Number: 890123456789012345678901234567890123456789012345678901234567 ++ ++ + +++++ + + + + ++ + ++++++++ ++ ++ ++ ++ D47128- CCAACAACCUUGACAGCAUCCUUAGUCAUGCUUUUAGUCCAUUAUGCAAUAAUAGGCCCA PTTLTASLVMLLVHTAIIGP D17139- CCGCUGACGCUGACAGCGGCGGUAUUUAUGCUAGUGGCUCAUUAUGCCAUAAUUGGACCC PLTLTAAVPMLVAHTAIIGP D27135- CCUAUAACCCUCACAGCGGCUCUUCUUUUAUUGGUAGCACAUUAUGCCAUCAUAGGACCG PITLTAALLLLVAHTAIIGP D37130- CCACUAACUCUCACAGCGGCAGUUCUCCUGCUAGUCACGCAUUAUGCUAUUAUAGGUCCA PLTLTAAVLLLVTHTAIIGP + + + + + + + + + + + + + D4 = rDEN4 Dl = rDENl(WP) D2 = rDEN2(Tonga/74) D3 = rDEN3(Sleman/78) +Homology among all four serotypes Nucleotides are underlined in even multiples of 10.
(107) Evaluation of the replication, immunogenicity, and protective efficacy of rDEN130 and wild-type parental rDEN1 virus (derived from the pRS424DEN1WP cDNA) in juvenile rhesus monkeys was performed as follows. Dengue virus-seronegative monkeys were injected subcutaneously with 5.0 log.sub.10PFU of virus in a 1 ml dose divided between two injections in each side of the upper shoulder area. Monkeys were observed daily and blood was collected on days 0-10 and 28 and serum was stored at 70 C. Titer of virus in serum samples was determined by plaque assay in Vero cells as described previously (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). Plaque reduction neutralization titers were determined for the day 28 serum samples as previously described (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). All monkeys were challenged on day 28 with a single dose of 5.0 log.sub.10PFU of wild-type rDEN1 and blood was collected for 10 days. Virus titer in post-challenge sera was determined by plaque assay in Vero cells. Monkeys inoculated with full-length wild-type rDEN1 were viremic for 2-3 days with a mean peak titer of 2.1 log.sub.10PFU/ml (Table 16), and monkeys inoculated with rDEN130 were viremic for less than 1 day with a mean peak titer of 0.8 log.sub.10PFU/ml, indicating that the 30 mutation is capable of attenuating DEN1. As expected for an attenuated virus, the immune response, as measured by neutralizing antibody titer, was lower following inoculation with rDEN130 compared to inoculation with wild-type rDEN1 (Table 16), yet sufficiently high to protect the animals against wild-type DEN1 virus challenge. Wild-type rDEN1 virus was not detected in any serum sample collected following virus challenge, indicating that monkeys were completely protected following immunization with either full-length wild-type rDEN1 or recombinant virus rDEN130. The level of attenuation specified by the 30 mutation was comparable in both the DEN1 and DEN4 genetic backgrounds (
(108) TABLE-US-00019 TABLE 16 The 30 mutation attenuates rDEN1 for rhesus monkeys Mean no. Mean peak Mean Mean peak days with titer neutralization titer of Virus* n viremia (log.sub.10 pfu/ml) titer challenge virus rDEN1 4 2.8 2.1 1230 <0.7 rDEN130 4 0.5 0.8 780 <0.7 *Rhesus monkeys were inoculated subcuateously with 5.0 log.sub.10 PFU of virus. Serum samples were collected daily for 10 days. Serum for neutralization assay was collected on day 28. All monkeys were challenged on day 28 with 5.0 log.sub.10 PFU of rDEN1.
(109) As previously reported, rDEN4 virus replicated to greater than 6.0 log.sub.10PFU/ml serum in SCID-HuH-7 mice, while the replication of rDEN4 virus bearing the 30 mutation was reduced by about 10-fold (Blaney, J. E. Jr. et al. 2002 Virology 300:125-139). The replication of rDEN130 was compared to that of wt rDEN1 in SCID-HuH-7 mice (Table 17). rDEN130 replicated to a level approximately 100-fold less than its wt rDEN1 parent. This result further validates the use of the SCID-HuH-7 mouse model for the evaluation of attenuated strains of DEN virus, with results correlating closely with those observed in rhesus monkeys.
(110) TABLE-US-00020 TABLE 17 The 30 mutation attenuates rDEN1 for HuH-7-SCID mice No. of Mean peak virus titer.sup.6 Virus Mice.sup.5 (log.sub.10 pfu/ml SE) wt rDEN1 9 7.3 0.2 rDEN130 8 5.0 0.3 .sup.5Groups of HuH-7-SCID mice were inoculated directly into the tumor with 4.0 log.sub.10 pfu virus. Serum was collected on day 6 and 7, and virus titer was determined by plaque assay in Vero cells. .sup.6Significant difference was found between rDEN1 and rDEN130 viruses, Tukey-Kramer test (P < 0.005).
(111) Finally, the infectivity of rDEN1 and rDEN130 for mosquitoes was assessed, using the methods described in detail in Example 5. Previously, the 30 mutation was shown to decrease the ability of rDEN4 to cross the mosquito midgut barrier and establish a salivary gland infection (Troyer, J. M. et al. 2001 Am J Trop Med Hyg 65:414-419). However neither rDEN1 nor rDEN130 was able to infect the midgut of Aedes aegypti mosquitoes efficiently via an artificial bloodmeal (Table 18), so it was not possible to determine whether 30 might further block salivary gland infection. A previous study also showed that the 30 had no effect on the infectivity of rDEN4 for Toxorhynchites splendens mosquitoes infected via intrathoracic inoculation (Troyer, J. M. et al. 2001 Am J Trop Med Hyg 65:414-419), and a similar pattern was seen for rDEN1 and rDEN130 (Table 18). The genetic basis for the inability of rDEN1 to infect the mosquito midgut has not been defined at this time. However, this important property of restricted infectivity for the mosquito midgut is highly desirable in a vaccine candidate since it would serve to greatly restrict transmission of the vaccine virus from a vaccinee to a mosquito vector.
(112) TABLE-US-00021 TABLE 18 DEN1 and DEN130 viruses are both highly infectious for Toxorhynchites splendens, but do not infect Aedes aegypti efficiently. Toxorhynchites splendens (intrathoracic inoculation) Aedes aegypti (oral infection) Dose.sup.a Dose.sup.c % infected.sup.d Virus (log.sub.10 pfu) No. tested % infected.sup.b (log.sub.10 pfu) No. tested Midgut Head rDEN1 3.5 7 100 4.0 26 11 0 2.5 8 75 1.5 7 71 0.5 5 60 MID.sub.50 < 0.5 MID.sub.50 4.4 rDEN1 2.7 8 100 3.2 20 10 0 30 1.7 7 100 0.7 6 83 MID.sub.50 < 0.7 MID.sub.50 3.6 .sup.aAmount of virus present in 0.22 l inoculum. .sup.bPercentage of mosquitoes with IFA detectable antigen in head tissue prepared 14 days after inoculation. .sup.cVirus titer ingested, assuming a 2 l bloodmeal. .sup.dPercentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection. When virus infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MID.sub.50 was estimated by assuming that a 10-fold more concentrated virus dose would infect 100% of the mosquitoes.
(113) Thus, the 30 mutation, first described in DEN4, was successfully transferred to rDEN1. The resulting virus, rDEN130, was shown to be attenuated in monkeys and SCID-HuH-7 mice to levels similar to recombinant virus rDEN430, thereby establishing the conservation of the attenuation phenotype specified by the 30 mutation in a different DEN virus background. Based on the favorable results of rDEN430 in recent clinical trials (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13), it is predicted that rDEN130 will be suitably attenuated in humans. To complete the tetravalent vaccine, attenuated rDEN2 and rDEN3 recombinant viruses bearing the 30 mutation are contemplated as being prepared (See Examples 3 and 4 below). The demonstration that the 30 mutation specifies a phenotype that is transportable to another DEN serotype has important implications for development of the tetravalent vaccine. This indicates that the 30 mutation is expected to have a corresponding effect on DEN2 and DEN3 wild-type viruses.
EXAMPLE 3
Generation and Characterization of a Recombinant DEN2 Virus Containing the 30 Mutation
(114) Evaluation of rDEN130 showed that it was satisfactorily attenuated. Based on this result, we sought to extend our technology to the creation of a DEN2 vaccine candidate. To do this, the 30 mutation was introduced into the cDNA of DEN2. A DEN2 virus isolate from a 1974 dengue epidemic in the Kingdom of Tonga (Tonga/74) (Gubler, D. J. et al. 1978 Am J Trop Med Hyg 27:581-589) was chosen to represent wt DEN2. The genome of DEN2 (Tonga/74) was sequenced in its entirety and served as consensus sequence for the construction of a full-length cDNA clone (Appendix 1). cDNA fragments of DEN2 (Tonga/74) were generated by reverse-transcription of the genome as indicated in
(115) For transcription and generation of infectious virus, cDNA (p2 and p230) was linearized with Acc65I (isoschizomer of KpnI which cleaves leaving only a single 3 nucleotide) and used as template in a transcription reaction using SP6 RNA polymerase as previously described (Blaney, J. E. et. al. 2002 Virology 300:125-139). Transcripts were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection and cell culture supernatants were harvested on day 7.
(116) rDEN2 virus was recovered from the p2 cDNA in both Vero and C6/36 cells, while rDEN230 was recovered from the p230 cDNA clone in only C6/36 cells (Table 19). The level of infectious virus recovered in C6/36 cells was comparable for the p2 and p230 cDNA clones when assayed by plaque titration and immunostaining in Vero or C6/36 cells. As previously observed, the efficiency of transfection in C6/36 cells was higher than that in Vero cells. Two rDEN230 viruses were recovered from independent cDNA clones, #2 and #10.
(117) TABLE-US-00022 TABLE 19 rDEN2 virus is recovered in Vero and C6/36 cells, but rDEN230 virus is recovered only in C6/36 cells. Virus titer of transfection harvest (day 7) determined in the indicated Transfection cDNA cell type (log.sub.10 PFU/ml) cell type construct Clone Virus Vero cells C6/36 cells Vero cells p2 #8A rDEN2 3.1 4.3 p230 #2 rDEN230 <0.7 <0.7 p230 #10 rDEN230 <0.7 <0.7 C6/36 cells p2 #8A rDEN2 5.5 7.5 p230 #2 rDEN230 4.8 7.6 p230 #10 rDEN230 4.6 7.5
(118) To produce working stocks of rDEN2 and rDEN230 viruses, transfection harvests were passaged and terminally diluted in Vero cells, and genomic sequences of the viruses were determined. The Vero cell transfection harvest of rDEN2 virus was terminally diluted once in Vero cells, and individual virus clones were passaged once in Vero cells. To assess whether any homologous Vero cell adaptation mutations identified in the rDEN4 NS4B 7100-7200 region were present in these virus clones, seven independent terminally diluted clones were sequenced over this region. Each of the seven rDEN2 viruses contained a single nucleotide substitution in this region at nucleotide 7169 (U>C) resulting in a Val>Ala amino acid change. This nucleotide corresponds to the 7162 mutation identified in rDEN4 (Blaney, J. E. et. al. 2002 Virology 300:125-139), which has a known Vero cell adaptation phenotype suggesting that this mutation may confer a replication enhancement phenotype in rDEN2 virus. One rDEN2 virus clone was completely sequenced and in addition to the 7169 mutation, a missense mutation (Glu>Ala) was found in NS5 at residue 3051 (Table 20).
(119) TABLE-US-00023 TABLE 20 Missense mutations which accumulate in rDEN2 and rDEN230 viruses after transfection or passage in Vero cells. Nucleotide Nucleotide Amino acid Amino acid Virus Gene position substitution position.sup.a change rDEN2.sup.b NS4B .sup.7169.sup.c U > C 2358 Val > Ala (Vero) NS5 9248 A > C 3051 Glu > Ala rDEN230.sup.d NS3 4946 A > G 1617 Lys > Arg (Vero) NS4B .sup.7169.sup.c U > C 2358 Val > Ala .sup.aAmino acid position in DEN2 polyprotein beginning with the methionine residue of the C protein (nucleotides 97-99) as position 1. .sup.bVirus was recovered in Vero cells and terminally-diluted once in Vero cells. Virus stock was prepared in Vero cells. .sup.cSame nucleotide position as 7162 in rDEN4. .sup.dVirus was recovered in C6/36 cells and passaged three times in Vero cells. Virus was then terminally diluted and a stock was prepared in Vero cells.
(120) Because both rDEN2 and rDEN230 viruses grown in Vero cells acquired the same mutation at nucleotide 7169, which corresponds to the Vero cell adaptation mutation previously identified in rDEN4 at nucleotide 7162, it was reasoned that this mutation is associated with growth adaptation of rDEN2 and rDEN230 in Vero cells. In anticipation that the 7169 mutation may allow rDEN230 to be recovered directly in Vero cells, the mutation was introduced into the rDEN230 cDNA plasmid to create p230-7169. Transcripts synthesized from p230-7169, as well as p2 and p230 were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection as described above. Virus rDEN230-7169 was recovered from the p230-7169 cDNA in both Vero and C6/36 cells, while rDEN230 was recovered from the p230 cDNA clone in only C6/36 cells (Table 21). The 7169 mutation is both necessary and sufficient for the recovery of rDEN230 in Vero cells.
(121) TABLE-US-00024 TABLE 21 rDEN230-7169 virus containing the 7169 Vero cell adaptation mutation is recovered in both Vero and C6/36 cells Virus titer of trans- fection harvest (day 14) determined Transfection cDNA in C6/36 cells cell type construct Clone Virus (log.sub.10 PFU/ml) Vero cells p2 #8A rDEN2 6.8 p230 #2 rDEN230 <0.7 p230-7169.sup.a #37 rDEN230-7169 5.1 C6/36 cells p2 #8A rDEN2 6.9 p230 #2 rDEN230 7.1 p230-7169 #37 rDEN230-7169 7.2 .sup.aNucleotide 7169 in rDEN2 corresponds to nucleotide 7162 in rDEN4 which has been shown to be associated with growth adaptation in Vero cells.
(122) To initially assess the ability of the 30 mutation to attenuate rDEN2 virus in an animal model, the replication of DEN2 (Tonga/74), rDEN2, and rDEN230 viruses was evaluated in SCID-HuH-7 mice. Previously, attenuation of vaccine candidates in SCID-HuH-7 mice has been demonstrated to be predictive of attenuation in the rhesus monkey model of infection (Examples 1 and 2). The recombinant viruses tested in this experiment were recovered in C6/36 cells. The DEN2 Tonga/74 virus isolate, rDEN2, and two independent rDEN230 viruses, (clones 20A and 21A) which were derived from two independent p230 cDNA clones, were terminally diluted twice in C6/36 cells prior to production of a working stock in C6/36 cells. These viruses should not contain any Vero cell adaptation mutations. DEN2 Tonga/74 virus replicated to a mean virus titer of 6.2 log.sub.10PFU/ml in the serum of SCID-HuH-7 mice, and rDEN2 virus replicated to a similar level, 5.6 log.sub.10 PFU/ml (Table 22). Both rDEN230 viruses were greater than 100-fold restricted in replication compared to rDEN2 virus. These results indicate that the 30 mutation has an attenuating effect on replication of rDEN2 virus similar to that observed for rDEN4 and rDEN1 viruses.
(123) TABLE-US-00025 TABLE 22 The 30 mutation restricts rDEN2 virus replication in SCID-HuH-7 mice. Mean virus Mean log.sub.10-unit No. of titer SE (log.sub.10 reduction from Virus mice PFU/ml serum).sup.a value for wt.sup.b DEN2 (Tonga/74) 8 6.2 0.3 rDEN2 9 5.6 0.2 rDEN230 (clone 20A) 9 3.1 0.2 2.5 rDEN230 (clone 21A) 9 2.9 0.3 2.7 .sup.aGroups of SCID-HuH-7 mice were inoculated directly into the tumor with 10.sup.4 PFU virus grown in C6/36 cells. Serum was collected on day 7 and titered in C6/36 cells. .sup.bComparison of mean virus titers of mice inoculated with mutant virus and concurrent rDEN2 control.
(124) DEN2 virus replication in SCID-HuH-7 mice was also determined using DEN2 (Tonga/74), rDEN2, and rDEN230 which were passaged in Vero cells (see Table 20, footnotes b and d). Both rDEN2 and rDEN230 had acquired a mutation in NS4B, nucleotide 7169, corresponding to the 7162 mutation identified in rDEN4 as Vero cell adaptation mutation. In the presence of the 7169 mutation, the 30 mutation reduced replication of rDEN230 by 1.0 log.sub.10PFU/ml (Table 23). Previously, using virus grown in C6/36 cells and lacking the 7169 mutation, the 30 mutation reduced replication of rDEN230 by about 2.5 log.sub.10PFU/ml (Table 22). These results indicate that Vero cell growth adaptation in DEN2 may also confer a slight growth advantage in HuH-7 liver cells. Nevertheless, the attenuation conferred by the 30 mutation is still discernible in these Vero cell growth adapted viruses.
(125) TABLE-US-00026 TABLE 23 The 30 mutation restricts Vero cell adapted rDEN2 virus replication in SCID-HuH-7 mice. Mean log.sub.10-unit No. Mean virus titer SE reduction from Virus of mice (log.sub.10 PFU/ml serum).sup.a value for wt.sup.b DEN2 (Tonga/74) 6 5.9 0.3 rDEN2 7 5.9 0.2 rDEN230 9 4.9 0.3 1.0 .sup.aGroups of SCID-HuH-7 mice were inoculated directly into the tumor with 10.sup.4 PFU virus. Serum was collected on day 7 and titered in C6/36 cells. .sup.bComparison of mean virus titers of mice inoculated with rDEN230 and rDEN2 control.
(126) Evaluation of the replication, immunogenicity, and protective efficacy of rDEN230 and wild-type parental rDEN2 virus in juvenile rhesus monkeys was performed as follows. Dengue virus-seronegative monkeys were injected subcutaneously with 5.0 log.sub.10PFU of virus in a 1 ml dose divided between two injections in each side of the upper shoulder area. Monkeys were observed daily and blood was collected on days 0-10 and 28 and serum was stored at 70 C. Viruses used in this experiment were passaged in Vero cells, and recombinant viruses contained the mutations shown in Table 20 (See footnotes b and d). Titer of virus in serum samples was determined by plaque assay in Vero cells as described previously (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). Plaque reduction neutralization titers were determined for the day 28 serum samples as previously described (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). All monkeys were challenged on day 28 with a single dose of 5.0 log.sub.10PFU of wt DEN2 (Tonga/74) and blood was collected for 10 days. Virus titer in post-challenge sera was determined by plaque assay in Vero cells. Monkeys inoculated with wt DEN2 (Tonga/74) or rDEN2 were viremic for 4-5 days with a mean peak titer of 2.1 or 1.9 log.sub.10PFU/ml, respectively.
(127) Monkeys inoculated with rDEN230 were viremic for 2-3 days with a mean peak titer of 1.7 log.sub.10PFU/ml (Table 24,
(128) TABLE-US-00027 TABLE 24 rDEN230 is slightly more attenuated for rhesus monkeys than rDEN2 Geometric mean serum neutralizing No. of Mean no. antibody titer monkeys of viremic Mean peak (reciprocal No. of with days per virus titer dilution) Virus.sup.a monkeys viremia monkey.sup.b (log.sub.10 PFU/ml SE) Day 0 Day 28 mock 2 0 0 <0.7 <10 <10 DEN2 (Tonga/74) 4 4 4.5 2.1 0.3 <10 311 rDEN2 (Vero) 4 4 4.0 1.9 0.1 <10 173 rDEN230 (Vero) 4 4 2.8 1.7 0.2 <10 91 .sup.aGroups of rhesus monkeys were inoculated subcutaneously with 10.sup.5 PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 12, and 28. Virus titer was determined by plaque assay in Vero cells. .sup.bViremia was not detected in any monkey after day 8.
(129) TABLE-US-00028 TABLE 25 rDEN230 protects rhesus monkeys from wt DENT2 virus challenge Geometric mean Mean no. of serum neutralizing viremic days per Mean peak antibody titer No. of monkey after virus titer (reciprocal dilution) Virus.sup.a monkeys DEN2 challenge (log.sub.10 PFU/ml SE) Day 28 Day 56 Mock 2 4.0 2.1 0.1 <10 338 DEN2 (Tonga/74) 4 0 <0.7 311 334 rDEN2 (Vero) 4 0 <0.7 173 318 rDEN230 (Vero) 4 0 <0.7 91 267 .sup.a28 days after inoculation with the indicated viruses, monkeys were challenged subcutaneously with 10.sup.5 PFU DEN2 (Tonga/74) in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Virus titer was determined by plaque assay in Vero cells.
(130) The infectivity of DEN2 (Tonga/74), rDEN2 and rDEN230 for Aedes aegypti mosquitoes via an artificial bloodmeal was evaluated using the methods described in detail in Example 5. However at doses of 3.3 to 3.5 log.sub.10pfu ingested, none of these three viruses infected any mosquitoes, indicating that DEN2 (Tonga/74) is poorly infectious for Aedes aegypti. As with rDEN1, the genetic basis for this lack of infectivity remains to be defined. The important property of restricted infectivity for the mosquito midgut is highly desirable in a vaccine candidate because it would serve to greatly restrict transmission of the virus from a vaccinee to a mosquito vector.
(131) Several missense mutation identified in rDEN4 have been demonstrated to confer attenuated replication in suckling mouse brain and/or SCID-HuH-7 mice (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol 75:9731-9740). In addition, missense mutations that enhance replication of rDEN4 virus in Vero cells have been characterized. The significant sequence conservation among the DEN virus serotypes provides a strategy by which the mutations identified in rDEN4 viruses are contemplated as being used to confer similar phenotypes upon rDEN2 virus. Six mutations identified in rDEN4 virus that are at a site conserved in rDEN2 virus are being introduced into the p2 and p230 cDNA clones (Table 26). Specifically, two rDEN4 mutations, NS3 4891 and 4995, which confer Vero cell adaptation phenotypes and decreased replication in mouse brain, one mutation, NS4B 7182, which confers a Vero cell adaptation phenotype, and three mutations, NS1 2650, NS3 5097, and 3 UTR 10634 which confer decreased replication in mouse brain and SCID-HuH-7 mice are being evaluated. These mutations have been introduced into sub-cloned fragments of the p2 and p230 cDNA clones, and have been used to generate mutant full-length cDNA clones (Table 26), from which virus has been recovered in C6/36 cells (Table 27). The evaluation of these mutant rDEN2 viruses is contemplated as determining that such point mutations can be transported into a different DEN virus serotype and confer a similar useful phenotype, as has been demonstrated for the 30 deletion mutation.
(132) TABLE-US-00029 TABLE26 Introductionofconservedpointmutationscharacterizedin rDEN4virusesintorDEN2Tonga/74virus. Phenotypein rDEN4virus MutationinrDEN4virus MutationintroducedintoDEN2virus Vero Mouse SCID- Nucleo- Amino Amino Nucleo- Amino Amino RE Adap- brain HuH-7 tide acid acid tide acid acid site/mutagenic tation.sup.a att.sup.b att.sup.c Gene/region position position.sup.d change position position.sup.d change region.sup.e + + NS3 4891 1597 Ile> Thr 4889 1598 Ile> Thr NarI CCAcgGGcGCCGT + + NS3 4995 1632 Ser> Pro 4993 1633 Ser> Pro StuI AAGGccTGGA + NS4b 7182 2361 Gly> Ser 7189 2365 Gly> Ser XmaI TAtccCCGGGAC + + NS1 2650 850 Asn> Ser 2648 851 Asn> Ser SacI AGAgcTctcTC + + NS3 5097 1666 Asp> Asn 5095 1667 Asp> Asn XmaI GaATCTCCACCCgGA + + 3 UTR 10634 n/a.sup.f n/a 10698 n/a n/a none CTGTcGAATC .sup.aPresence of the indicated mutation increases plaque size in Vero cells two-fold or greater than rDEN4 virus. .sup.bPresence of the indicated mutation restricts replication in 7-day-old mouse brain greater than 100-fold compared to rDEN4 virus. .sup.cPresence of the indicated mutation restricts replication in SCID-HuH-7 mice greater than 100-fold compared to rDEN4 virus. .sup.dAmino acid position in DEN4 or DEN2 polyprotein beginning with the methionine residue of the C protein (nucleotides 102-104 or 97-99, respectively) as position 1. .sup.ePrimers were engineered which introduced (underline) translationally-silent restriction enzyme (RE) sites. Lowercase letters indicate nt changes and bold letters indicate the site of the 5-FU mutation, which in some oligonucleotides differs from the original nucleotide substitution change in order to create a unique RE site. The change preserves the codon for the amino acid substitution. .sup.fNucleotide substitution in the 3 UTR is U > C in DEN4 and DEN2 virus.
(133) TABLE-US-00030 TABLE 27 rDEN2 viruses containing conserved 5-FU mutations are recovered in C6/36 cells. Virus Nucleotide Virus titer of transfection (nucleotide position in harvest (day 7) determined position in rDEN2) rDEN4 in C6/36 cells (log.sub.10 PFU/ml) rDEN2-4889 4891 7.6 rDEN2-4993 4995 7.2 rDEN2-7189 7182 3.5 rDEN2-2648 2650 .sup.a rDEN2-5095 5097 .sup.a rDEN2-10698 10634 7.7 .sup.aTransfection has not yet been attempted.
EXAMPLE 4
Generation and Characterization of a Recombinant DEN3 Virus Containing the 30 Mutation
(134) Because rDEN130 was satisfactorily attenuated, we sought to extend our technology to the creation of a DEN3 vaccine candidate. To do this, the 30 mutation was introduced into the cDNA of DEN3, similar to the method used to create rDEN230. A DEN3 virus isolate from a 1978 dengue epidemic in rural Sleman, Central Indonesia (Sleman/78) (Gubler, D. J. et al. 1981 Am J Trop Med Hyg 30:1094-1099) was chosen to represent wt DEN3. The genome of DEN3 (Sleman/78) was sequenced in its entirety and served as consensus sequence for the construction of a full-length cDNA clone (Appendix 2). cDNA fragments of DEN3 (Sleman/78) were generated by reverse-transcription of the genome as indicated in
(135) For transcription and generation of infectious virus, cDNA plasmids p3 and p330 were digested with SpeI and religated to remove the linker sequence, linearized with Acc65I (isoschizomer of KpnI which cleaves leaving only a single 3 nucleotide), and used as templates in a transcription reaction using SP6 RNA polymerase as previously described (Blaney, J. E. et. al. 2002 Virology 300:125-139). Transcripts were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection and cell culture supernatants were harvested on day 14.
(136) rDEN3 virus was recovered from the p3 cDNA in both Vero and C6/36 cells, while rDEN330 was recovered from the p330 cDNA clone in only C6/36 cells (Table 28). The level of infectious virus recovered in C6/36 cells was comparable for the p3 and p330 cDNA clones when assayed by plaque titration in Vero or C6/36 cells. As previously observed, the efficiency of transfection in C6/36 cells was higher than that in Vero cells. Two rDEN330 viruses were recovered from independent cDNA clones, #22 and #41.
(137) TABLE-US-00031 TABLE 28 rDEN3 virus is recovered in Vero and C6/36 cells, but rDEN330 virus is recovered only in C6/36 cells. Virus titer of transfection harvest (day 14) determined in the indicated Transfection cDNA cell type (log.sub.10 PFU/ml) cell type construct Clone Virus Vero cells C6/36 cells Vero cells p3 #50 rDEN3 5.2 6.3 p330 #22 rDEN330 <0.7 <0.7 p330 #41 rDEN330 <0.7 <0.7 C6/36 cells p3 #50 rDEN3 5.2 6.0 p330 #22 rDEN330 5.9 6.9 p330 #41 rDEN330 5.1 7.2
(138) To produce working stocks of viruses, transfection harvests will be passaged and terminally diluted in Vero cells, and genomic sequences of the viruses will be determined. To improve virus yield in Vero cells, the Vero cell adaptation mutation previously identified in rDEN4 at nucleotide 7162 was introduced into the homologous NS4B region of p3 and p330 to create p3-7164 and p330-7164. This mutation creates a Val to Ala substitution at amino acid position 2357. As demonstrated for rDEN230, this mutation allowed for the direct recovery of virus in Vero cells (Table 27) and is anticipated to have the same effect for rDEN330.
(139) To initially assess the ability of the 30 mutation to attenuate rDEN3 virus in an animal model, the replication of DEN3 (Sleman/78), rDEN3, and rDEN330 viruses will be evaluated in SCID-HuH-7 mice and rhesus monkeys. Previously, attenuation of vaccine candidates in SCID-HuH-7 mice has been demonstrated to be predictive of attenuation in the rhesus monkey model of infection (Examples 1 and 2). The evaluation of these mutant rDEN3 viruses is contemplated as determining that the 30 deletion mutations can be transported into the DEN3 virus serotype and confer a similar useful phenotype, as has been demonstrated for DEN1, DEN2, and DEN4.
(140) In summary, the strategy of introducing the 30 mutation into wild-type DEN viruses of each serotype to generate a suitably attenuated tetravalent vaccine formulation is a unique and attractive approach for several reasons. First, the mutation responsible for attenuation is a 30-nucleotide deletion in the 3 UTR, thus assuring that all of the structural and non-structural proteins expressed by each of the four components of the tetravalent vaccine are authentic wild-type proteins. Such wild-type proteins should elicit an antibody response that is broad based, rather than based solely on the M and E proteins that are present in chimeric dengue virus vaccine candidates (Guirakhoo, F. et al. 2001 J Virol 75:7290-304; Huang, C. Y. et al. 2000 J Virol 74:3020-8). The uniqueness of this approach derives from the fact that other live attenuated dengue virus vaccines have mutations in their structural or non-structural proteins (Butrapet, S. et al. 2000 J Virol 74:3011-9; Puri, B. et al. 1997 J Gen Virol 78:2287-91), therefore the immune response induced by these viruses will be to a mutant protein, rather than a wild-type protein. Second, deletion mutations are genetically more stable than point mutations, and reversion of the attenuation phenotype is unlikely. In humans, DEN430 present in serum of vaccinees retained its 30 mutation, confirming its genetic stability in vivo (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-13). The attenuating mutations in other existing dengue live attenuated vaccine candidates are based on less stable point mutations (Butrapet, S. et al. 2000 J Virol 74:3011-9: Puri, B. et al. 1997 J Gen Virol 78:2287-91). Third, since the 30 mutation is common to each of the four viruses of the tetravalent vaccine, recombination between any of the four vaccine serotypes would not lead to loss of the attenuating mutation or reversion to a wild-type phenotype. Recombination between components of the trivalent polio vaccine has been observed (Guillot, S. et al. 2000 J Virol 74:8434-43), and naturally occurring recombinant dengue viruses have been described (Worobey, M. et al. 1999 PNAS USA 96:7352-7) indicating the ability of this flavivirus to exchange genetic elements between two different viruses. Clearly, gene exchange is readily achieved between different DEN virus serotypes using recombinant cDNA techniques (Bray, M. and Lai, C. J. 1991 PNAS USA 88:10342-6). Fourth, viruses with wild-type structural proteins appear more infectious than viruses with altered structural proteins (Huang, C. Y. et al. 2000 J Virol 74:3020-80). This permits the use of a low quantity of each of the four virus components in the final vaccine, contributing to the low cost of manufacture. Low-cost manufacture is an essential element in defining the ultimate utility of a dengue virus vaccine.
EXAMPLE 5
Generation and Characterization of Intertypic Chimeric DEN2 Viruses Containing the 30 Mutation
(141) The four serotypes of dengue virus are defined by antibody responses induced by the structural proteins of the virus, primarily by a neutralizing antibody response to the envelope (E) protein. These structural proteins include the E glycoprotein, a membrane protein (M), and a capsid (C) protein. The mature virus particle consists of a well-organized outer protein shell surrounding a lipid bilayer membrane and a less-well-defined inner nucleocapsid core (Kuhn, R. J. et al. 2002 Cell 108:717-25). The E glycoprotein is the major protective antigen and readily induces virus neutralizing antibodies that confer protection against dengue virus infection. An effective dengue vaccine must therefore minimally contain the E protein of all four serotypes, namely DEN1, DEN2, DEN3, and DEN4, thereby inducing broad immunity and precluding the possibility of developing the more serious illnesses DHF/DSS, which occur in humans during secondary infection with a heterotypic wild-type dengue virus. Based on a previously reported strategy (Bray, M. and Lai, C. J. 1991 PNAS USA 88:10342-6), a recombinant cDNA technology is being used to develop a live attenuated tetravalent dengue virus vaccine composed of a set of intertypic chimeric dengue viruses bearing the structural proteins of each serotype.
(142) Following the identification of a suitably attenuated and immunogenic DEN4 recombinant virus, namely DEN430 (Durbin, A. P et al. 2001 Am J Trop Med Hyg 65:405-13), chimeric viruses based on the DEN4 cDNA have been generated in which the C-M-E (CME) or M-E (ME) genes have been replaced with the corresponding genes derived from the prototypic DEN2 New Guinea C (NGC) strain (
(143) For transcription and generation of virus, chimeric cDNA clones were linearized and used as template in a transcription reaction using SP6 RNA polymerase as described (Durbin, A. P et al. 2001 Am J Trop Med Hyg 65:405-13). Transcripts were introduced into Vero cells using liposome-mediated transfection and recombinant dengue virus was harvested on day 7. The genomes of the resulting viruses were confirmed by sequence analysis of viral RNA isolated from recovered virus as previously described (Durbin, A. P et al. 2001 Am J Trop Med Hyg 65:405-13). Incidental mutations arising from virus passage in tissue culture were identified in all viruses and are listed in Table 29. Notably, each virus contained a missense mutation in NS4B corresponding to a previously identified mutation from rDEN4 and associated with adaptation to replication in Vero cells (See Table 30 for correlation of nucleotide positions between rDEN4 and chimeric viruses). All viruses replicated in Vero cells to titers in excess of 6.0 log.sub.10PFU/ml, indicating that the chimeric viruses, even those containing the 30 mutation, replicate efficiently in cell culture, a property essential for manufacture of the vaccine.
(144) TABLE-US-00032 TABLE 29 Missense mutations observed among the Vero cell-grown chimeric DEN2/4 viruses Amino Amino Nucleotide Nucleotide acid acid Virus Gene position change position change rDEN2/4(CME) NS4B 7161.sup.a A > U 2355 Leu > Phe rDEN2/430(CME) M 743 G > A 216 Gly > Glu E 1493 C > U 466 Ser > Phe NS4B 7544.sup.b C > T 2483 Ala > Val rDEN2/4(ME) E 1065 U > C 322 Phe > Leu NS4B 7163.sup.a A > U 2354 Leu > Phe rDEN2/430(ME) NS4B 7163.sup.a A > C 2354 Leu > Phe .sup.aSame nucleotide position as 7163 in rDEN4. .sup.bSame nucleotide position as 7546 in rDEN4.
(145) TABLE-US-00033 TABLE 30 Nucleotide (nt) length differences for DEN chimeric viruses compared to rDEN4. rDEN nt difference Amino chimeric from rDEN4 ORF start acid length virus (following CME region) (nt position) C M E 1/4 ME 0 102 113 166 495 1/4 CME +3 102 114 166 495 2/4 ME 0 102 113 166 495 2/4 CME 2 97 114 166 495 3/4 ME 6 102 113 166 493 3/4 CME 3 102 114 166 493 rDEN4 102 113 166 495
(146) Results of a safety, immunogenicity, and efficacy study in monkeys are presented in Table 31. Monkeys inoculated with wild-type DEN2 were viremic for approximately 5 days with a mean peak titer of 2.1 log.sub.10PFU/ml, while monkeys inoculated with any of the chimeric DEN2 viruses were viremic for 1.2 days or less and had a mean peak titer of less than 1.0 log.sub.10PFU/ml. This reduction in the magnitude and duration of viremia clearly indicates that the chimeric viruses containing either the CME or ME proteins of DEN2 were more attenuated than the parental DEN2 NGC virus. Neither the animals receiving the wild-type DEN2 nor the DEN2/4 chimeric viruses were ill. The decreased replication of the attenuated viruses in monkeys is accompanied by a reduction in the immune response of inoculated monkeys. This is indicated in Table 31 by approximately a 5-fold reduction in the level of neutralizing antibody following inoculation with the chimeric viruses in comparison to titers achieved in animals inoculated with wild-type virus. Addition of the 30 mutation to the CME chimeric virus further attenuated the virus, such that rDEN2/430(CME) did not replicate in monkeys to a detectable level and did not induce a detectable immune response. This virus appeared over-attenuated, and if similar results were seen in humans, this virus would not be suitable for use as a vaccine. However, addition of the 30 mutation to the ME chimeric virus did not further attenuate this chimeric virus and the resulting rDEN2/430(ME) virus appears satisfactorily attenuated and immunogenic for use as a vaccine.
(147) TABLE-US-00034 TABLE 31 Chimerization between dengue virus types 2 and 4 results in recombinant viruses which are attenuated for rhesus monkeys. Mean peak Geometric mean Mean no. virus titer neutralizing days with (log.sub.10 antibody titer Group* Virus n viremia pfu/ml) (reciprocal) 1 rDEN2/4 (CME) 6 1.2 0.9 50 2 rDEN2/430 (CME) 8 0 <0.7 <5 3 rDEN2/4 (ME) 4 1.0 0.8 76 4 rDEN2/430 (ME) 4 0.3 0.7 62 5 DEN2 NGC 6 5.5 2.1 312 *Rhesus monkeys were inoculated subcutaneously with 5.0 log.sub.10 PFU of virus. Serum samples were collected daily for 10 days. Serum for neutralization assay was collected on day 28. Serum samples obtained before virus inoculation had a neutralizing antibody titer of <5.
(148) As described in the previous examples, SCID mice transplanted with the HuH-7 cells are a sensitive model for the evaluation of dengue virus attenuation. Each chimeric DEN2/4 virus was inoculated into groups of SCID-HuH-7 mice and levels of virus in the serum were determined (Table 32). Chimeric viruses replicated to levels between 20- and 150-fold lower than either of the parental viruses (rDEN4 and DEN2-NGC). CME chimeric viruses were slightly more attenuated than the comparable ME chimeric viruses, with the 30 mutation providing a 0.5 log.sub.10 reduction in replication. This level of attenuation by the 30 mutation was similar to that observed previously for rDEN430.
(149) TABLE-US-00035 TABLE 32 Chimerization between dengue virus types 2 and 4 results in recombinant viruses which are attenuated for HuH-7-SCID mice. No. of Mean peak virus titer Statistical Virus.sup.a mice (log.sub.10 pfu/ml SE) group.sup.b rDEN4 32 6.3 0.2 A DEN2-NGC 9 6.1 0.2 A rDEN2/4 (CME) 7 4.4 0.3 B rDEN2/430 (CME) 7 3.9 0.3 B rDEN2/4 (ME) 6 4.8 0.5 B rDEN2/430 (ME) 9 4.3 0.2 B .sup.aGroups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log.sub.10 PFU of the indicated virus. Serum was collected on day 7 and virus titer was determined in Vero cells. .sup.bMean peak titers were assigned to statistical groups using the Tukey post-hoc test (P < 0.05). Groups with the same letter designation are not significantly different.
(150) To evaluate the replication levels of each DEN2/4 chimeric virus in mosquitoes, two different genera of mosquitoes were experimentally infected. Aedes aegypti were infected by ingesting a virus-containing blood meal. By evaluating the presence of virus antigen in both the midgut and head tissue, infectivity could be determined for the local tissues (midgut), and the ability of virus to disseminate and replicate in tissues beyond the midgut barrier (head) could also be measured. The presence of virus in the head is limited by the ability of the ingested virus to replicate in the midgut and then disseminate to the salivary glands in the head, as well as the innate ability of the virus to replicate in the salivary glands. Intrathoracic inoculation of virus into Toxorhynchites splendens bypasses the mosquito midgut barrier. Parental viruses rDEN4 and DEN2-NGC readily infect Ae. aegypti and T splendens (Table 33), with DEN2-NGC appearing to be much more infectious in T. splendens. Each of the rDEN2/4 chimeric viruses was also tested in both mosquito types. In many cases it was not possible to inoculate Ae. aegypti with an undiluted virus stock of sufficient titer to achieve a detectable infection due to the very low infectivity of several of the viruses. Nevertheless, it is clear that the rDEN2/4 chimeric viruses are less infectious for the midgut and head. Parental viruses rDEN4 and DEN2-NGC, administered at a maximum dose of approximately 4.0 log.sub.10PFU, were detectable in 74% and 94% of midgut preparations, and 32% and 71% of head preparations, respectively. Among the chimeric viruses, the highest level of infectivity, as observed for rDEN2/430 (CME), resulted in only 26% infected midgut samples and 6% head samples. In the more permissive T. splendens, the rDEN2/4 chimeric viruses were generally less infectious than either parental virus, with CME chimeric viruses being less infectious than ME viruses. It has previously been reported for DEN4 that the 30 mutation does not have a discernable effect on virus infectivity in T. splendens similar to that observed here for the rDEN2/4 chimeric viruses (Troyer, J. M. et al. 2001 Am J Trop Med Hyg 65:414-419).
(151) TABLE-US-00036 TABLE 33 Dengue 2/4 chimeric viruses are less infectious compared to either parental virus strain in mosquitoes Toxorhynchites splendens Aedes aegypti (intrathoracic inoculation) (oral infection) Dose.sup.a No. % Dose.sup.c No. % infected.sup.d Virus log.sub.10 pfu tested infected.sup.b log.sub.10 pfu tested Midgut Head rDEN4 3.3 6 83 3.8 38 74 32 2.3 7 57 2.8 15 26 6 1.3 6 0 1.8 20 10 5 MID.sub.50 = 2.2 MID.sub.50 = 3.4 MID.sub.50 4.1 DEN2-NGC 2.5 5 100 4.0 17 94 71 1.2 15 93 3.0 25 36 16 0.2 4 75 2.0 30 0 0 0.02 8 38 MID.sub.50 = 3.2 MID.sub.50 = 3.6 MID.sub.50 = 0.5 rDEN2/4 (CME) 3.9 9 11 4.4 11 9 0 2.9 5 0 3.4 10 0 0 MID.sub.50 4.3 MID.sub.50 4.9 Nc.sup.e rDEN2/430 3.5 6 17 4.0 15 26 6 (CME) 2.5 6 17 3.0 10 0 0 MID.sub.50 3.9 MID.sub.50 4.3 MID.sub.50 4.5 rDEN2/4 (ME) 3.4 6 100 3.9 23 4 0 2.4 5 20 MID.sub.50 4.4 Nc 1.4 5 0 MID.sub.50 = 2.8 rDEN2/430 2.6 11 9 3.1 30 0 0 (ME) MID.sub.50 3.0 nc Nc .sup.aAmount of virus present in 0.22 l inoculum. .sup.bPercentage of mosquitoes with IFA detectable antigen in head tissue prepared 14 days after inoculation. .sup.cVirus titer ingested, assuming a 2 l bloodmeal. .sup.dPercentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection. When virus infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MID.sub.50 was estimated by assuming that a 10-fold more concentrated virus dose would infect 100% of the mosquitoes. .sup.enc = not calculated, since virus antigen was not detected.
(152) Chimerization of the DEN2 structural genes with rDEN430 virus resulted in a virus, rDEN2/430(CME), that had decreased replication in Vero cells compared to either parent virus. To evaluate Vero cell adaptation mutations (Blaney, J. E. et al. 2002 Virology 300:125-139) as a means of increasing the virus yield of a DEN vaccine candidate in Vero cells, selected mutations were introduced into this chimeric virus. Accordingly, rDEN2/430(CME) viruses bearing adaptation mutations were recovered, terminally diluted, and propagated in C6/36 cells to determine if the virus yield in Vero cells could be increased.
(153) rDEN2/430(CME) viruses bearing Vero cell adaptation mutations were generated as follows. DNA fragments were excised from rDEN4 cDNA constructs encompassing single or double DEN4 Vero cell adaptation mutations and introduced into the cDNA clone of rDEN2/430(CME). The presence of the Vero cell adaptation mutation was confirmed by sequence analysis, and RNA transcripts derived from the mutant cDNA clones were transfected, terminally diluted, and propagated in C6/36 cells.
(154) For evaluation of growth kinetics, Vero cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01. Confluent cell monolayers in duplicate 25-cm.sup.2 tissue culture flasks were washed and overlaid with a 1 ml inoculum containing the indicated virus. After a two hour incubation at 37 C., cells were washed three times in MEM and 5 ml of MEM supplemented with 2% FBS was added. A 1 ml aliquot of tissue culture medium was removed, replaced with fresh medium, and designated the day 0 time-point. At the indicated time points post-infection, 1 ml samples of tissue culture medium were removed, clarified by centrifugation, and frozen at 80 C. The level of virus replication was assayed by plaque titration in C6/36 cells and visualized by immunoperoxidase staining. The limit of detection was <0.7 log.sub.10PFU/ml.
(155) The growth properties of rDEN2/430(CME) viruses bearing single Vero cell adaptation mutations at NS4B-7153, -7162, -7163, -7182, NS5-7630 or three combinations of mutations were compared in Vero cells with rDEN2/430 (CME) virus (
(156) These results have particular significance for the development of a live attenuated dengue virus vaccine. First, it is clear that chimerization leads to attenuation of the resulting virus, as indicated by studies in rhesus monkeys, HuH7-SCID mice and mosquitoes. Although this conclusion was not made in the previous study with DEN2/DEN4 or DEN1/DEN4 chimeric viruses (Bray, M. et al. 1996 J Virol 70:4162-6), careful examination of the data would suggest that the chimeric viruses are more attenuated in monkeys compared to the wild-type parent viruses. Second, the 30 mutation can further augment this attenuation in a chimeric-dependent manner. Specifically, in this example, chimeric viruses bearing the CME region of DEN2 were over-attenuated by the addition of 30, whereas the attenuation phenotype of chimeric viruses bearing just the ME region of DEN2 was unaltered by the addition of the 30 mutation. This unexpected finding indicates that in a tetravalent vaccine comprised of individual component viruses bearing a shared attenuating mutation, such as the 30 mutation, only ME chimeric viruses can be utilized since CME chimeric viruses bearing the 30 mutation can be over-attenuated in rhesus monkeys and might provide only limited immunogenicity in humans.
EXAMPLE 6
Generation and Characterization of Intertypic Chimeric DEN3 Viruses Containing the 30 Mutation
(157) Chimeric viruses based on the DEN4 cDNA have been generated in which the CME or ME genes have been replaced with the corresponding genes derived from DEN3 (Sleman/78), a virus isolate from the 1978 dengue outbreak in the Sleman region of Indonesia (Gubler, D. J. et al. 1981 Am J Trop Med Hyg 30:1094-1099) (Appendix 2). As described in Example 5 for the DEN2 chimeric viruses, CME chimeric viruses for DEN3 were generated by replacing the BglII/XhoI region of the cDNA for either rDEN4 or rDEN430 with a similar region derived from DEN3 (Sleman/78) (
(158) TABLE-US-00037 TABLE 34 Missense mutations observed among Vero cell-grown chimeric DEN3/4 viruses Amino Amino Nucleotide Nucleotide acid acid Virus Gene position change position change rDEN3/430 M 825 T > C 242 Phe > Leu (CME) E 1641 C > T 514 Leu > Phe E 2113 A > G 671 Lys > Arg NS4B 7159.sup.a T > C 2353 Leu > Ser rDEN3/4(ME) M 460 A > G 120 Asp > Gly NS4B 7177.sup.b G > U 2359 Gly > Val NS5 7702 C > U 2534 Ser > Phe rDEN3/430 E 1432 A > U 444 Gln > Leu (ME) NS4B 7156.sup.a U > C 2352 Leu > Ser NS5 8692 A > C 2864 Asn > His .sup.aSame nucleotide position as 7162 in rDEN4. .sup.bSame nucleotide position as 7183 in rDEN4.
(159) As described in the previous examples, SCID mice transplanted with HuH-7 cells are a sensitive model for the evaluation of dengue virus attenuation. Each chimeric DEN3/4 virus was inoculated into groups of SCID-HuH-7 mice and levels of virus in the serum were determined (Table 35). While chimeric virus rDEN3/4 (CME) was not attenuated, the remaining chimeric viruses replicated to levels between 40- and 400-fold lower than either of the parental viruses (rDEN4 and DEN3-Sleman/78). In the CME chimeric virus, the 30 mutation providing a remarkable 2.7 log.sub.10 reduction in replication. This level of attenuation conferred by the 30 mutation in the CME chimeric virus was much greater than that observed previously for rDEN430. The rDEN3/4 (ME) virus was 100-fold reduced in replication compared to either parent virus indicating that the ME chimerization was attenuating per se. Addition of the 30 mutation to rDEN3/4 (ME) did not result in additional attenuation.
(160) TABLE-US-00038 TABLE 35 Chimerization between dengue virus types 3 and 4 results in recombinant viruses which are attenuated for HuH-7-SCID mice. No. of Mean peak virus titer Statistical Virus.sup.a mice (log.sub.10 pfu/ml SE) group.sup.b rDEN4 32 6.3 0.2 A DEN3-Sleman/78 23 6.4 0.2 A rDEN3/4 (CME) 7 6.4 0.6 A rDEN3/430 (CME) 5 3.7 0.4 B rDEN3/4 (ME) 6 4.2 0.7 B rDEN3/430 (ME) 7 4.7 0.4 A, B .sup.aGroups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log.sub.10 PFU of the indicated virus. Serum was collected on day 7 and virus titer was determined in Vero cells. .sup.bMean peak titers were assigned to statistical groups using the Tukey post-hoc test (P < 0.05). Groups with the same letter designation are not significantly different.
(161) Evaluation of the replication and immunogenicity of the DEN3 chimeric recombinant viruses and wild-type DEN3 virus in monkeys was performed as described in Example 5. Results of this safety and immunogenicity study in monkeys are presented in Table 36. Monkeys inoculated with rDEN3/4(CME) and wild-type DEN (Sleman/78) were viremic for approximately 2 days with a mean peak titer of between 1.6 and 1.8 log.sub.10PFU/ml, respectively, indicating that chimerization of the CME structural genes of DEN3 did not lead to attenuation of virus replication, a different pattern than that observed for DEN2 chimerization (Table 31). However, chimerization of the ME structural genes resulted in attenuated viruses with undetectable viremia in monkeys, although all monkeys seroconverted with a greater than 10-fold increase in serum antibody levels. As expected for an attenuated virus, the immune response, as measured by neutralizing antibody titer, was lower following inoculation with any of the chimeric viruses compared to inoculation with wt DEN3 (Sleman/78), yet sufficiently high to protect the animals against wtDEN3 virus challenge (Table 37). It is clear that addition of the 30 mutation to rDEN3/4(CME) was capable of further attenuating the resulting virus rDEN3/430(CME).
(162) TABLE-US-00039 TABLE 36 The 30 mutation further attenuates rDEN3/4(CME) for rhesus monkeys Geometric mean Mean peak serum neutralizing Mean no. virus titer antibody of viremic (log.sub.10 titer (reciprocal No. of days per PFU/ml dilution) Virus.sup.a monkeys monkey.sup.b SE) Day 0 Day 28 DEN3 4 2.3 1.8 <5 707 (Sleman/78) rDEN3/4 4 2.0 1.6 <5 211 (CME) rDEN3/430 4 0 <1.0 <5 53 (CME) rDEN3/4 4 0 <1.0 <5 70 (ME) rDEN3/430 4 0 <1.0 <5 58 (ME) .sup.aGroups of rhesus monkeys were inoculated subcutaneously with 10.sup.5 PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 12, and 28. Virus titer was determined by plaque assay in Vero cells. .sup.bViremia was not detected in any monkey after day 4.
(163) TABLE-US-00040 TABLE 37 rDEN3/4 chimeric viruses protect rhesus monkeys from wt DEN3 virus challenge Geometric mean serum neutralizing Mean no. of Mean peak antibody titer viremic days virus titer (reciprocal per monkey (log.sub.10 dilution) No. of after rDEN3 PFU/ml Day Day Virus.sup.a monkeys challenge SE) 28 56 Mock 2 5.0 2.5 0.4 <5 372 DEN3 (Sleman/78) 4 0 <1.0 707 779 rDEN3/4 (CME) 4 0 <1.0 211 695 rDEN3/430 (CME) 4 0.8 1.1 0.2 53 364 rDEN3/4 (ME) 4 0 <1.0 70 678 rDEN3/430 (ME) 4 0 <1.0 58 694 .sup.a28 days after primary inoculation with the indicated viruses, rhesus monkeys were challenged subcutaneously with 10 PFU DEN3 (Sleman/78) virus in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Virus titer was determined by plaque assay in Vero cells.
(164) To evaluate the replication levels of each DEN3/4 chimeric virus in mosquitoes, Aedes aegypti were infected by ingesting a virus-containing blood meal (Table 38). Parental viruses rDEN4 and DEN3 (Sleman/78) readily infect Ae. aegypti. Each of the rDEN3/4 chimeric viruses was also tested. In many cases it was not possible to infect Ae. aegypti with an undiluted virus stock of sufficient titer to achieve a detectable infection due to the very low infectivity of several of the viruses. At a dose of approximately 2.8-2.9 log.sub.10PFU, rDEN4, DEN3 (Sleman/78), and rDEN3/4(CME) were equally infectious and disseminated to the head with equal efficiency. For the remaining chimeric viruses, infection was not detectable even at a dose of 3.4 log.sub.10PFU, indicating that replication of rDEN3/4(ME) and rDEN3/430(CME) is restricted in Ae. aegypti. By comparing infectivity of rDEN3/4(CME) and rDEN3/430(CME), it is clear that the 30 mutation is capable of further attenuating the chimeric virus for mosquitoes.
(165) TABLE-US-00041 TABLE 38 Ability of DEN3/4 chimeric viruses to infect Aedes aegypti fed an infectious bloodmeal. No. Dose Mosqui- No. (%) No. (%) Ingested toes Midgut Disseminated Virus Tested (log.sub.10 pfu).sup.a Tested Infections.sup.b,c,d Infections.sup.e rDEN4 3.8 18 14 (77%) 2 (14%) 2.8 20 7 (34%) 2 (10%) 1.8 18 0 0 MID.sub.50 = 3.4 MID.sub.50 4.4 DEN3 2.9 16 3 (18%) 2 (12%) (Sleman) 1.9 10 1 (10%) 0 MID.sub.50 3.5 MID.sub.50+0 3.5 rDEN3/4 3.9 20 6 (30%) 2 (10%) (CME) 2.9 18 4 (22%) 0 1.9 13 1 (7%) 0 MID.sub.50 4.2 MID.sub.50 4.5 DEN3/430 3.3 20 0 0 (CME) MID.sub.50 4.3 MID.sub.50 4.3 DEN3/4 3.4 15 0 0 (ME) MID.sub.50 4.4 MID.sub.50 4.4 aAmount of virus ingested, assuming a 2 bloodmeal. .sup.bNumber (percentage) of mosquitoes with detectable dengue virus in midgut tissue; mosquitoes were assayed 21 days post feed, and dengue virus antigen was identified by IFA. .sup.cWhen infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MID.sub.50 was estimated by assuming that a 10-fold more concentrated virus dose would infect 100% of the mosquitoes. .sup.dWhen no infection was detected, the MID.sub.50 was assumed to be greater than a 10-fold higher dose of virus than the one used. .sup.eNumber (percentage) of mosquitoes with detectable dengue virus antigen in both midgut and head tissue.
EXAMPLE 7
Generation and Characterization of Intertypic Chimeric DEN1 Viruses Containing the 30 Mutation
(166) Chimeric viruses based on the DEN4 cDNA have been generated in which the CME or ME genes have been replaced with the corresponding genes derived from DEN1 (Puerto Rico/94), a virus isolate from a 1994 dengue outbreak in Puerto Rico (Appendices 3 and 4). As described in Example 4 for the DEN2 chimeric viruses, CME chimeric viruses for DEN1 were generated by replacing the BglII/XhoI region of the cDNA for either rDEN4 or rDEN430 with a similar region derived from DEN1 (Puerto Rico/94) (
(167) For transcription and generation of virus, chimeric cDNA clones were linearized and used as template in a transcription reaction using SP6 RNA polymerase as described. Transcripts were introduced into C6/36 mosquito cells using liposome-mediated transfection and recombinant dengue virus was harvested between day 7 and 14. Viruses were subsequently grown in Vero cells and biologically cloned by terminal dilution in Vero cells. All viruses replicated in Vero cells to titers in excess of 6.0 log.sub.10PFU/ml, indicating that the chimeric viruses, even those containing the 30 mutation, replicate efficiently in cell culture. Genomic sequence analysis is currently underway to identify incidental mutations arising from virus passage in tissue culture.
(168) To evaluate the replication levels of DEN1/4(CME) and rDEN1/430(CME) chimeric virus in mosquitoes, Aedes aegypti were infected by ingesting a virus-containing blood meal (Table 39). Parental virus rDEN4 infects Ae. aegypti with an MID50 of 4.0 log.sub.10PFU. However, parental virus DEN1(Puerto Rico/94), is unable to infect Ae. aegypti at a dose of up to 3.4 log.sub.10PFU. Thus CME chimeric viruses DEN1/4 and rDEN1/430 share this inability to infect Ae. aegypti. Therefore, it is unnecessary in Ae. aegypti to evaluate the effect of the 30 mutation on the infectivity of the DEN1/4 chimeric viruses, in a manner similar to that used for the DEN2/4 and DEN3/4 chimeric viruses.
(169) TABLE-US-00042 TABLE 39 Inability of DEN1/4 chimeric viruses to infect Aedes aegypti fed an infectious bloodmeal. No. Dose Mosqui- No. (%) No. (%) Virus ingested toes Midgut Disseminated tested (log.sub.10 pfu).sup.a Tested Infections.sup.b,c,d Infections.sup.e rDEN4 4.3 21 18 (85%) 8 (44%) 3.3 15 3 (20%) 0 2.3 20 0 0 MID.sub.50 = 4.0 MID.sub.50 4.3 DEN1 3.4 21 0 0 (Puerto Rico/94) MID.sub.50 4.4 MID.sub.50 4.4 rDEN 1/4 3.8 20 0 0 (CME) MID.sub.50 4.8 MID.sub.50 4.8 rDEN1/430 2.8 20 0 0 (CME) MID.sub.50 3.8 MID.sub.50 3.8 .sup.aAmount of virus ingested, assuming a 2 bloodmeal. .sup.bNumber (percentage) of mosquitoes with detectable dengue virus in midgut tissue; mosquitoes were assayed 21 days post feed, and dengue virus antigen was identified by IFA. .sup.cWhen infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MID.sub.50 was estimated by assuming that a 10-fold more concentrated virus dose would infect 100% of the mosquitoes. .sup.dWhen no infection was detected, the MID.sub.50 was assumed to be greater than a 10-fold higher dose of virus than the one used. .sup.eNumber (percentage) of mosquitoes with detectable dengue virus antigen in both midgut and head tissue.
(170) As described in the previous examples, SCID mice transplanted with the HuH-7 cells are a sensitive model for the evaluation of dengue virus attenuation. Each chimeric DEN1/4 virus was inoculated into groups of SCID-HuH-7 mice and levels of virus in the serum were determined (Table 40). Chimeric viruses replicated to levels between 15- and 250-fold lower than either of the parental viruses, rDEN4 and DEN1 (Puerto Rico/94). CME chimeric viruses were more attenuated than the comparable ME chimeric viruses, with the 30 mutation providing a 0.8 log.sub.10 reduction in replication. This level of attenuation exerted by the 30 mutation in the CME chimeric viruses was similar to that observed previously for rDEN430. However, the attenuating effect of the 30 mutation in the ME chimeric viruses is indiscernible.
(171) TABLE-US-00043 TABLE 40 Chimerization between dengue virus types 1 and 4 results in recombinant viruses which are attenuated for HuH-7-SCID mice. No. of Mean peak virus titer Statistical Virus.sup.a mice (log.sub.10 pfu/ml SE) group.sup.b rDEN4 32 6.3 0.2 A DEN1 4 6.4 0.2 A (Puerto Rico/94) rDEN1/4 (CME) 8 4.7 0.2 B, C rDEN1/430 (CME) 6 3.9 0.4 C rDEN1/4 (ME) 6 5.0 0.2 B rDEN1/430 (ME) 6 5.1 0.3 B .sup.aGroups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log.sub.10 PFU of the indicated virus. Serum was collected on day 7 and virus titer was determined in Vero cells. .sup.bMean peak titers were assigned to statistical groups using the Tukey post-hoc test (P < 0.05). Groups with the same letter designation are not significantly different.
(172) TABLE-US-00044 APPENDIX1 NucleotideandaminoacidsequenceofDEN2(Tonga/74)cDNAplasmidp2 102030405060708090 100 AGTTGTTAGTCTACGTGGACCGACAAAGACAGATTCTTTGAGGGAGCTAAGCTCAACGTAGTTCTAACTGTTTTTTGATTAGAGAGCAGATCTCTGATGA Met> 110120130140150160170180190 200 ATAACCAACGGAAAAAGGCGAGAAACACGCCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTGTCAACTGTACAACAGTTGACAAAGAGATTCTCACT AsnAsnGlnArgLysLysAlaArgAsnThrProPheAsnMetLeuLysArgGluArgAsnArgValSerThrValGlnGlnLeuThrLysArgPheSerLeu> 210220230240250260270280290 300 TGGAATGCTGCAGGGACGAGGACCACTAAAATTGTTCATGGCCCTGGTGGCATTCCTTCGTTTCCTAACAATCCCACCAACAGCAGGGATATTAAAAAGA GlyMetLeuGlnGlyArgGlyProLeuLysLeuPheMetAlaLeuValAlaPheLeuArgPheLeuThrIleProProThrAlaGlyIleLeuLysArg> 310320330340350360370380390 400 TGGGGAACAATTAAAAAATCAAAGGCTATTAATGTTCTGAGAGGCTTCAGGAAGAGATTTGGAAGGATGCTGAATATCTTAAACAGGAGACGTAGAACTG TrpGlyThrIleLysLysSerLysAlaIleAsnValLeuArgGlyPheArgLysGluIleGlyArgMetLeuAsnIleLeuAsnArgARgArgArgThr> 410420430440450460470480490 500 TAGGCATGATCATCATGCTGACTCCAACAGTGATGGCGTTTCATCTGACCACACGCAACGGAGAACCACACATGATTGTCAGTAGACAAGAAAAAGGGAA ValGlyMetIleIleMetLeuThrProThrValMetAlaPheHisLeuThrThrArgAsnGlyGluProHisMetIleValSerArgGlnGluLysGlyLys> 510520530540550560570580590 600 AAGCCTTCTGTTCAAGACAAAGGATGGCACGAACATGTGTACCCTCATGGCCATGGACCTTGGTGAGTTGTGTGAAGACACAATCACGTATAAATGTCCT SerLeuLeuPheLysThrLysAspGlyThrAsnMetCysThrLeuMetAlaMetAspLeuGlyGluLeuCysGluAspThrIleThrTyrLysCysPro> 610620630640650660670680690 700 TTTCTCAAGCAGAACGAACCAGAAGACATAGATTGTTGGTGCAACTCCACGTCCACATGGGTAACTTATGGGACATGTACCACCACAGGAGAGCACAGAA PheLeuLysGlnAsnGluProGluAspIleAspCysTrpCysAsnSerThrSerThrTrpValThrTyrGlyThrCysThrThrThrGlyGluHisArg> 710720730740750760770780790 800 GAGAAAAAAGATCAGTGGCGCTTGTTCCACACGTGGGAATGGGATTGGAGACACGAACTGAAACATGGATCTCATCAGAAGGGGCCTGGAAACATGCCCA ArgGluLysArgSerValAlaLeuValProHisValGlyMetGlyLeuGluThrArgThrGluThrTrpMetSerSerGluGlyAlaTrpLysHisAlaGln> 810820830840850860870880890 900 GAGAATTGAAACTTGGATTCTGAGACATCCAGGCTTTACCATAATGGCCGCAATCCTGGCATACACCATAGGGACGACGCATTTCCAAAGAGTCCTGATA ArgIleGluThrTrpIleLeuArgHisProGlyPheThrIleMetAlaAlaIleLeuAlaTyrThrIleGlyThrThrHisPheGlnArgValLeuIle> 910920930940950960970980990 1000 TTCATCCTACTGACAGCCATCGCTCCTTCAATGACAATGCGCTGCATAGGAATATCAAATAGGGACTTTGTGGAAGGAGTGTCAGGAGGGAGTTGGGTTG PheIleLeuLeuThrAlaIleAlaProSerMetThrMetArgCysIleGlyIleSerAsnArgAspPheValGluGlyValSerGlyGlySerTrpVal> 101010201030104010501060107010801090 1100 ACATAGTTTTAGAACATGGAAGTTGTGTGACGACGATGGCAAAAAACAAACCAACACTGGACTTTGAACTGATAAAAACAGAAGCCAAACAACCTGCCAC AspIleValLeuGluHisGlySerCysValThrThrMetAlaLysAsnLysProThrLeuAspPheGluLeuIleLysThrGluAlaLysGlnProAlaThr> 111011201130114011501160117011801190 1200 CTTAAGGAAGTACTGTATAGAGGCCAAACTGACCAACACGACAACAGACTCGCGCTGCCCAACACAAGGGGAACCCACCCTGAATGAAGAGCAGGACAAA LeuArgLysTyrCysIleGluAlaLysLeuThrAsnThrThrThrAspSerArgCysProThrGlnGlyGluProThrLeuAsnGluGluGlnAspLys> 121012201230124012501260127012801290 1300 AGGTTTGTCTGCAAACATTCCATGGTAGACAGAGGATGGGGAAATGGATGTGGATTGTTTGGAAAAGGAGGCATCGTGACCTGTGCTATGTTCACATGCA ArgPheValCysLysHisSerMetValAspArgGlyTrpGlyAsnGlyCysGlyLeuPheGlyLysGlyGlyIleValThrCysAlaMetPheThrCys> 131013201330134013501360137013801390 1400 AAAAGAACATGGAAGGAAAAATTGTTCAGCCAGAAAACCTGGAATACACTGTCGTGATAACACCTCATTCAGGGGAAGAACATGCAGTGGGAAATGACAC LysLysAsnMetGluGlyLysIleValGlnProGluAsnLeuGluTyrThrValValIleThrProHisSerGlyGluGluHisAlaValGlyAsnAspThr> 141014201430144014501460147014801490 1500 AGGAAAACATGGTAAAGAAGTCAAGATAACACCACAGAGCTCCATCACAGAGGCGGAACTGACAGGCTATGGCACTGTTACGATGGAGTGCTCTCCAAGA GlyLysHisGlyLysGluValLysIleThrProGlnSerSerIleThrGluAlaGluLeuThrGlyTyrGlyThrValThrMetGluCysSerProArg> 151015201530154015501560157015801590 1600 ACGGGCCTCGACTTCAATGAGATGGTGTTGCTGCAAATGGAAGACAAAGCCTGGCTGGTGCACAGACAATGGTTCCTAGACCTACCGTTGCCATGGCTGC ThrGlyLeuAspPheAsnGluMetValLeuLeuGlnMetGluAspLysAlaTrpLeuValHisArgGlnTrpPheLeuAspLeuProLeuProTrpLeu> 161016201630164016501660167016801690 1700 CCGGAGCAGACACACAAGGATCAAATTGGATACAGAAAGAAACACTGGTCACCTTCAAAAATCCCCATGCGAAAAAACAGGATGTTGTTGTCTTAGGATC ProGlyAlaAspThrGlnGlySerAsnTrplleGlnLysGluThrLeuValThrPheLysAsnProHisAlaLysLysGlnAspValValValLeuGlySer> 171017201730174017501760177017801790 1800 CCAAGAGGGGGCCATGCATACAGCACTCACAGGGGCTACGGAAATCCAGATGTCATCAGGAAACCTGCTGTTCACAGGACATCTCAAGTGCAGGCTGAGA GlnGluGlyAlaMetHisThrAlaLeuThrGlyAlaThrGluIleGlnMetSerSerGlyAsnLeuLeuPheThrGlyHisLeuLysCysArgLeuArg> 181018201830184018501860187018801890 1900 ATGGACAAATTACAACTTAAAGGGATGTCATACTCCATGTGCACAGGAAAGTTTAAAATTGTGAAGGAAATAGCAGAAACACAACATGGAACAATAGTCA MetAspLysLeuGlnLeuLysGlyMetSerTyrSerMetCysThrGlyLysPheLysIleValLysGluIleAlaGluThrGlnHisGlyThrIleVal> 191019201930194019501960197019801990 2000 TTAGAGTACAATATGAAGGAGACGGCTCTCCATGCAAGATCCCCTTTGAGATAATGGATCTGGAAAAAAGACATGTTTTGGGCCGCCTGATCACAGTCAA IleArgValGlnTyrGluGlyAspGlySerProCysLysIleProPheGluIleMetAspLeuGluLysArgHisValLeuGlyArgLeuIleThrValAsn> 201020202030204020502060207020802090 2100 CCCAATTGTAACAGAAAAGGACAGTCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATAGGAGTGGAACCAGGACAATTGAAG ProIleValThrGluLysAspSerProValAsnIleGluAlaGluProProPheGlyAspSerTyrIleIleIleGlyValGluProGlyGlnLeuLys> 211021202130214021502160217021802190 2200 CTGGACTGGTTCAAGAAAGGAAGTTCCATCGGCCAAATGTTTGAGACAACAATGAGGGGAGCGAAAAGAATGGCCATTTTGGGTGACACAGCCTGGGATT LeuAspTrpPheLysLysGlySerSerIleGlyGlnMetPheGluThrThrMetArgGlyAlaLysArgMetAlaIleLeuGlyAspThrAlaTrpAsp> 221022202230224022502260227022802290 2300 TTGGATCTCTGGGAGGAGTGTTCACATCAATAGGAAAGGCTCTCCACCAGGTTTTTGGAGCAATCTACGGGGCTGCTTTCAGTGGGGTCTCATGGACTAT PheGlySerLeuGlyGlyValPheThrSerIleGlyLysAlaLeuHisGlnValPheGlyAlaIleTyrGlyAlaAlaPheSerGlyValSerTrpThrMet> 231023202330234023502360237023802390 2400 GAAGATCCTCATAGGAGTTATCATCACATGGATAGGAATGAACTCACGTAGCACTAGTCTGAGCGTGTCACTGGTGTTAGTGGGAATCGTGACACTTTAC LysIleLeuIleGlyValIleIleThrTrpIleGlyMetAsnSerArgSerThrSerLeuSerValSerLeuValLeuValGlyIleValThrLeuTyr> 241024202430244024502460247024802490 2500 TTGGGAGTTATGGTGCAGGCCGATAGTGGTTGCGTTGTGAGCTGGAAGAACAAAGAACTAAAATGTGGCAGTGGAATATTCGTCACAGATAACGTGCATA LeuGlyValMetValGlnAlaAspSerGlyCysValValSerTrpLysAsnLysGluLeuLysCysGlySerGlyIlePheValThrAspAsnValHis> 251025202530254025502560257025802590 2600 CATGGACAGAACAATACAAGTTCCAACCAGAATCCCCTTCAAAACTGGCCTCAGCCATCCAGAAAGCGCATGAAGAGGGCATCTGTGGAATCCGCTCAGT ThrTrpThrGluGlnTyrLysPheGlnProGluSerProSerLysLeuAlaSerAlaIleGlnLysAlaHisGluGluGlyIleCysGlyIleArgSerVal> 261026202630264026502660267026802690 2700 AACAAGACTGGAAAATCTTATGTGGAAACAGATAACATCAGAATTGAATCATATTCTATCAGAAAATGAAGTGAAACTGACCATCATGACAGGAGACATC ThrArgLeuGluAsnLeuMetTrpLysGlnIleThrSerGluLeuAsnHisIleLeuSerGluAsnGluValLysLeuThrIleMetThrGlyAspIle> 271027202730274027502760277027802790 2800 AAAGGAATCATGCAGGTAGGAAAACGATCTTTGCGGCCTCAACCCACTGAGTTGAGGTATTCATGGAAAACATGGGGTAAAGCGAAAATGCTCTCCACAG LysGlyIleMetGlnValGlyLysArgSerLeuArgProGlnProThrGluLeuArgTyrserTrpLysThrTrpGlyLysAlaLysMetLeuSerThr> 281028202830284028502860287028802890 2900 AACTCCACAATCAGACCTTCCTCATTGATGGTCCCGAAACAGCAGAATGCCCCAACACAAACAGAGCTTGGAATTCACTGGAAGTTGAGGACTACGGCTT GluLeuHisAsnGlnThrPheLeuIleAspGlyProGluThrAlaGluCysProAsnThrAsnArgAlaTrpAsnSerLeuGluValGluAspTyrGlyPhe> 291029202930294029502960297029802990 3000 TGGAGTATTCACTACCAATATATGGCTAAGATTGAGAGAAAAGCAGGATGTATTTTGTGACTCAAACTCATGTGCAGCGGCCATAAAGGACAACAGAGCC GlyValPheThrThrAsnIleTrpLeuArgLeuArgGluLysGlnAspValPheCysAspSerLysLeuMetSreAlaAlaIleLysAspAsnArgAla> 301030203030304030503060307030803090 3100 GTCCATGCTGATATGGGTTATTGGATAGAAAGCGCACTCAATGATACATGGAAGATAGAGAAAGCTTCTTTCATTGAAGTCAAAAGTTGCCACTGGCCAA ValHisAlaAspMetGlyTryTrpIleGluSerAlaLeuAsnAspThrTrpLysIleGluLysAlaSerPheIleGluValLysSerCysHisTrpPro> 311031203130314031503160317031803190 3200 AGTCACACACCCTATGGAGTAATGGAGTGCTAGAAAGCGAGATGGTCATTCCAAAGAATTTCGCTGGACCAGTGTCACAACATAATAACAGACCAGGCTA LysSerHisThrLeuTrpSerAsnGlyValLeuGluSerGluMetValIleProLysAsnPheAlaGlyProValSerGlnHisAsnAsnArgProGlyTyr> 321032203230324032503260327032803290 3300 TTACACACAAACAGCAGGACCTTGGCATCTAGGCAAGCTTGAGATGGACTTTGATTTCTGCGAAGGGACTACAGTGGTGGTAACCGAGAACTGTGGAAAC TyrThrGlnThrAlaGlyProTrpHisLeuGlyLysLeuGluMetAspPheAspPheCysGluGlyThrThrValValValThrGluAsnCysGlyAsn> 331033203330334033503360337033803390 3400 AGAGGGCCCTCTTTAAGAACAACCACTGCCTCAGGAAAACTCATAACGGAATGGTGTTGTCGATCTTGCACACTACCACCACTAAGATACAGAGGTGAGG ArgGlyProSerLeuArgThrThrThrAlaSerGlyLysLeuIleThrGluTrpCysCysArgSerCysThrLeuProProLeuArgTyrArgGlyGlu> 341034203430344034503460347034803490 3500 ATGGATGTTGGTACGGGATGGAAATCAGACCATTGAAAGAGAAAGAAGAAAATCTGGTCAGTTCTCTGGTTACAGCCGGACATGGGCAGATTGACAATTT AspGlyCysTrpTyrGlyMetGluIleArgProLeuLysGluLysGluGluAsnLeuValSerSerLeuValThrAlaGlyHisGlyGlnIleAspAsnPhe> 351035203530354035503560357035803590 3600 CTCATTAGGAATCTTGGGAATGGCACTGTTCCTTGAAGAAATGCTCAGGACTCGAGTAGGAACAAAACATGCAATATTACTCGTCGCAGTTTCTTTCGTG SerLeuGlyIleLeuGlyMetAlaLeuPheLeuGluGluMetLeuArgThrArgValGlyThrLysHisAlaIleLeuLeuValAlaValSerPheVal> 361036203630364036503660367036803690 3700 ACGCTAATCACAGGGAACATGTCTTTTAGAGACCTGGGAAGAGTGATGGTTATGGTGGGTGCCACCATGACAGATGACATAGGCATGGGTGTGACTTATC ThrLeuIleThrGlyAsnMetSerPheArgAspLeuGlyArgValMetValMetValGlyAlaThrMetThrAspAspIleGlyMetGlyValThrTyr> 371037203730374037503760377037803790 3800 TCGCTCTACTAGCAGCTTTTAGAGTCAGACCAACCTTTGCAGCTGGACTGCTCTTGAGAAAACTGACCTCCAAGGAATTAATGATGACTACCATAGGAAT LeuAlaLeuLeuAlaAlaPheArgValArgProThrPheAlaAlaGlyLeuLeuLeuArgLysLeuThrSerLysGluLeuMetMetThrThrIleGlyIle> 381038203830384038503860387038803890 3900 CGTTCTTCTCTCCCAGAGTAGCATACCAGAGACCATTCTTGAACTGACCGACGCGTTAGCTCTAGGCATGATGGTCCTCAAGATGGTGAGAAACATGGAA ValLeuLeuSerGlnSerSerIleProGluThrIleLeuGluLeuThrAspAlaLeuAlaLeuGlyMetMetValLeuLysMetValArgAsnMetGlu> 391039203930394039503960397039803990 4000 AAATATCAGCTGGCAGTGACCATCATGGCTATTTTGTGCGTCCCAAATGCTGTGATATTACAGAACGCATGGAAAGTGAGTTGCACAATATTGGCAGTGG LysTyrGlnLeuAlaValThrIleMetAlaIleLeuCysValProAsnAlaValIleLeuGlnAsnAlaTrpLysValSerCysThrIleLeuAlaVal> 401040204030404040504060407040804090 4100 TGTCTGTTTCCCCCCTGCTCTTAACATCCTCACAACAGAAAGCGGACTGGATACCATTAGCGTTGACGATCAAAGGTCTTAATCCAACAGCCATTTTTCT ValSerValSerProLeuLeuLeuThrSerSerGlnGlnLysAlaAspTrpIleProLeuAlaLeuThrIleLysGlyLeuAsnProThrAlaIlePheLeu> 411041204130414041504160417041804190 4200 AACAACCCTCTCAAGAACCAACAAGAAAAGGAGCTGGCCTTTAAATGAGGCCATCATGGCGGTTGGGATGGTGAGTATCTTGGCCAGCTCTCTCTTAAAG ThrThrLeuSerArgThrAsnLysLysArgSerTrpProLeuAsnGluAlaIleMetAlaValGlyMetValSerIleLeuAlaSerSerLeuLeuLys> 421042204230424042504260427042804290 4300 AATGACATCCCCATGACAGGACCATTAGTGGCTGGAGGGCTCCTTACTGTGTGCTACGTGCTAACTGGGCGGTCAGCCGATCTGGAATTAGAGAGAGCTA AsnAspIleProMetThrGlyProLeuValAlaGlyGlyLeuLeuThrValCysTyrValLeuThrGlyArgSerAlaAspLeuGluLeuGluArgAla> 431043204330434043504360437043804390 4400 CCGATGTCAAATGGGATGACCAGGCAGAGATATCAGGTAGCAGTCCAATCCTGTCAAGAACAATATCAGAAGATGGCAGCATGTCAATAAAGAATGAAGA ThrAspValLysTrpAspAspGlnAlaGluIleSerGlySerSerProIleLeuSerIleThrIleSerGluAspGlySerMetSerlleLysAsnGluGlu> 441044204430444044504460447044804490 4500 GGAAGAGCAAACACTGACTATACTCATTAGAACAGGATTGCTTGTGATCTCAGGACTCTTTCCGGTATCAATACCAATTACAGCAGCAGCATGGTATCTG GluGluGlnThrLeuTheIleLeuIleArgThrGlyLeuLeuValIleSerGlyLeuPheProValSerIleProIleThrAlaAlaAlaTrpTyrLeu> 451045204530454045504560457045804590 4600 TGGGAAGTAAAGAAACAACGGGCTGGAGTGCTGTGGGATGTCCCCTCACCACCACCCGTGGGAAAAGCTGAATTGGAAGATGGAGCCTACAGAATCAAGC TrpGluValLysLysGlnArgAlaGlyValLeuTrpAspValProSerProProProValGlyLysAlaGluLeuGluAspGlyAlaTyrArgIleLys> 461046204630464046504660467046804690 4700 AAAAAGGAATCCTTGGATATTCCCAGATCGGAGCTGGAGTTTACAAAGAAGGAACATTTCACACAATGTGGCACGTCACACGTGGCGCTGTCCTAATGCA GlnLysGlyIleLeuGlyTyrSerGlnIleGlyAlaGlyValTyrLysGluGlyThrPheHisThrMetTrpHisValThrArgGlyAlaValLeuMetHis> 471047204730474047504760477047804790 4800 TAAGGGGAAGAGGATTGAACCATCATGGGCGGACGTCAAGAAAGACTTAATATCATATGGAGGAGGTTGGAAGCTAGAAGGAGAATGGAAAGAAGGAGAA LysGlyLysArgIleGluProSerTrpAlaAspValLysLysAspLeuIleSerTyrGlyGlyGlyTrpLysLeuGluGlyGluTrpLysGluGlyGlu> 481048204830484048504860487048804890 4900 GAAGTCCAGGTCTTGGCATTGGAGCCAGGGAAAAATCCAAGAGCCGTCCAAACAAAGCCTGGCCTTTTTAGAACCAACACTGGAACCATAGGTGCCGTAT GluValGlnValLeuAlaLeuGluProGlyLysAsnProArgAlaValGlnThrLysProGlyLeuPheArgThrAsnThrGlyThrIleGlyAlaVal> 491049204930494049504960497049804990 5000 CTCTGGACTTTTCCCCTGGGACGTCAGGATCTCCAATCGTCGACAAAAAAGGAAAAGTTGTAGGTCTCTATGGCAATGGTGTCGTTACAAGGAGTGGAGC SerLeuAspPheSerProGlyThrSerGlySerProIleValAspLysLysGlyLysValValGlyLeuTyrGlyAsnGlyValValThrArgSerGlyAla> 501050205030504050505060507050805090 5100 ATATGTGAGTGCCATAGCTCAGACTGAAAAAAGCATTGAAGACAATCCAGAGATTGAAGATGACATCTTTCGAAAGAGAAGATTGACTATCATGGATCTC TyrValSerAlaIleAlaGlnThrGluLysSerIleGluAspAsnProGluIleGluAspAspIlePheArgLysArgArgLeuThrIleMetAspLeu> 511051205130514051505160517051805190 5200 CACCCAGGAGCAGGAAAGACAAAGAGATACCTCCCGGCCATAGTCAGAGAGGCCATAAAAAGAGGCTTGAGAACACTAATCCTAGCCCCCACTAGAGTCG HisProGlyAlaGlyLysThrLysArgTyrLeuProAlaIleValArgGluAlaIleLysArgGlyLeuArgThrLeuIleLeuAlaProThrArgVal> 521052205230524052505260527052805290 5300 TGGCAGCTGAAATGGAGGAAGCCCTTAGAGGACTTCCAATAAGATACCAAACTCCAGCTATCAGGGCTGAGCACACCGGGCGGGAGATTGTAGACTTAAT ValAlaAlaGluMetGluGluAlaLeuArgGlyLeuProIleArgTyrGlnThrProAlaIleArgAlaGluHisThrGlyArgGluIleValAspLeuMet> 531053205330534053505360537053805390 5400 GTGTCATGCCACATTTACCATGAGGCTGCTATCACCAATCAGGGTGCCAAATTACAACCTGATCATCATGGACGAAGCCCATTTTACAGATCCAGCAAGC CysHisAlaThrPheThrMetArgLeuLeuSerProIleArgValProAsnTyrAsnLeuIleIleMetAspGluAlaHisPheThrAspProAlaSer> 541054205430544054505460547054805490 5500 ATAGCAGCTAGGGGATACATCTCAACTCGAGTGGAGATGGGGGAGGCAGCTGGAATTTTTATGACAGCCACTCCTCCGGGTAGTAGAGATCCATTTCCTC IleAlaAlaArgGlyTyrIleSerThrArgValGluMetGlyGluAlaAlaGlyIlePheMetThrAlaThrProProGlySerArgAspProPhePro> 551055205530554055505560557055805590 5600 AGAGCAATGCACCAATTATGGACGAAGAAAGAGAAATTCCGGAACGTTCATGGAACTCTGGGCACGAGTGGGTCACGGATTTTAAAGGAAAGACTGTCTG GlnSerAsnAlaProIleMetAspGluGluArgGluIleProGluArgSerTrpAsnSerGlyHisGluTrpValThrAspPheLysGlyLysThrValTrp> 561056205630564056505660567056805690 5700 GTTTGTTCCAAGCATAAAAACCGGAAATGACATAGCAGCCTGCCTGAGAAAGAATGGAAAGAGGGTGATACAACTCAGTAGGAAGACCTTTGATTCTGAA PheValProSerIleLysThrGlyAsnAspIleAlaAlaCysLeuArgLysAsnGlyLysArgValIleGlnLeuSerArgLysThrPheAspSerGlu> 571057205730574057505760577057805790 5800 TATGTCAAGACTAGAACCAATGACTGGGATTTCGTGGTTACAACTGACATCTCGGAAATGGGCGCCAACTTTAAAGCTGAGAGGGTCATAGACCCCAGAC TyrValLysThrArgThrAsnAspTrpAspPheValValThrThrAspIleSerGluMetGlyAlaAsnPheLysAlaGluArgValIleAspProArg> 581058205830584058505860587058805890 5900 GCTGCATGAAACCAGTTATATTGACAGACGGCGAAGAGCGGGTGATTCTGGCAGGACCCATGCCAGTGACCCACTCTAGTGCAGCACAAAGAAGAGGGAG ArgCysMetLysProValIleLeuThrAspGlyGluGluArgValIleLeuAlaGluProMetProValThrHisSerSerAlaAlaGlnArgArgGlyArg> 591059205930594059505960597059805990 6000 AATAGGAAGGAATCCAAGGAATGAAAATGATCAATATATATATATGGGGGAACCACTGGAAAATGATGAAGACTGTGCGCACTGGAAGGAAGCTAAGATG IleGlyArgAsnProArgAsnGluAsnAspGlnTyrIleTyrMetGlyGluProLeuGluAsnAspGluAspCysAlaHisTrpLysGluAlaLysMet> 601060206030604060506060607060806090 6100 CTCCTAGATAATATCAACACACCTGAAGGAATCATTCCCAGCTTGTTCGAGCCAGAGCGTGAAAAGGTGGATGCCATTGACGGTGAATATCGCTTGAGAG LeuLeuAspAsnIleAsnThrProGluGlyIleIleProSerLeuPheGluProGluArgGluLysValAspAlaIleAspGlyGluTyrArgLeuArg> 611061206130614061506160617061806190 6200 GAGAAGCACGGAAAACTTTTGTGGACCTAATGAGAAGAGGAGACCTACCAGTCTGGTTGGCTTATAAAGTGGCAGCTGAAGGTATCAACTACGCAGACAG GlyGluAlaArgLysThrPheValAspLeuMetArgArgGlyAspLeuProValTrpLeuAlaTyrLysValAlaAlaGluGlyIleAsnTyrAlaAspArg> 621062206230624062506260627062806290 6300 AAGATGGTGTTTTGACGGAACCAGAAACAATCAAATCTTGGAAGAAAATGTGGAAGTGGAAATCTGGACAAAGGAAGGGGAAAGGAAAAAATTGAAACCT ArgTrpCysPheAspGlyThrArgAsnAsnGlnIleLeuGluGluAsnValGluValGluIleTrpThrLysGluGlyGluArgLysLysLeuLysPro> 631063206330634063506360637063806390 6400 AGATGGTTAGATGCTAGGATCTACTCCGACCCACTGGCGCTAAAAGAGTTCAAGGAATTTGCAGCCGGAAGAAAGTCCCTAACCCTGAACCTAATTACAG ArgTrpLeuAspAlaArgIleTyrSerAspProLeuAlaLeuLysGluPheLysGluPheAlaAlaGlyArgLysSerLeuThrLeuAsnLeuIleThr> 641064206430644064506460647064806490 6500 AGATGGGCAGACTCCCAACTTTTATGACTCAGAAGGCCAGAGATGCACTAGACAACTTGGCGGTGCTGCACACGGCTGAAGCGGGTGGAAAGGCATACAA GluMetGlyArgLeuProThrPheMetThrGlnLysAlaArgAspAlaLeuAspAsnLeuAlaValLeuHisThrAlaGluAlaGlyGlyLysAlaTyrAsn> 651065206530654065506560657065806590 6600 TCATGCTCTCAGTGAATTACCGGAGACCCTGGAGACATTGCTTTTGCTGACACTGTTGGCCACAGTCACGGGAGGAATCTTCCTATTCCTGATGAGCGGA HisAlaLeuSerGluLeuProGluThrLeuGluThrLeuLeuLeuLeuThrLeuLeuAlaThrValThrGlyGlyIlePheLeuPheLeuMetSerGly> 661066206630664066506660667066806690 6700 AGGGGTATGGGGAAGATGACCCTGGGAATGTGCTGCATAATCACGGCCAGCATCCTCTTATGGTATGCACAAATACAGCCACATTGGATAGCAGCCTCAA ArgGlyMetGlyLysMetThrLeuGlyMetCysCysIleIleThrAlaSerIleLeuLeuTryTyrAlaGlnIleGlnProHisTrpIleAlaAlaSer> 671067206730674067506760677067806790 6800 TAATATTGGAGTTCTTTCTCATAGTCTTGCTCATTCCAGAACCAGAAAAGCAGAGGACACCTCAGGATAATCAATTGACTTATGTCATCATAGCCATCCT IleIleLeuGluPhePheLeuIleValLeuLeuIleProGluProGluLysGlnArgThrProGlnAspAsnGlnLeuThrTyrVallleIleAlaIleLeu> 681068206830684068506860687068806890 6900 CACAGTGGTGGCCGCAACCATGGCAAACGAAATGGGTTTTCTGGAAAAAACAAAGAAAGACCTCGGACTGGGAAACATTGCAACTCAGCAACCTGAGAGC ThrValValAlaAlaThrMetAlaAsnGluMetGlyPheLeuGluLysThrLysLysAspLeuGlyLeuGlyAsnIleAlaThrGlnGlnProGluSer> 691069206930694069506960697069806990 7000 AACATTCTGGACATAGATCTACGTCCTGCATCAGCATGGACGTTGTATGCCGTGGCTACAACATTTATCACACCAATGTTGAGACATAGCATTGAAAATT AsnIleLeuAspIleAspLeuArgProAlaSerAlaTrpThrLeuTyrAlaValAlaThrThrPheIleThrProMetLeuArgHisSerIleGluAsn> 701070207030704070507060707070807090 7100 CCTCAGTAAATGTGTCCCTAACAGCCATAGCTAACCAAGCCACAGTGCTAATGGGTCTCGGAAAAGGATGGCCATTGTCAAAGATGGACATTGGAGTTCC SerSerValAsnValSerLeuThrAlaIleAlaAsnGlnAlaThrValLeuMetGlyLeuGlyLysGlyTrpProLeuSerLysMetAspIleGlyValPro> 711071207130714071507160717071807190 7200 CCTCCTTGCTATTGGGTGTTACTCACAAGTCAACCCTATAACCCTCACAGCGGCTCTTCTTTTATTGGTAGCACATTATGCCATCATAGGACCGGGACTT LeuLeuAlaIleGlyCysTyrSerGlnValAsnProIleThrLeuThrAlaAlaLeuLeuLeuLeuValAlaHisTyrAlaIleIleGlyProGlyLeu> 721072207230724072507260727072807290 7300 CAAGCCAAAGCAACTAGAGAAGCTCAGAAAAGAGCAGCAGCGGGCATCATGAAAAACCCAACTGTGGATGGAATAACAGTGATAGATCTAGATCCAATAC GlnAlaLysAlaThrArgGluAlaGlnLysArgAlaAlaAlaGlyIleMetLysAsnProThrValAspGlyIleThrValIleAspLeuAspProIle> 731073207330734073507360737073807390 7400 CCTATGATCCAAAGTTTGAAAAGCAGTTGGGACAAGTAATGCTCCTAGTCCTCTGCGTGACCCAAGTGCTGATGATGAGGACTACGTGGGCTTTGTGTGA ProTyrAspProLysPheGluLysGlnLeuGlyGlnValMetLeuLeuValLeuCysValThrGlnValLeuMetMetArgThrThrTrpAlaLeuCysGlu> 741074207430744074507460747074807490 7500 AGCCTTAACTCTAGCAACTGGACCCGTGTCCACATTGTGGGAAGGAAATCCAGGGAGATTCTGGAACACAACCATTGCAGTGTCAATGGCAAACATCTTT AlaLeuThrLeuAlaThrGlyProValSerThrLeuTrpGluGlyAsnProGlyArgPheTrpAsnThrThrIleAlaValSerMetAlaAsnIlePhe> 751075207530754075507560757075807590 7600 AGAGGGAGTTACCTGGCTGGAGCTGGACTTCTCTTTTCTATCATGAAGAACACAACCAGCACGAGAAGAGGAACTGGCAATATAGGAGAAACGTTAGGAG ArgGlySerTyrLeuAlaGlyAlaGlyLeuLeuPheSerIleMetLysAsnThrThrSerThrArgArgGlyThrGlyAsnIleGlyGluThrLeuGly> 761076207630764076507660767076807690 7700 AGAAATGGAAAAGCAGACTGAACGCATTGGGGAAAAGTGAATTCCAGATCTACAAAAAAAGTGGAATTCAAGAAGTGGACAGAACCTTAGCAAAAGAAGG GluLysTrpLysSerArgLeuAsnAlaLeuGlyLysSerGluPheGlnIleTyrLysLysSerGlyIleGlnGluValAspArgThrLeuAlaLysGluGly> 771077207730774077507760777077807790 7800 CATTAAAAGAGGAGAAACGGATCATCACGCTGTGTCGCGAGGCTCAGCAAAACTGAGATGGTTCGTTGAAAGGAATTTGGTCACACCAGAAGGGAAAGTA IleLysArgGlyGluThrAspHisHisAlaValSerArgGlySerAlaLysLeuArgTrpPheValGluArgAsnLeuValThrProGluGlyLysVal> 781078207830784078507860787078807890 7900 GTGGACCTTGGTTGTGGCAGAGGGGGCTGGTCATACTATTGTGGAGGATTAAAGAATGTAAGAGAAGTTAAAGGCTTAACAAAAGGAGGACCAGGACACG ValAspLeuGlyCysGlyArgGlyGlyTrpSerTyrTyrCysGlyGlyLeuLysAsnValArgGluValLysGlyLeuThrLysGlyGlyProGlyHis> 791079207930794079507960797079807990 8000 AAGAACCTATCCCTATGTCAACATATGGGTGGAATCTAGTACGCTTACAGAGCGGAGTTGATGTTTTTTTTGTTCCACCAGAGAAGTGTGACACATTGTT GluGluProIleProMetSerThrTyrGlyTrpAsnLeuValArgLeuGlnSerGlyValAspValPhePheValProProGluLysCysAspThrLeuLeu> 801080208030804080508060807080808090 8100 GTGTGACATAGGGGAATCATCACCAAATCCCACGGTAGAAGCGGGACGAACACTCAGAGTCCTCAACCTAGTGGAAAATTGGCTGAACAATAACACCCAA CysAspIleGlyGluSerSerProAsnProThrValGluAlaGlyArgThrLeuArgValLeuAsnLeuValGluAsnTrpLeuAsnAsnAsnThrGln> 811081208130814081508160817081808190 8200 TTTTGCGTAAAGGTTCTTAACCCGTACATGCCCTCAGTCATTGAAAGAATGGAAACCTTACAACGGAAATACGGAGGAGCCTTGGTGAGAAATCCACTCT PheCysValLysValLeuAsnProTyrMetProSerValIleGluArgMetGluThrLeuGlnArgLysTyrGlyGlyAlaLeuValArgAsnProLeu> 821082208230824082508260827082808290 8300 CACGGAATTCCACACATGAGATGTACTGGGTGTCCAATGCTTCCGGGAACATAGTGTCATCAGTGAACATGATTTCAAGAATGCTGATCAACAGATTCAC SerArgAsnSerThrHisGluMetTyrTrpValSerAsnAlaSerGlyAsnIleValSerSerValAsnMetIleSerArgMetLeuIleAsnArgPheThr> 831083208330834083508360837083808390 8400 TATGAGACACAAGAAGGCCACCTATGAGCCAGATGTCGACCTCGGAAGCGGAACCCGCAATATTGGAATTGAAAGTGAGACACCGAACCTAGACATAATT MetArgHisLysLysAlaThrTyrGluProAspValAspLeuGlySerGlyThrArgAsnIleGlyIleGluSerGluThrProAsnLeuAspIleIle> 841084208430844084508460847084808490 8500 GGGAAAAGAATAGAAAAAATAAAACAAGAGCATGAAACGTCATGGCACTATGATCAAGACCACCCATACAAAACATGGGCTTACCATGGCAGCTATGAAA GlyLysArgIleGluLysIleLysGlnGluHisGluThrSerTrpHisTyrAspGlnAspHisProTyrLysThrTrpAlaTyrHisGlySerTyrGlu> 851085208530854085508560857085808590 8600 CAAAACAGACTGGATCAGCATCATCCATGGTGAACGGAGTAGTCAGATTGCTGACAAAACCCTGGGACGTTGTTCCAATGGTGACACAGATGGCAATGAC ThrLysGlnThrGlySerAlaSerSerMetValAsnGlyValValArgLeuLeuThrLysProTrpAspValValProMetValThrGlnMetAlaMetThr> 861086208630864086508660867086808690 8700 AGACACAACTCCTTTTGGACAACAGCGCGTCTTCAAAGAGAAGGTGGATACGAGAACCCAAGAACCAAAAGAAGGCACAAAAAAACTAATGAAAATCACG AspThrThrPropheGlyGlnGlnArgValPheLysGluLysValAspThrArgThrGlnGluProLysGluGlyThrLysLysLeuMetLysIleThr> 871087208730874087508760877087808790 8800 GCAGAGTGGCTCTGGAAAGAACTAGGAAAGAAAAAGACACCTAGAATGTGTACCAGAGAAGAATTCACAAAAAAGGTGAGAAGCAATGCAGCCTTGGGGG AlaGluTrpLeuTrpLysGluLeuGlyLysLysLysThrProArgMetCysThrArgGluGluPheThrLysLysValArgSerAsnAlaAlaLeuGly> 881088208830884088508860887088808890 8900 CCATATTCACCGATGAGAACAAGTGGAAATCGGCGCGTGAAGCCGTTGAAGATAGTAGGTTTTGGGAGCTGGTTGACAAGGAAAGGAACCTCCATCTTGA AlaIlePheThrAspGluAsnLysTrpLysSerAlaArgGluAlaValGluAspSerArgPheTrpGluLeuValAspLySGluArgAsnLeuHisLeuGlu> 891089208930894089508960897089808990 9000 AGGGAAATGTGAAACATGTGTATACAACATGATGGGGAAAAGAGAGAAAAAACTAGGAGAGTTTGGTAAAGCAAAAGGCAGCAGAGCCATATGGTACATG GlyLysCysGluThrCysValTyrAsnMetMetGlyLysArgGluLysLysLeuGlyGluPheGlyLysAlaLysGlySerArgAlaIleTrpTyrMet> 901090209030904090509060907090809090 9100 TGGCTCGGAGCACGCTTCTTAGAGTTTGAAGCCCTAGGATTTTTGAATGAAGACCATTGGTTCTCCAGAGAGAACTCCCTGAGTGGAGTGGAAGGAGAAG TrpLeuGlyAlaArgPheLeuGluPheGluAlaLeuGlyPheLeuAsnGluAspHisTrpPheSerArgGluAsnSerLeuSerGlyValGluGlyGlu> 911091209130914091509160917091809190 9200 GGCTGCATAAGCTAGGTTACATCTTAAGAGAGGTGAGCAAGAAAGAAGGAGGAGCAATGTATGCCGATGACACCGCAGGCTGGGACACAAGAATCACAAT GlyLeuHisLysLeuGlyTyrIleLeuArgGluValSerLysLysGluGlyGlyAlaMetTyrAlaAspAspThrAlaGlyTrpAspThrArgIleThrIle> 921092209230924092509260927092809290 9300 AGAGGATTTGAAAAATGAAGAAATGATAACGAACCACATGGCAGGAGAACACAAGAAACTTGCCGAGGCCATTTTTAAATTGACGTACCAAAACAAGGTG GluAspLeuLysAsnGluGluMetIleThrAsnHisMetAlaGlyGluHisLysLysLeuAlaGluAlaIlePheLysLeuThrTyrGlnAsnLysVal> 931093209330934093509360937093809390 9400 GTGCGTGTGCAAAGACCAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGGGGTAGTGGACAAGTTGGCACCTATGGCCTCAACA ValArgValGlnArgProThrProArgGlyThrValMetAspIleIleSerArgArgAspGlnArgGlySerGlyGlnValGlyThrTyrGlyLeuAsn> 941094209430944094509460947094809490 9500 CTTTCACCAACATGGAAGCACAACTAATTAGGCAAATGGAGGGGGAAGGAATCTTCAAAAGCATCCAGCACTTGACAGCCTCAGAAGAAATCGCTGTGCA ThrPheThrAsnMetGluAlaGlnLeuIleArgGlnMetGluGlyGluGlyIlePheLysSerIleGlnHisLeuThrAlaSerGluGluIleAlaValGln> 951095209530954095509560957095809590 9600 AGATTGGCTAGTAAGAGTAGGGCGTGAAAGGTTGTCAAGAATGGCCATCAGTGGAGATGATTGTGTTGTGAAACCTTTAGATGATAGATTTGCAAGAGCT AspTrpLeuValArgValGlyArgGluArgLeuSerArgMetAlaIleSerGlyAspAspCysValValLysProLeuAspAspArgPheAlaArgAla> 961096209630964096509660967096809690 9700 CTAACAGCTCTAAATGACATGGGAAAGGTTAGGAAGGACATACAGCAATGGGAGCCCTCAAGAGGATGGAACGACTGGACGCAGGTGCCCTTCTGTTCAC LeuThrAlaLeuAsnAspMetGlyLysValArgLysAspIleGlnGlnTrpGluProSerArgGlyTrpAsnAspTrpThrGlnValProPheCysSer> 971097209730974097509760977097809790 9800 ACCATTTTCACGAGTTAATTATGAAAGATGGTCGCACACTCGTAGTTCCATGCAGAAACCAAGATGAATTGATCGGCAGAGCCCGAATTTCCCAGGGAGC HisHisPheHisGluLeuIleMetLysAspGlyArgThrLeuValValProCysArgAsnGlnAspGluLeuIleGlyArgAlaArgIleSerGlnGlyAla> 981098209830984098509860987098809890 9900 TGGGTGGTCTTTACGGGAGACGGCCTGTTTGGGGAAGTCTTACGCCCAAATGTGGAGCTTGATGTACTTCCACAGACGTGATCTCAGGCTAGCGGCAAAT GlyTrpSerLeuArgGluThrAlaCysLeuGlyLysSerTyrAlaGlnMetTrpSerLeuMetTyrPheHisArgArgAspLeuArgLeuAlaAlaAsn> 991099209930994099509960997099809990 10000 GCCATCTGCTCGGCAGTCCCATCACACTGGATTCCAACAAGCCGGACAACCTGGTCCATACACGCCAGCCATGAATGGATGACGACGGAAGACATGTTGA AlaIleCysSerAlaValProSerHisTrpIleProThrSerArgThrThrTrpSerIleHisAlaSerHisGluTrpMetThrThrGluAspMetLeu> 100101002010030100401005010060100701008010090 10100 CAGTTTGGAACAGAGTGTGGATCCTAGAAAATCCATGGATGGAAGACAAAACTCCAGTGGAATCATGGGAGGAAATCCCATACCTGGGAAAAAGAGAAGA ThrValTrpAsnArgValTrpIleLeuGluAsnProTrpMetGluAspLysThrProValGluSerTrpGluGluIleProTyrLeuGlyLysArgGluAsp> 101101012010130101401015010160101701018010190 10200 CCAATGGTGCGGCTCGCTGATTGGGCTGACAAGCAGAGCCACCTGGGCGAAGAATATCCAGACAGCAATAAACCAAGTCAGATCCCTCATTGGCAATGAG GlnTrpCysGlySerLeuIleGlyLeuThrSerArgAlaThrTrpAlaLysAsnIleGlnThrAlaIleAsnGlnValArgSerLeuIleGlyAsnGlu> 102101022010230102401025010260102701028010290 10300 GAATACACAGATTACATGCCATCCATGAAAAGATTCAGAAGAGAAGAGGAAGAGGCAGGAGTTTTGTGGTAGAAAAACATGAAACAAAACAGAAGTCAGG GluTyrThrAspTyrMetProSerMetLysArgPheArgArgGluGluGluGluAlaGlyValLeuTrp***> 103101032010330103401035010360103701038010390 10400 TCGGATTAAGCCATAGTACGGGAAAAACTATGCTACCTGTGAGCCCCGTCCAAGGACGTTAAAAGAAGTCAGGCCATTTTGATGCCATAGCTTGAGCAAA 104101042010430104401045010460104701048010490 10500 CTGTGCAGCCTGTAGCTCCACCTGAGAAGGTGTAAAAAATCCGGGAGGCCACAAACCATGGAAGCTGTACGCATGGCGTAGTGGACTAGCGGTTAGAGGA 105101052010530105401055010560105701058010590 10600 GACCCCTCCCTTACAGATCGCAGCAACAATGGGGGCCCAAGGTGAGATGAAGCTGTAGTCTCACTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCAAAA 106101062010630106401065010660106701068010690 10700 CAAAAAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAGGACGCCAGAAAATGGAATGGTGCTGTTGA 107101072010730107401075010760107701078010790 10800 ATCAACAGGTTCTGGTACCGGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG 108101082010830108401085010860108701088010890 10900 ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA 109101092010930109401095010960109701098010990 11000 CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA 110101102011030110401105011060110701108011090 11100 GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC 111101112011130111401115011160111701118011190 11200 AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA 112101122011230112401125011260112701128011290 11300 AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC 113101132011330113401135011360113701138011390 11400 AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT 114101142011430114401145011460114701148011490 11500 CTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGA 115101152011530115401155011560115701158011590 11600 TAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTC 116101162011630116401165011660116701168011690 11700 ACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGC 117101172011730117401175011760117701178011790 11800 TGCTGGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACT 118101182011830118401185011860118701188011890 11900 TGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCG 119101192011930119401195011960119701198011990 12000 GTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCC 120101202012030120401205012060120701208012090 12100 CCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAAT 121101212012130121401215012160121701218012190 12200 GCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCA 122101222012230122401225012260122701228012290 12300 CTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGA 123101232012330123401235012360123701238012390 12400 TCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTAT 124101242012430124401245012460124701248012490 12500 CGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGC 125101252012530125401255012560125701258012590 12600 ATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTT 126101262012630126401265012660126701268012690 12700 CGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTG 127101272012730127401275012760127701278012790 12800 CCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAG 128101282012830128401285012860128701288012890 12900 CCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTC 129101292012930129401295012960129701298012990 13000 GGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAA 130101302013030130401305013060130701308013090 13100 TCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTC 131101312013130131401315013160131701318013190 13200 TGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAA 132101322013230132401325013260132701328013290 13300 GCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCA 133101332013330133401335013360133701338013390 13400 TCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGG 134101342013430134401345013460134701348013490 13500 AAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTG 135101352013530135401355013560135701358013590 13600 TGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG 136101362013630136401365013660136701368013690 13700 GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCAC 137101372013730137401375013760137701378013790 13800 GTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTA 138101382013830138401385013860138701388013890 13900 AGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG 139101392013930139401395013960139701398013990 14000 CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT 140101402014030140401405014060140701408014090 14100 GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG 141101412014130141401415014160141701418014190 14200 TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC 142101422014230142401425014260142701428014290 14300 ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATC 143101432014330143401435014360143701438014390 14400 CGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT 144101442014430144401445014460144701448014490 14500 GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAATCCAGTTACCTTCGGAAAAAGAG 145101452014530145401455014560145701458014590 14600 TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA 146101462014630146401465014660146701468014690 14700 TCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC 147101472014730147401475014760147701478014790 14800 CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAG 148101482014830148401485014860148701488014890 14900 CGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT 149101492014930149401495014960149701498014990 15000 ACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC 150101502015030150401505015060150701508015090 15100 ATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAAGATCTGGCTAGCGAT 1511015120151301514015150 GACCCTGCTGATTGGTTCGCTGACCATTTCCGGGCGCGCCGATTTAGGTGACACTATAG Bases 1 to 10713: DEN2 virus genome cDNA Bases 97 to 10269: DEN2 polyprotein ORF Bases 97 to 438: C protein ORF Bases 439 to 936: prM protein ORF Bases 937 to 2421: E protein ORF Bases 2422 to 3477: NS1 protein ORF Bases 3478 to 4131: NS2A protein ORF Bases 4132 to 4521: NS2B protein ORF Bases 4522 to 6375: NS3 protein ORF Bases 6376 to 6756: NS4A protein ORF Bases 6757 to 6825: 2K protein ORF Bases 6826 to 7569: NS4B protein ORF Bases 7570 to 10269: NS5 protein ORF
(173) TABLE-US-00045 APPENDIX2 NucleotideandaminoacidsequenceofDEN3(S1eman/78)cDNAplasmidp3 102030405060708090100 AGTTGTTAGTCTACGTGGACCGACAAGAACAGTTTCGACTCGGAAGCTTGCTTAACGTAGTACTGACAGTTTTTTATTAGAGAGCAGATCTCTGATGAACMet Asn> 110120130140150160170180190200 AACCAACGGAAAAAGACGGGAAAACCGTCTATCAATATGCTGAAACGCGTGAGAAACCGTGTGTCAACTGGATCACAGTTGGCGAAGAGATTCTCAAGAG AsnGlnArgLysLysThrGlyLysProSerIleAsnMetLeuLysArgValArgAsnArgValSerThrGlySerGlnLeuAlaLysArgPheSerArg> 210220230240250260270280290300 GACTGCTGAACGGCCAAGGACCAATGAAATTGGTTATGGCGTTCATAGCTTTCCTCAGATTTCTAGCCATTCCACCGACAGCAGGAGTCTTGGCTAGATG GlyLeuLeuAsnGlyGlnGlyProMetLysLeuValMetAlaPheIleAlaPheLeuArgPheLeuAlaIleProProThrAlaGlyValLeuAlaArgTrp> 310320330340350360370380390400 GGGAACCTTTAAGAAGTCGGGGGCTATTAAGGTCCTGAGAGGCTTCAAGAAGGAGATCTCAAACATGCTGAGCATTATCAACAGACGGAAAAAGACATCG GlyThrPheLysLysSerGlyAlaIleLysValLeuArgGlyPheLysLysGluIleSerAsnMetLeuSerIleIleAsnArgArgLysLysThrSer> 410420430440450460470480490500 CTCTGTCTCATGATGATGTTACCAGCAACACTTGCTTTCCACTTGACTTCACGAGATGGAAGCCGCGCATGATTGTGGGGAAGAATGAAAGAGGAAAAT LeuCysLeuMetMetLeuProAlaThrLeuAlaPheHisLeuThrSerArgAspGlyGluProArgMetIleValGlyLysAsnGluArgGlyLys> 510520530540550560570580590600 CCCTACTTTTTAAGACAGCCTCTGGAATCAACATGTGCACACTCATAGCCATGGATTTGGGAGAGATGTGTGATGACACGGTCACCTACAAATGCCCCCT SerLeuLeuPheLysThrAlaSerGlyIleAsnMetCysThrLeuIleAlaMetAspLeuGlyGluMetCysAspAspThrValThrTyrLysCysProLeu> 610620630640650660670680690700 CATTACTGAAGTGGAGCCTGAAGACATTGACTGCTGGTGCAACCTTACATCGACATGGGTGACCTACGGAACGTGCAATCAAGCTGGAGAGCACAGACGC IleThrGluValGluProGluAspIleAspCysTrpCysAsnLeuThrSerThrTrpValThrTyrGlyThrCysAsnGlnAlaGlyGluHisArgArg> 710720730740750760770780790800 GACAAAAGATCGGTGGCGTTAGCTCCCCATGTCGGCATGGGACTGGACACACGCACCCAAACCTGGATGTCGGCTGAAGGAGCTTGGAGACAGGTCGAGA AspLysArgSerValAlaLeuAlaProHisValGlyMetGlyLeuAspThrArgThrGlnThrTrpMetSerAlaGluGlyAlaTrpArgGlnValGlu> 810820830840850860870880890900 AGGTAGAGACATGGGCCTTTAGGCACCCAGGGTTCACAATACTAGCCCTATTTCTTGCCCATTACATAGGCACTTCCTTGACCCAGAAAGTGGTTATTTT LysValGluThrTrpAlaPheArgHisProGlyPheThrIleLeuAlaLeuPheLeuAlaHisTyrIleGlyThrSerLeuThrGlnLysValValIlePhe> 9109209309409509609709809901000 CATACTACTAATGCTGGTCACCCCATCCATGACAATGAGATGCGTGGGAGTAGGAAACAGAGATTTTGTGGAAGGCCTATCAGGAGCTACGTGGGTTGAC IleLeuLeuMetLeuValThrProSerMetThrMetArgCysValGlyValGlyAsnArgAspPheValGluGlyLeuSerGlyAlaThrTrpValAsp> 1010102010301040105010601070108010901100 GTGGTGCTCGAGCACGGTGGGTGTGTGACTACCATGGCTAAGAACAAGCCCACGCTGGATATAGAGCTCCAGAAGACCGAGGCCACCCAACTGGCGACCC ValValLeuGluHisGlyGlyCysValThrThrMetAlaLysAsnLysProThrLeuAspIleGluLeuGlnLysThrGluAlaThrGlnLeuAlaThr> 1110112011301140115011601170118011901200 TAAGGAAACTATGTATTGAGGGAAAAATTACCAACGTAACAACCGACTCAAGGTGCCCCACCCAAGGGGAAGCGATTTTACCTGAGGAGCAGGACCAGAA LeuArgLysLeuCysIleGluGlyLysIleThrAsnValThrThrAspSerArgCysProThrGlnGlyGluAlaIleLeuProGluGluGlnAspGlnAsn> 1210122012301240125012601270128012901300 CCACGTGTGCAAGCACACATACGTGGACAGAGGCTGGGGAAACGGTTGTGGTTTGTTTGGCAAGGGAAGCCTGGTAACATGCGCGAAATTTCAATGTTTG HisValCysLysHisThrTyrValAspArgGlyTrpGlyAsnGlyCysGlyLeuPheGlyLysGlySerLeuValThrCysAlaLysPheGlnCysLeu> 1310132013301340135013601370138013901400 GAATCAATAGAGGGAAAAGTGGTGCAGCATGAGAACCTCAAATACACCGTCATCATCACAGTGCACACAGGAGATCAACACCAGGTGGGAAATGAAACGC GluSerIleGluGlyLysValValGlnHisGluAsnLeuLysTyrThrValIleIleThrValHisThrGlyAspGlnHisGlnValGlyAsnGluThr> 1410142014301440145014601470148014901500 AGGGAGTCACGGCTGAGATAACACCCCAGGCATCAACCGTTGAAGCCATCTTACCTGAATATGGAACCCTTGGGCTAGAATGCTCACCACGGACAGGTTT GlnGlyValThrAlaGluIleThrProGlnAlaSerThrValGluAlaIleLeuProGluTyrGlyThrLeuGlyLeuGluCysSerProArgThrGlyLeu> 1510152015301540155015601570158015901600 AGATTTCAATGAAATGATTTTGTTGACAATGAAGAACAAAGCATGGATGGTACATAGACAATGGTTTTTTGACCTACCTTTACCATGGACATCAGGAGCT AspPheAsnGluMetIleLeuLeuThrMetLysAsnLysAlaTrpMetValHisArgGlnTrpPhePheAspLeuProLeuProTrpThrSerGlyAla> 1610162016301640165016601670168016901700 ACAACAGAAACACCAACCTGGAATAAGAAAGAGCTTCTTGTGACATTCAAAAACGCACATGCAAAAAAGCAAGAAGTAGTAGTCCTTGGATCGCAAGAGG ThrThrGluThrProThrTrpAsnLysLysGluLeuLeuValThrPheLysAsnAlaHisAlaLysLysGlnGluValValValLeuGlySerGlnGlu> 1710172017301740175017601770178017901800 GAGCAATGCACACAGCACTGACAGGAGCTACAGAGATCCAAACCTCAGGAGGCACAAGTATTTTTGCGGGGCACTTAAAATGTAGACTCAAGATGGACAA GlyAlaMetHisThrAlaLeuThrGlyAlaThrGluIleGlnThrSerGlyGlyThrSerIlePheAlaGlyHisLeuLysCysArgLeuLysMetAspLys> 1810182018301840185018601870188018901900 ATTGGAACTCAAGGGGATGAGCTATGCAATGTGCTTGAATGCCTTTGTGTTGAAGAAAGAAGTCTCCGAAACGCAACATGGGACAATACTCATCAAGGTT LeuGluLeuLysGlyMetSerTyrAlaMetCysLeuAsnAlaPheValLeuLysLysGluValSerGluThrGlnHisGlyThrIleLeuIleLysVal> 1910192019301940195019601970198019902000 GAGTACAAAGGGGAAGATGCACCTTGCAAGATTCCTTTCTCCACGGAGGATGGACAAGGGAAAGCCCACAATGGCAGACTGATCACAGCTAACCCAGTGG GluTyrLysGlyGluAspAlaProCysLysIleProPheSerThrGluAspGlyGlnGlyLysAlaHisAsnGlyArgLeuIleThrAlaAsnProVal> 2010202020302040205020602070208020902100 TGACCAAGAAGGAGGAGCCTGTCAATATTGAGGCAGAACCTCCTTTTGGGGAAAGCAATATAGTAATTGGAATTGGAGACAAAGCCTTGAAAATCAACTG ValThrLysLysGluGluProValAsnIleGluAlaGluProProPheGlyGluSerAsnIleValIleGlyIleGlyAspLysAlaLeuLysIleAsnTrp> 2110212021302140215021602170218021902200 GTACAAGAAGGGAAGCTCGATTGGGAAGATGTTCGAGGCCACTGCCAGAGGTGCAAGGCGCATGGCCATCTTGGGAGACACAGCCTGGGACTTTGGATCA TyrLysLysGlySerSerIleGlyLysMetPheGluAlaThrAlaArgGlyAlaArgArgMetAlaIleLeuGlyAspThrAlaTrpAspPheGlySer> 2210222022302240225022602270228022902300 GTAGGTGGTGTTTTAAATTCATTAGGAAAAATGGTGCACCAAATATTTGGAAGTGCTTACACAGCCCTATTTAGTGGAGTCTCCTGGATAATGAAAATTG ValGlyGlyValLeuAsnSerLeuGlyLysMetValHisGlnIlePheGlySerAlaTyrThrAlaLeuPheSerGlyValSerTrpIleMetLysIle> 2310232023302340235023602370238023902400 GAATAGGTGTCCTTTTAACCTGGATAGGGTTGAATTCAAAAAACACTAGTATGAGCTTTAGCTGCATTGTGATAGGAATCATTACACTCTATCTGGGAGC GlyIleGlyValLeuLeuThrTrpIleGlyLeuAsnSerLysAsnThrSerMetSerPheSerCysIleValIleGlyIleIleThrLeuTyrLeuGlyAla> 2410242024302440245024602470248024902500 CGTGGTGCAAGCTGACATGGGGTGTGTCATAAACTGGAAAGGCAAAGAACTCAAATGTGGAAGTGGAATTTTCGTCACTAATGAGGTCCACACCTGGACA ValValGlnAlaAspMetGlyCysValIleAsnTrpLysGlyLysGluLeuLysCysGlySerGlyIlePheValThrAsnGluValHisThrTrpThr> 2510252025302540255025602570258025902600 GAGCAATACAAATTTCAAGCAGACTCCCCCAAAAGACTGGCGACAGCCATTGCAGGCGCTTGGGAGAATGGAGTGTGCGGAATCAGGTCGACAACCAGAA GluGlnTyrLysPheGlnAlaAspSerProLysArgLeuAlaThrAlaIleAlaGlyAlaTrpGluAsnGlyValCysGlyIleArgSerThrThrArg> 2610262026302640265026602670268026902700 TGGAGAACCTCTTGTGGAAGCAAATAGCCAATGAACTGAACTACATATTATGGGAAAACAACATCAAATTAACGGTAGTTGTGGGTGATATAATTGGGGT MetGluAsnLeuLeuTrpLysGlnIleAlaAsnGluLeuAsnTyrIleLeuTrpGluAsnAsnIleLysLeuThrValValValGlyAspIleIleGlyVal> 2710272027302740275027602770278027902800 CTTAGAGCAAGGGAAAAGAACACTAACACCACAACCCATGGAACTAAAATATTCATGGAAAACATGGGGAAAGGCGAAGATAGTGACAGCTGAAACACAA LeuGluGlnGlyLysArgThrLeuThrProGlnProMetGluLeuLysTyrSerTrpLysThrTrpGlyLysAlaLysIleValThrAlaGluThrGln> 2810282028302840285028602870288028902900 AATTCCTCTTTCATAATAGATGGGCCAAACACACCAGAGTGTCCAAGTGCCTCAAGAGCATGGAATGTGTGGGAGGTGGAAGATTACGGGTTCGGAGTCT AsnSerSerPheIleIleAspGlyProAsnThrProGluCysProSerAlaSerArgAlaTrpAsnValTrpGluValGluAspTyrGlyPheGlyVal> 2910292029302940295029602970298029903000 TCACAACTAACATATGGCTGAAACTCCGAGAGATGTACACCCAACTATGTGACCACAGGCTAATGTCGGCAGCCGTTAAGGATGAGAGGGCCGTACACGC PheThrThrAsnIleTrpLeuLysLeuArgGluMetTyrThrGlnLeuCysAspHisArgLeuMetSerAlaAlaValLysAspGluArgAlaValHisAla> 3010302030303040305030603070308030903100 CGACATGGGCTATTGGATAGAAAGCCAAAAGAATGGAAGTTGGAAGCTAGAAAAGGCATCCCTCATAGAGGTAAAAACCTGCACATGGCCAAAATCACAC AspMetGlyTyrTrpIleGluSerGlnLysAsnGlySerTrpLysLeuGluLysAlaSerLeuIleGluValLysThrCysThrTrpProLysSerHis> 3110312031303140315031603170318031903200 ACTCTTTGGAGCAATGGTGTGCTAGAGAGTGACATGATCATCCCAAAGAGTCTGGCTGGTCCCATTTCGCAACACAACTACAGGCCCGGATACCACACCC ThrLeuTrpSerAsnGlyValLeuGluSerAspMetIleIleProLysSerLeuAlaGlyProIleSerGlnHisAsnTyrArgProGlyTyrHisThr> 3210322032303240325032603270328032903300 AAACGGCAGGACCCTGGCACTTAGGAAAATTGGAGCTGGACTTCAACTATTGTGAAGGAACAACAGTTGTCATCACAGAAAATTGTGGGACAAGAGGCCC GlnThrAlaGlyProTrpHisLeuGlyLysLeuGluLeuAspPheAsnTyrCysGluGlyThrThrValValIleThrGluAsnCysGlyThrArgGlyPro> 3310332033303340335033603370338033903400 ATCACTGAGAACAACAACAGTGTCAGGGAAGTTGATACACGAATGGTGTTGCCGCTCGTGTACACTTCCTCCCCTGCGATACATGGGAGAAGACGGCTGC SerLeuArgThrThrThrValSerGlyLysLeuIleHisGluTrpCysCysArgSerCysThrLeuProProLeuArgTyrMetGlyGluAspGlyCys> 3410342034303440345034603470348034903500 TrpTyrGlyMetGluIleArgProIleAsnGluLysGluGluAsnMetValLysSerLeuValSerAlaGlySerGlyLysValAspAsnPheThrMet> TGGTATGGCATGGAAATTAGACCCATTAATGAGAAAGAAGAGAACATGGTAAAGTCTTTAGTCTCAGCAGGGAGTGGAAAGGTGGATAACTTCACAATGG 3510352035303540355035603570358035903600 GTGTCTTGTGTTTGGCAATCCTTTTTGAAGAGGTGATGAGAGGAPAATTTGGGAPAAAGCACATGATTGCAGGGGTTCTCTTCACGTTTGTACTCCTCT GlyValLeuCysLeuAlaIleLeuPheGluGluValMetArgGlyLysPheGlyLysLysHisMetIleAlaGlyValLeuPheThrPheValLeuLeuLeu> 3610362036303640365036603670368036903700 CTCAGGGCAAATAACATGGAGAGACATGGCGCACACACTCATAATGATTGGGTCCAACGCCTCTGACAGAATGGGAATGGGCGTCACTTACCTAGCATTG SerGlyGlnIleThrTrpArgAspMetAlaHisThrLeuIleMetIleGlySerAsnAlaSerAspArgMetGlyMetGlyValThrTyrLeuAlaLeu> 3710372037303740375037603770378037903800 ATTGCAACATTTAAAATTCAGCCATTTTTGGCTTTGGGATTCTTCCTGAGGAAACTGACATCTAGAGAAAATTTATTGTTGGGAGTTGGGTTGGCCATGG IleAlaThrPheLysIleGlnProPheLeuAlaLeuGlyPhePheLeuArgLysLeuThrSerArgGluAsnLeuLeuLeuGlyValGlyLeuAlaMet> 3810382038303840385038603870388038903900 CAACAACGTTACAACTGCCAGAGGACATTGAACAAATGGCGAATGGAATAGCTTTAGGGCTCATGGCTCTTAAATTAATAACACAATTTGAAACATACCA AlaThrThrLeuGlnLeuProGluAspIleGluGlnMetAlaAsnGlyIleAlaLeuGlyLeuMetAlaLeuLysLeuIleThrGlnPheGluThrTyrGln> 3910392039303940395039603970398039904000 ACATGGACGGCATTAGTCTCCCTAATGTGTTCAAATACAATTTTCACGTTGACTGTTGCCTGGAGAACAGCCACCCTGATTTTGGCCGGAATTTCTCTT LeuTrpThrAlaLeuValSerLeuMetCysSerAsnThrIlePheThrLeuThrValAlaTrpArgThrAlaThrLeuIleLeuAlaGlyIleSerLeu> 4010402040304040405040604070408040904100 TTGCCAGTGTGCCAGTCTTCGAGCATGAGGAAAACAGATTGGCTCCCAATGGCTGTGGCAGCTATGGGAGTTCCACCCCTACCACTTTTTATTTTCAGTT LeuProValCysGlnSerSerSerMetArgLysThrAspTrpLeuProMetAlaValAlaAlaMetGlyValProProLeuProLeuPheIlePheSer> 4110412041304140415041604170418041904200 TGAAAGATACGCTCAAAAGGAGAAGCTGGCCACTGAATGAGGGGGTGATGGCTGTTGGACTTGTGAGTATTCTAGCTAGTTCTCTCCTTAGGAATGACGT LeuLysAspThrLeuLysArgArgSerTrpProLeuAsnGluGlyValMetAlaValGlyLeuValSerIleLeuAlaSerSerLeuLeuArgAsnAspVal> 4210422042304240425042604270428042904300 GCCCATGGCTGGACCATTAGTGGCTGGGGGCTTGCTGATAGCGTGCTACGTCATAACTGGCACGTCAGCAGACCTCACTGTAGAAAAAGCAGCAGATGTC ProMetAlaGlyProLeuValAlaGlyGlyLeuLeuIleAlaCysTyrValIleThrGlyThrSerAlaAspLeuThrValGluLysAlaAlaAspVal> 4310432043304340435043604370438043904400 ACATGGGAGGAAGAGGCTGAGCAAACAGGAGTGTCCCACAATTTAATGATCACAGTTGATGACGATGGAACAATGAGAATAAAAGATGATGAGACTGAGA ThrTrpGluGluGluAlaGluGlnThrGlyValSerHisAsnLeuMetIleThrValAspAspAspGlyThrMetArgIleLysAspAspGluThrGlu> 4410442044304440445044604470448044904500 ACATCTTAACAGTGCTTTTGAAAACAGCATTACTAATAGTGTCAGGCATTTTTCCATACTCCATACCCGCAACACTGTTGGTCTGGCACACTTGGCAAAA AsnIleLeuThrValLeuLeuLysThrAlaLeuLeuIleValSerGlyIlePheProTyrSerIleProAlaThrLeuLeuValTrpHisThrTrpGlnLys> 4510452045304540455045604570458045904600 GCAAACCCAAAGATCCGGTGTCCTATGGGACGTTCCCAGCCCCCCAGAGACACAGAAAGCAGAACTGGAAGAAGGGGTTTATAGGATCAAGCAGCAAGGA GlnThrGlnArgSerGlyValLeuTrpAspValProSerProProGluThrGlnLysAlaGluLeuGluGluGlyValTyrArgIleLysGlnGlnGly> 4610462046304640465046604670468046904700 ATTTTTGGGAAAACCCAAGTGGGGGTTGGAGTACAAAAAGAAGGAGTTTTCCACACCATGTGGCACGTCACAAGAGGAGCAGTGTTGACACACAATGGGA IlePheGlyLysThrGlnValGlyValGlyValGlnLysGluGlyValPheHisThrMetTrpHisValThrArgGlyAlaValLeuThrHisAsnGly> 4710472047304740475047604770478047904800 AAAGACTGGAACCAAACTGGGCTAGCGTGAAAAAAGATCTGATTTCATACGGAGGAGGATGGAAATTGAGTGCACAATGGCAAAAAGGAGAGGAGGTGCA LysArgLeuGluProAsnTrpAlaSerValLysLysAspLeuIleSerTyrGlyGlyGlyTrpLysLeuSerAlaGlnTrpGlnLysGlyGluGluValGln> 4810482048304840485048604870488048904900 GGTTATTGCCGTAGAGCCTGGGAAGAACCCAAAGAACTTTCAAACCATGCCAGGCATTTTCCAGACAACAACAGGGGGAGATAGGAGCGATTGCACTGGAC ValIleAlaValGluProGlyLysAsnProLysAsnPheGlnThrMetProGlyIlePheGlnThrThrThrGlyGluIleGlyAlaIleAlaLeuAsp> 4910492049304940495049604970498049905000 TTCAAGCCTGGAACTTCAGGATCTCCCATCATAAACAGAGAGGGAAAGGTACTGGGATTGTATGGCAATGGAGTGGTCACAAAGAATGGTGGCTATGTCA PheLysProGlyThrSerGlySrProIleIleAsnArgGluGlyLysValLeuGlyLeuTyrGlyAsnGlyValValThrLysAsnGlyGlyTyrVal> 5010502050305040505050605070508050905100 GTGGAATAGCACAAACAAATGCAGAACCAGACGGACCGACACCAGAGTTGGAAGAAGAGATGTTCAAAAAGCGAAATCTAACCATAATGGATCTCCATCC SerGlyIleAlaGlnThrAsnAlaGluProAspGlyProThrProGluLeuGluGluGluMetPheLysLysArgAsnLeuThrIleMetAspLeuHisPro> 5110512051305140515051605170518051905200 CGGGTCAGGAAAGACGCGGAAATATCTTCCAGCTATTGTTAGAGAGGCAATCAAGAGACGCTTAAGGACTCTAATTTTGGCACCAACAAGGGTAGTTGCA GlySerGlyLysThrArgLysTyrLeuProAlaIleValArgGluAlaIleLysArgArgLeuArgThrLeuIleLeuAlaProThrArgValValAla> 5210522052305240525052605270528052905300 GCTGAGATGGAAGAAGCATTGAAAGGGCTCCCAATAAGGTATCAAACAACTGCAACAAAATCTGAACACACAGGGAGAGAGATTGTTGATCTAATGTGCC AlaGluyMetGluGluAlaLeuLysGlyLeuProIleArgTyrGlnThrThrAlaThrLysSerGluHisThrGlyArgGluIleValAspLeuMetCys> 5310532053305340535053605370538053905400 ACGCAACGTTCACAATGCGTTTGCTGTCACCAGTCAGGGTTCCAAACTACAACTTGATAATAATGGATGAGGCTCATTTCACAGACCCAGCCAGTATAGC HisAlaThrPheThrMetArgLeuLeuSerProValArgValProAsnTyrAsnLeuIleIleMetAspGluAlaHisPheThrAspProAlaSerIleAla> 5410542054305440545054605470548054905500 GGCTAGAGGGTACATATCAACTCGTGTAGGAATGGGAGAGGCAGCCGCAATTTTCATGACAGCCACACCCCCTGGAACAGCTGATGCCTTTCCTCAGAGC AlaArgGlyTyrIleSerThrArgValGlyMetGlyGluAlaAlaAlaIlePheMetThrAlaThrProProGlyThrAlaAspAlaPheProGlnSer> 5510552055305540555055605570558055905600 AACGCTCCAATTCAAGATGAAGAAAGAGACATACCAGAACGCTCATGGAATTCAGGCAATGAATGGATTACCGACTTTGCCGGGAAGACGGTGTGGTTTG AsnAlaProIleGlnAspGluGluArgAspIleProGluArgSerTrpAsnSerGlyAsnGluTrpIleThrAspPheAlaGlyLysThrValTrpPhe> 5610562056305640565056605670568056905700 TCCCTAGCATCAAAGCTGGAAATGACATAGCAAACTGCTTGCGGAAAAATGGAAAAAAGGTCATTCAACTTAGTAGGAAGACTTTTGACACAGAATATCA ValProSerIleLysAlaGlyAsnAspIleAlaAsnCysLeuArgLysAsnGlyLysLysValIleGlnLeuSerArgLysThrPheAspThrGluTyrGln> 5710572057305740575057605770578057905800 AAAGACTAAACTAAATGATTGGGACTTTGTGGTGACAACAGACATTTCAGAAATGGGAGCCAATTTCAAAGCAGACAGAGTGATCGACCCAAGAAGATGT LysThrLysLeuAsnAspTrpAspPheValValThrThrAspIleSerGluMetGlyAlaAsnPheLysAlaAspArgValIleAspProArgArgCys> 5810582058305840585058605870588058905900 CTCAAGCCAGTGATTTTGACAGACGGACCCGAGCGCGTGATCCTGGCGGGACCAATGCCAGTCACCGTAGCGAGCGCTGCGCAAAGGAGAGGGAGAGTTG LeuLysProValIleLeuThrAspGlyProGluArgValIleLeuAlaGlyProMetProValThrValAlaSerAlaAlaGlnArgArgGlyArgVal> 5910592059305940595059605970598059906000 GCAGGAACCCACAAAAAGAAAATGACCAATACATATTCATGGGCCAGCCCCTCAATAATGATGAAGACCATGCTCACTGGACAGAAGCAAAAATGCTGCT GlyArgAsnProGlnLysGluAsnAspGlnTyrIlePheMetGlyGlnProLeuAsnAsnAspGluAspHisAlaHisTrpThrGluAlaLysMetLeuLeu> 6010602060306040605060606070608060906100 AGACAACATCAACACACCAGAAGGGATCATACCAGCTCTCTTTGAACCAGAAAGGGAGAAGTCAGCCGCCATAGACGGCGAATACCGCCTGAAGGGTGAG AspAsnIleAsnThrProGluGlyIleIleProAlaLeuPheGluProGluArgGluLysSerAlaAlaIleAspGlyGluTyrArgLeuLysGlyGlu> 6110612061306140615061606170618061906200 TCCAGGAAGACCTTCGTGGAACTCATGAGGAGGGGTGACCTCCCAGTTTGGCTAGCCCATAAAGTAGCATCAGAAGGGATCAAATATACAGATAGAAAGT SerArgLysThrPheValGluLeuMetArgArgGlyAspLeuProValTrpLeuAlaHisLysValAlaSerGluGlyIleLysTyrThrAspArgLys> 6210622062306240625062606270628062906300 GGTGTTTTGATGGAGAACGCAACAATCAAATTTTAGAGGAGAATATGGATGTGGAAATCTGGACAAAGGAAGGAGAAAAGAAAAAATTGAGACCTAGGTG TrpCysPheAspGlyGluArgAsnAsnGlnIleLeuGluGluAsnMetAspValGluIleTrpThrLysGluGlyGluLysLysLysLeuArgProArgTrp> 6310632063306340635063606370638063906400 GCTTGATGCCCGCACTTATTCAGATCCCTTAGCGCTCAAGGAATTCAAGGACTTTGCGGCTGGTAGAAAGTCAATTGCCCTTGATCTTGTGACAGAAATA LeuAspAlaArgThrTyrSerAspProLeuAlaLeuLysGluPheLysAspPheAlaAlaGlyArgLysSerIleAlaLeuAspLeuValThrGluIle> 6410642064306440645064606470648064906500 GGAAGAGTGCCTTCACACTTAGCTCACAGAACGAGAAACGCCCTGGACAATCTGGTGATGTTGCACACGTCAGAACATGGCGGGAGGGCCTACAGGCATG GlyArgValProSerHisLeuAlaHisArgThrArgAsnAAlaLeuAspAsnLeuValMetLeuHisThrSerGLuHisGlyGlyArgAlaTyrArgHis> 6510652065306540655065606570658065906600 CAGTGGAGGAACTACCAGAAACAATGGAAACACTCTTACTCCTGGGACTCATGATCCTGTTAACAGGTGGAGCAATGCTTTTCTTGATATCAGGTAAAGG AlaValGluGluLeuProGluThrMetGluThrLeuLeuLeuLeuGlyLeuMetIleLeuLeuThrGlyGlyAlaMetLeuPheLeuIleSerGlyLysGly> 6610662066306640665066606670668066906700 GATTGGAAAGACTTCAATAGGACTCATTTGTGTAGCTGCTTCCAGCGGTATGTTATGGATGGCTGATGTCCCACTCCAATGGATCGCGTCTGCCATAGTC IleGlyLysThrSerIleGLyLeuIleCysValAlaAlaSerSerGlyMetLeuTrpMetAlaAspValProLeuGlnTrpIleAlaSerAlaIleVal> 6710672067306740675067606770678067906800 CTGGAGTTTTTTATGATGGTGTTACTTATACCAGAACCAGAAAAGCAGAGAACTCCCCAAGACAATCAACTCGCATATGTCGTGATAGGCATACTCACAC LeuGluPhePheMetMetValLeuLeuIleProGluProGluLysGlnArgThrProGlnAspAsnGlnLeuAlaTyrValValIleGlyIleLeuThr> 6810682068306840685068606870688068906900 TGGCTGCAATAGTAGCAGCCAATGAAATGGGACTGTTGGAAACCACAAAGAGAGATTTAGGAATGTCCAAAGAACCAGGTGTTGTTTCTCCAACCAGCTA LeuAlaAlaIleValAlaAlaAsnGluMetGlyLeuLeuGluThrThrLysArgAspLeuGlyMetSerLysGluProGlyValValSerProThrSerTyr> 6910692069306940695069606970698069907000 TTTGGATGTGGACTTGCACCCAGCATCAGCCTGGACATTGTACGCTGTGGCCACAACAGTAATAACACCAATGTTGAGACATACCATAGAGAATTCCACA LeuAspValAspLeuHisProAlaSerAlaTrpThrLeuTyrAlaValAlaThrThrValIleThrProMetLeuArgHisThrIleGluAsnSerThr> 7010702070307040705070607070708070907100 GCAAATGTGTCCCTGGCAGCTATAGCCAACCAGGCAGTGGTCCTGATGGGTTTAGACAAAGGATGGCCGATATCGAAAATGGACTTAGGCGTGCCACTAT AlaAsnValSerLeuAlaAlaIleAlaAsnGlnAlaValValLeuMetGlyLeuAspLysGlyTrpProIleSerLysMetAspLeuGLyValProLeu> 7110712071307140715071607170718071907200 TGGCACTGGGTTGTTATTCACAAGTGAACCCACTAACTCTCACAGCGGCAGTTCTCCTGCTAGTCACGCATTATGCTATTATAGGTCCAGGATTGCAGGC LeuAlaLeuGlyCysTyrSerGlnValAsnProLeuThrLeuThrAlaAlaValLeuLeuLeuValThrHisTyrAlaIleIleGlyProGlyLeuGlnAla> 7210722072307240725072607270728072907300 AAAAGCCACTCGTGAAGCTCAAAAAAGGACAGCTGCTGGAATAATGAAGAATCCAACGGTGGATGGGATAATGACAATAGACCTAGATCCTGTAATATAC LysAlaThrArgGluAlaGlnLysArgThrAlaAlaGlyIleMetLysAsnProThrValAspGlyIleMetThrIleAspLeuAspProValIleTyr> 7310732073307340735073607370738073907400 GATTCAAAATTTGAAAAGCAACTAGGACAGGTTATGCTCCTGGTTCTGTGTGCAGTTCAACTTTTGTTAATGAGAACATCATGGGCTTTTTGTGAAGCTC AspSerLysPheGluLysGlnLeuGlyGlnValMetLeuLeuValLeuCysAlaValGlnLeuLeuLeuMetArgThrSerTrpAlaPheCysGluAla> 7410742074307440745074607470748074907500 TAACCCTAGCCACAGGACCAATAACAACACTCTGGGAAGGATCACCTGGGAAGTTCTGGAACACCACGATAGCTGTTTCCATGGCGAACATCTTTAGAGG LeuThrLeuAlaThrGlyProIleThrThrLeuTrpGluGlySerProGlyLysPheTrpAsnThrThrIleAlaValSerMetAlaAsnIlePheArgGly> 7510752075307540755075607570758075907600 GAGCTATTTAGCAGGAGCTGGGCTTGCTTTTTCTATCATGAAATCAGTTGGAACAGGAAAGAGAGGGACAGGGTCACAGGGTGAAACCTTGGGAGAAAAG SerTyrLeuAlaGlyAlaGlyLeuAlaPheSerIleMetLysSerValGlyThrGlyLysArgGlyThrGlySerGlnGlyGluThrLeuGlyGluLys> 7610762076307640765076607670768076907700 TGGAAAAAGAAATTGAATCAATTACCCCGGAAAGAGTTTGACCTTTACAAGAAATCCGGAATCACTGAAGTGGATAGAACAGAAGCCAAAGAAGGGTTGA TrpLysLysLysLeuAsnGlnLeuProArgLysGluPheAspLeuTyrLysLysSerGlyIleThrGluValAspArgThrGluAlaLysGluGlyLeu> 7710772077307740775077607770778077907800 AAAGAGGAGAAATAACACACCATGCCGTGTCCAGAGGCAGCGCAAAACTTCAATGGTTCGTGGAGAGAAACATGGTCATCCCCGAAGGAAGAGTCATAGA LysArgGlyGluIleThrHisHisAlaValSerArgGlySerAlaLysLeuGlnTrpPheValGluArgAsnMetValIleProGluGlyArgValIleAsp> 7810782078307840785078607870788078907900 CTTAGGCTGTGGAAGAGGAGGCTGGTCATATTATTGTGCAGGACTGAAAAAAGTTACAGAAGTGCGAGGATACACAAAAGGCGGCCCAGGACATGAAGAA LeuGlyCysGlyArgGlyGlyTrpSerTyrTyrCysAlaGlyLeuLysLysValThrGluValArgGlyTyrThrLysGlyGlyProGlyHisGluGlu> 7910792079307940795079607970798079908000 CCAGTACCTATGTCTACATACGGATGGAACATAGTCAAGTTAATGAGTGGAAAGGATGTGTTTTATCTTCCACCTGAAAAGTGTGATACTCTATTGTGTG ProValProMetSerThrTyrGlyTrpAsnIleValLysLeuMetSerGlyLysAspValPheTyrLeuProProGluLysCysAspThrLeuLeuCys> 8010802080308040805080608070808080908100 ACATTGGAGAATCTTCACCAAGCCCAACAGTGGAAGAAAGCAGAACCATAAGAGTCTTGAAGATGGTTGAACCATGGCTAAAAAATAACCAGTTTTGCAT AspIleGlyGluSerSerProSerProThrValGluGluSerArgThrIleArgValLeuLysMetValGluProTrpLeuLysAsnAsnGlnPheCysIle> 8110812081308140815081608170818081908200 TAAAGTATTGAACCCTTACATGCCAACTGTGATTGAGCACCTAGAAAGACTACAAAGGAAACATGGAGGAATGCTTGTGAGAAATCCACTCTCACGAAAC LysValLeuAsnProTyrMetProThrValIleGluHisLeuGluArgLeuGlnArgLysHisGlyGlyMetLeuValArgAsnProLeuSerArgAsn> 8210822082308240825082608270828082908300 TCCACGCACGAAATGTACTGGATATCTAATGGCACAGGCAATATCGTTTCTTCAGTCAACATGGTATCCAGATTGCTACTTAACAGATTCACAATGACAC SerThrHisGLuMetTyrTrpIleSerAsnGlyThrGlyAsnIleValSerSerValAsnMetValSerArgLeuLeuLeuAsnArgPheThrMetThr> 8310832083308340835083608370838083908400 ATAGGAGACCCACCATAGAGAAAGATGTGGATTTAGGAGCGGGGACCCGACATGTCAATGCGGAACCAGAAACACCCAACATGGATGTCATTGGGGAAAG HisArgArgProThrIleGluLysAspValAspLeuGlyAlaGlyThrArgHisValAsnAlaGluProGluThrProAsnMetAspValIleGlyGluArg> 8410842084308440845084608470848084908500 AATAAGAAGGATCAAGGAGGAGCATAGTTCAACATGGCACTATGATGATGAAAATCCTTATAAAACGTGGGCTTACCATGGATCCTATGAAGTTAAGGCC IleArgArgIleLysGluGluHisSerSerThrTrpHisTyrAspAspGluAsnProTyrLysThrTrpAlaTyrHisGlySerTyrGluValLysAla> 8510852085308540855085608570858085908600 ACAGGCTCAGCCTCCTCCATGATAAATGGAGTCGTGAAACTCCTCACGAAACCATGGGATGTGGTGCCCATGGTGACACAGATGGCAATGACGGATACAA ThrGlySerAlaSerSerMetIleAsnGlyValValLysLeuLeuThrLysProTrpAspValValProMetValThrGlnMetAlaMetThrAspThr> 8610862086308640865086608670868086908700 CCCCATTCGGCCAGCAAAGGGTTTTTAAAGAGAAAGTGGACACCAGGACACCCAGACCTATGCCAGGAACAAGAAAGGTTATGGAGATCACAGCGGAATG ThrProPheGlyGlnGlnArgValPheLysGluLysValAspThrArgThrProArgProMetProGlyThrArgLysValMetGluIleThrAlaGluTrp> 8710872087308740875087608770878087908800 GCTTTGGAGAACCCTGGGAAGGAACAAAAGACCCAGATTATGTACGAGAGAGGAGTTCACAAAAAAGGTCAGAACCAACGCAGCTATGGGCGCCGTTTTT LeuTrpArgThrLeuGlyArgAsnLysArgProArgLeuCysThrArgGluGluPheThrLysLysValArgThrAsnAlaAlaMetGlyAlaValPhe> 8810882088308840885088608870888088908900 ACAGAGGAGAACCAATGGGACAGTGCTAGAGCTGCTGTTGAGGATGAAGAATTCTGGAAACTCGTGGACAGAGAACGTGAACTCCACAAATTGGGCAAGT ThrGluGluAsnGlnTrpAspSerAlaArgAlaAlaValGluAspGluGluPheTrpLysLeuValAspArgGluArgGluLeuHisLysLeuGlyLys 8910892089308940895089608970898089909000 GTGGAAGCTGCGTTTACAACATGATGGGCAAGAGAGAGAAGAAACTTGGAGAGTTTGGCAAAGCAAAAGGCAGTAGAGCCATATGGTACATGTGGTTGGG CysGlySerCysValTyrAsnMetMetGlyLysArgGluLysLysLeuGlyGluPheGlyLysAlaLysGlySerArgAlaIleTrpTyrMetTrpLeuGly> 9010902090309040905090609070908090909100 AGCCAGATACCTTGAGTTCGAAGCACTCGGATTCTTAAATGAAGACCATTGGTTCTCGCGTGAAAACTCTTACAGTGGAGTAGAAGGAGAAGGACTGCAC AlaArgTyrLeuGluPheGluAlaLeuGlyPheLeuAsnGluAspHisTrpPheSerArgGluAsnSerTyrSerGlyValGluGlyGluGlyLeuHis> 9110912091309140915091609170918091909200 AAGCTGGGATACATCTTAAGAGACATTTCCAGATACCCGGAGGAGCTATGTATGCTGATGACACAGCTGGTTGGGACACAAGAATTAACAGAAGATGACC LysLeuGlyTyrIleLeuArgAspIleSerLysIleProGlyGlyAlaMetTyrAlaAspAspThrAlaGlyTrpAspThrArgIleThrGluAspAsp> 9210922092309240925092609270928092909300 TGCACAATGAGGAAAAAATCACACAGCAAATGGACCCTGAACACAGGCAGTTAGCAAACGCTATATTCAAGCTCACATACCAAAACAAAGTGGTCAAAGT LeuHisAsnGluGluLysIleThrGlnGlnMetAspProGluHisArgGlnLeuAlaAsnAlaIlePheLysLeuThrTyrGlnAsnLysValValLysVal> 9310932093309340935093609370938093909400 TCAACGACCAACTCCAAAGGGCACGGTAATGGACATCATATCTAGGAAAGACCAAAGAGGCAGTGGACAGGTGGGAACTTATGGTCTGAATACATTCACC GlnArgProThrProLysGlyThrValMetAspIleIleSerArgLysAspGlnArgGlySerGlyGlnValGlyThrTyrGlyLeuAsnThrPheThr> 9410942094309440945094609470948094909500 AACATGGAAGCCCAGTTAATCAGACAAATGGAAGGAGAAGGTGTGTTGTCGAAGGCAGACCTCGAGAACCCTCATCTGCTAGAGAAGAAAGTTACACAAT AsnMetGluAlaGlnLeuIleArgGlnMetGluGlyGluGlyValLeuSerLysAlaAspLeuGluAsnProHisLeuLeuGluLysLysValThrGln> 9510952095309540955095609570958095909600 GGTTGGAAACAAAAGGAGTGGAGAGGTTAAAAAGAATGGCCATCAGCGGGGATGATTGCGTGGTGAAACCAATTGATGACAGGTTCGCCAATGCCCTGCT TrpLeuGluThrLysGlyValGluArgLeuLysArgMetAlaIleSerGlyAspAspCysValValLysProIleAspAspArgPheAlaAsnAlaLeuLeu> 9610962096309640965096609670968096909700 TGCCCTGAATGACATGGGAAAAGTTAGGAAGGACATACCTCAATGGCAGCCATCAAAGGGATGGCATGATTGGCAACAGGTCCCTTTCTGCTCCCACCAC AlaLeuAsnAspMetGlyLysValArgLysAspIleProGlnTrpGlnProSerLysGlyTrpHisAspTrpGLnGlnValProPheCysSerHisHis> 9710972097309740975097609770978097909800 TTTCATGAATTGATCATGAAAGATGGAAGAAAGTTGGTAGTTCCCTGCAGACCTCAGGATGAATTAATCGGGAGAGCGAGAATCTCTCAAGGAGCAGGAT PheHisGluLeuIleMetLysAspGlyArgLysLeuValValProCysArgProGlnAspGluLeuIleGlyArgAlaArgIleSerGlnGlyAlaGly> 9810982098309840985098609870988098909900 GGAGCCTTAGAGAAACTGCATGCCTAGGGAAAGCCTACGCCCAAATGTGGACTCTCATGTACTTTCACAGAAGAGATCTTAGACTAGCATCCAACGCCAT TrpSerLeuArgGluThrAlaCysLeuGlyLysAlaTyrAlaGlnMetTrpThrLeuMetTyrPheHisArgArgAspLeuArgLeuAlaSerAsnAlaIle> 99109920993099409950996099709980999010000 ATGTTCAGCAGTACCAGTCCATTGGGTCCCCACAAGCAGAACGACGTGGTCTATTCATGCTCACCATCAGTGGATGACTACAGAAGACATGCTTACTGTT CysSerAlaValProValHisTrpValProThrSerArgThrThrTrpSerIleHisAlaHisHisGlnTrpMetThrThrGluAspMetLeuThrVal> 10010100201003010040100501006010070100801009010100 TGGAACAGGGTGTGGATAGAGGATAATCCATGGATGGAAGACAAAACTCCAGTCAAAACCTGGGAAGATGTTCCATATCTAGGGAAGAGAGAAGACCAAT TrpAsnArgValTrpIleGluAspAsnProTrpMetGluAspLysThrProValLysThrTrpGluAspValProTyrLeuGlyLysArgGluAspGln> 10110101201013010140101501016010170101801019010200 GGTGCGGATCACTCATTGGTCTCACTTCCAGAGCAACCTGGGCCCAGAACATACTTACGGCAATCCAACAGGTGAGAAGCCTTATAGGCAATGAAGAGTT TrpCysGlySerLeuIleGlyLeuThrSerArgAlaThrTrpAlaGlnAsnIleLeuThrAlaIleGlnGlnValArgSerLeuIleGlyAsnGluGluPhe> 10210102201023010240102501026010270102801029010300 TCTGGACTACATGCCTTCGATGAAGAGATTCAGGAAGGAGGAGGAGTCAGAGGGAGCCATTTGGTAAACGTAGGAAGTGAAAAAGAGGCAAACTGTCAGG LeuAspTyrMetProSerMetLysArgPheArgLysGluGluGluSerGluGlyAlaIleTrp***> 10310103201033010340103501036010370103801039010400 CCACCTTAAGCCACAGTACGGAAGAAGCTGTGCAGCCTGTGAGCCCCGTCCAAGGACGTTAAAAGAAGAAGTCAGGCCCAAAAGCCACGGTTTGAGCAAA 10410104201043010440104501046010470104801049010500 CCGTGCTGCCTGTGGCTCCGTCGTGGGGACGTAAAACCTGGGAGGCTGCAAACTGTGGAAGCTGTACGCACGGTGTAGCAGACTAGCGGTTAGAGGAGAC 10510105201053010540105501056010570105801059010600 CCCTCCCATGACACAACGCAGCAGCGGGGCCCGAGCTCTGAGGGAAGCTGTACCTCCTTGCAAAGGACTAGAGGTTAGAGGAGACCCCCCGCAAATAAAA 10610106201063010640106501066010670106801069010700 ACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAGAACGCCAGAAAATGGAATGGTGCTGTTGAATCAAC 10710107201073010740107501076010770107801079010800 AGGTTCTGGTACCGGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC 10810108201083010840108501086010870108801089010900 CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT 10910109201093010940109501096010970109801099011000 AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT 11010110201103011040110501106011070110801109011100 CTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT 11110111201113011140111501116011170111801119011200 CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA 11210112201123011240112501126011270112801129011300 GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT 11310113201133011340113501136011370113801139011400 ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGA 11410114201143011440114501146011470114801149011500 AACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGATAAGCT 11510115201153011540115501156011570115801159011600 TTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTG 11610116201163011640116501166011670116801169011700 GATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTGG 11710117201173011740117501176011770117801179011800 CGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGC 11810118201183011840118501186011870118801189011900 CACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCT 11910119201193011940119501196011970119801199012000 GGCGCCTATATCGCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTTGGCAGGCCCCGTGG 12010120201203012040120501206012070120801209012100 CCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGA 12110121201213012140121501216012170121801219012200 GTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATG 12210122201223012240122501226012270122801229012300 ACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCC 12310123201233012340123501236012370123801239012400 TGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTCGGCGAGAAGCAGGCCATTATCGCCGG 12410124201243012440124501246012470124801249012500 CATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGG 12510125201253012540125501256012570125801259012600 ATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCA 12610126201263012640126501266012670126801269012700 CTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCC 12710127201273012740127501276012770127801279012800 CGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATC 12810128201283012840128501286012870128801289012900 AATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAG 12910129201293012940129501296012970129801299013000 CGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCG 13010130201303013040130501306013070130801309013100 ATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAA 13110131201313013140131501316013170131801319013200 CGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTG 13210132201323013240132501326013270132801329013300 GCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTA 13310133201333013340133501336013370133801339013400 ACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAA 13410134201343013440134501346013470134801349013500 CCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATC 13510135201353013540135501356013570135801359013600 GCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA 13610136201363013640136501366013670136801369013700 GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCG 13710137201373013740137501376013770137801379013800 ATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGA 13810138201383013840138501386013870138801389013900 AAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA 13910139201393013940139501396013970139801399014000 TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT 14010140201403014040140501406014070140801409014100 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC 14110141201413014140141501416014170141801419014200 CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT 14210142201423014240142501426014270142801429014300 CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA 14310143201433014340143501436014370143801439014400 CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA 14410144201443014440144501446014470144801449014500 GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA 14510145201453014540145501456014570145801459014600 GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT 14610146201463014640146501466014670146801469014700 GATCTTTTCTACGGGGTCTGACGCTCATTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA 14710147201473014740147501476014770147801479014800 AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCT 14810148201483014840148501486014870148801489014900 GTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG 14910149201493014940149501496014970149801499015000 AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG 15010150201503015040150501506015070150801509015100 TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAAGATCTGGCTAGCGATGACCCT 1511015120151301514015150 GCTGATTGGTTCGCTGACCATTTCCGGGCGCGCCGATTTAGGTGACACTATAG Bases 1 to 10707: DEN3 virus genome cDNA Bases 95 to 10264: DEN3 polyprotein ORF Bases 95 to 436: C protein ORF Bases 437 to 934: prM protein ORF Bases 935 to 2413: E protein ORF Bases 2414 to 3469: NS1 protein ORF Bases 3470 to 4123: NS2A protein ORF Bases 4124 to 4513: NS2B protein ORF Bases 4514 to 6370: NS3 protein ORF Bases 6371 to 6751: NS4A protein ORF Bases 6752 to 6820: 2K protein ORF Bases 6821 to 7564: NS4B protein ORF Bases 7575 to 10264 NS5 protein ORF
(174) TABLE-US-00046 APPENDIX3 NucleotideandaminoacidsequenceofDEN1(PuertoRico/94)CMEchimericregion 102030405060708090100 AGTTGTTAGTCTGTGTGGACCGACAAGGACAGTTCCAAATCGGAAGCTTGCTTAACACAGTTCTAACAGTTTGTTTGAATAGAGAGCAGATCTCTGGAAA 110120130140150160170180190200 AATGAACAACCAACGGAAAAAGACGGGTCGACCGTCTTTCAATATGCTGAAACGCGCGAGAAACCGCGTGTCAACTGGTTCACAGTTGGCGAAGAGATTC MetAsnAsnGlnArgLysLysThrGlyArgProSerPheAsnMetLeuLysArgAlaArgAsnArgValSerThrGlySerGlnLeuAlaLysArgPhe> 210220230240250260270280290300 TCAAAAGGATTGCTTTCAGGCCAAGGACCCATGAAATTGGTGATGGCTTTCATAGCATTTCTAAGATTTCTAGCCATACCCCCAACAGCAGGAATTTTGG SerLysGlyLeuLeuSerGlyGlnGlyProMetLysLeuValMetAlaPheIleAlaPheLeuArgPheLeuAlaIleProProThrAlaGlyIleLeu> 310320330340350360370380390400 CTAGATGGAGCTCATTCAAGAAGAATGGAGCGATCAAAGTGTTACGGGGTTTCAAAAAAGAGATCTCAAGCATGTTGAACATTATGAACAGGAGGAAAAA AlaArgTrpSerSerPheLysLysAsnGlyAlaIleLysValLeuArgGlyPheLysLysGluIleSerSerMetLeuAsnIleMetAsnArgArgLysLys> 410420430440450460470480490500 ATCTGTGACCATGCTCCTCATGCTGCTGCCCACAGCCCTGGCGTTCCATTTGACCACACGAGGGGGAGAGCCACACATGATAGTTAGTAAGCAGGAAAGA SerValThrMetLeuLeuMetLeuLeuProThrAlaLeuAlaPheHisLeuThrThrArgGlyGlyGluProHisMetIleValSerLysGlnGluArg> 510520530540550560570580590600 GGAAAGTCACTGTTGTTTAAGACCTCTGCAGGCATCAATATGTGCACTCTCATTGCGATGGATTTGGGAGAGTTATGCGAGGACACAATGACCTACAAAT GlyLysSerLeuLeuPheLysThrSerAlaGlyIleAsnMetCysThrLeuIleAlaMetAspLeuGlyGluLeuCysGluAspThrMetThrTyrLys> 610620630640650660670680690700 GCCCCCGGATCACTGAGGCGGAACCAGATGACGTTGACTGCTGGTGCAATGCCACAGACACATGGGTGACCTATGGGACGTGTTCTCAAACCGGCGAACA CysProArgIleThrGluAlaGluProAspAspValAspCysTrpCysAsnAlaThrAspThrTrpValThrTyrGlyThrCysSerGlnThrGlyGluHis> 710720730740750760770780790800 CCGACGAGACAAACGTTCCGTGGCACTGGCCCCACACGTGGGACTTGGTCTAGAAACAAGAACCGAAACATGGATGTCCTCTGAAGGTGCCTGGAAACAA ArgArgAspLysArgSerValAlaLeuAlaProHisValGlyLeuGlyLeuGluThrArgThrGluThrTrpMetSerSerGluGlyAlaTrpLysGln> 810820830840850860870880890900 GTACAAAAAGTGGAGACTTGGGCTTTGAGACACCCAGGATTCACGGTGACAGCCCTTTTTTTAGCACATGCCATAGGAACATCCATTACTCAGAAAGGGA ValGlnLysValGluThrTrpAlaLeuArgHisProGlyPheThrValThrAlaLeuPheLeuAlaHisAlaIleGlyThrSerIleThrGlnLysGly> 9109209309409509609709809901000 TCATTTTCATTCTGCTGATGCTAGTAACACCATCAATGGCCATGCGATGTGTGGGAATAGGCAACAGAGACTTCGTTGAAGGACTGTCAGGAGCAACGTG IleIlePheIleLeuLeuMetLeuValThrProSerMetAlaMetArgCysValGlyIleGlyAsnArgAspPheValGluGlyLeuSerGlyAlaThrTrp> 1010102010301040105010601070108010901100 GGTGGACGTGGTATTGGAGCATGGAAGCTGCGTCACCACCATGGCAAAAGATAAACCAACATTGGACATTGAACTCTTGAAGACGGAGGTCACAAACCCT ValAspValValLeuGluHisGlySerCysValThrThrMetAlaLysAspLysProThrLeuAspIleGluLeuLeuLysThrGluValThrAsnPro> 1110112011301140115011601170118011901200 GCCGTCTTGCGCAAACTGTGCATTGAAGCTAAAATATCAAACACCACCACCGATTCAAGGTGTCCAACACAAGGAGAGGCTACACTGGTGGAAGAACAGG AlaValLeuArgLysLeuCysIleGluAlaLysIleSerAsnThrThrThrAspSerArgCysProThrGlnGlyGluAlaThrLeuValGluGluGln> 1210122012301240125012601270128012901300 ACTCGAACTTTGTGTGTCGACGAACGTTTGTGGACAGAGGCTGGGGTAATGGCTGCGGACTATTTGGAAAAGGAAGCCTACTGACGTGTGCTAAGTTCAA AspSerAsnPheValCysArgArgThrPheValAspArgGlyTrpGlyAsnGlyCysGlyLeuPheGlyLysGlySerLeuLeuThrCysAlaLysPheLys> 1310132013301340135013601370138013901400 GTGTGTGACAAAACTAGAAGGAAAGATAGTTCAATATGAAAACTTAAAATATTCAGTGATAGTCACTGTCCACACTGGGGACCAGCACCAGGTGGGAAAC CysValThrLysLeuGluGlyLysIleValGlnTyrGluAsnLeuLysTyrSerValIleValThrValHisThrGlyAspGlnHisGlnValGlyAsn> 1410142014301440145014601470148014901500 GAGACTACAGAACATGGAACAATTGCAACCATAACACCTCAAGCTCCTACGTCGGAAATACAGCTGACTGACTACGGAGCCCTCACATTGGACTGCTCGC GluThrThrGluHisGlyThrIleAlaThrIleThrProGlnAlaProThrSerGluIleGlnLeuThrAspTyrGlyAlaLeuThrLeuAspCysSer> 1510152015301540155015601570158015901600 CTAGAACAGGGCTGGACTTTAATGAGATGGTTCTATTGACAATGAAAGAAAAATCATGGCTTGTCCACAAACAATGGTTTCTAGACTTACCACTGCCTTG ProArgThrGlyLeuAspPheAsnGluMetValLeuLeuThrMetLysGluLysSerTrpLeuValHisLysGlnTrpPheLeuAspLeuProLeuProTrp> 1610162016301640165016601670168016901700 GACTTCAGGAGCTTCAACATCTCAAGAGACTTGGAACAGACAAGATTTGCTGGTCACATTCAAGACAGCTCATGCAAAGAAACAGGAAGTAGTCGTACTG ThrSerGlyAlaSerThrSerGlnGluThrTrpAsnArgGlnAspLeuLeuValThrPheLysThrAlaHisAlaLysLysGlnGluValValValLeu> 1710172017301740175017601770178017901800 GGATCACAGGAAGGAGCAATGCACACTGCGTTGACTGGGGCGACAGAAATCCAGACGTCAGGAACGACAACAATCTTTGCAGGACACCTGAAATGCAGAC GlySerGlnGluGlyAlaMetHisThrAlaLeuThrGlyAlaThrGluIleGlnThrSerGlyThrThrThrIlePheAlaGlyHisLeuLysCysArg> 1810182018301840185018601870188018901900 TAAAAATGGATAAACTGACTTTAAAAGGGAGTCATATGTAATGTGCACAGGCTCATTTAAGCTAGAGAAGGAAGTGGCTGAGACCCAGCATGGAACTGT LeuLysMetAspLysLeuThrLeuLysGlyMetSerTyrValMetCysThrGlySerPheLysLeuGluLysGluValAlaGluThrGlnHisGlyThrVal> 1910192019301940195019601970198019902000 TTTAGTGCAGGTTAAATACGAAGGAACAGATGCGCCATGCAAGATCCCTTTTTCGGCCCAAGATGAGAAAGGAGTGACCCAGAATGGGAGATTGATAACA LeuValGlnValLysTyrGluGlyThrAspAlaProCysLysIleProPheSerAlaGlnAspGluLysGlyValThrGlnAsnGlyArgLeuIleThr> 2010202020302040205020602070208020902100 GCCAACCCCATAGTCACTGACAAAGAAAAACCAGTCAACATTGAGACAGAACCACCTTTTGGTGAGAGCTACATCGTGGTAGGGGCAGGTGAAAAAGCTT AlaAsnProIleValThrAspLysGluLysProValAsnIleGluThrGluProProPheGlyGluSerTyrIleValValGlyAlaGlyGluLysAla> 2110212021302140215021602170218021902200 TGAAACTGAGCTGGTTCAAGAAAGGGAGCAGCATAGGGAAAATGTTCGAAGCAACTGCCCGAGGAGCGCGAAGGATGGCTATCCTGGGAGACACCGCATG LeuLysLeuSerTrpPheLysLysGlySerSerIleGlyLysMetPheGluAlaThrAlaArgGlyAlaArgArgMetAlaIleLeuGlyAspThrAlaTrp> 2210222022302240225022602270228022902300 GGACTTTGGCTCTATAGGAGGAGTGTTCACATCAGTGGGAAAATTGGTACACCAGGTTTTTGGAGCCGCATATGGGGTTCTGTTCAGCGGTGTTTCTTGG AspPheGlySerIleGlyGlyValPheThrSerValGlyLysLeuValHisGlnValPheGlyAlaAlaTyrGlyValLeuPheSerGlyValSerTyr> 2310232023302340235023602370238023902400 ACCATGAAAATAGGAATAGGGATTCTGCTGACATGGCTAGGATTAAACTCGAGGAACACTTCAATGGCTATGACGTGCATAGCTGTTGGAGGAATCACTC ThrMetLysIleGlyIleGlyIleLeuLeuThrTrpLeuGlyLeuAsnSerArgAsnThrSerMetAlaMetThrCysIleAlaValGlyGlyIleThr> 24102420 TGTTTCTGGGCTTCACAGTTCAAGCA LeuPheLeuGlyPheThrValGlnAla> Bases 1 to 88 (BglII): DEN4 Bases 89 (BglII) to 2348 (XhoI): DEN1 Bases 2349 (XhoI) to 2426: DEN4 Bases 102 to 443: C protein ORF Bases 444 to 941: prM protein ORF Bases 942 to 2426: E protein ORF
(175) TABLE-US-00047 APPENDIX4 NucleotideandaminoacidsequenceofDEN1(PuertoRico/94)MEchimericregion 102030405060708090100 AGTTGTTAGTCTGTGTGGACCGACAAGGACAGTTCCAAATCGGAAGCTTGCTTAACACAGTTCTAACAGTTTGTTTGAATAGAGAGCAGATCTCTGGAAA 110120130140150160170180190200 AATGAACCAACGAAAAAAGGTGGTTAGACCACCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTATCAACCCCTCAAGGGTTGGTGAAGAGATTCTCA MetAsnGlnArgLysLysValValArgProProPheAsnMetLeuLysArgGluArgAsnArgValSerThrProGlnGlyLeuValLysArgPheSer> 210220230240250260270280290300 ACCGGACTTTTTTCTGGGAAAGGACCCTTACGGATGGTGCTAGCATTCATCACGTTTTTGCGAGTCCTTTCCATCCCACCAACAGCAGGGATTCTGAAGA ThrGlyLeuPheSerGlyLysGlyProLeuArgMetValLeuAlaPheIleThrPheLeuArgValLeuSerIleProProThrAlaGlyIleLeuLys> 310320330340350360370380390400 GATGGGGACAGTTGAAGAAAAATAAGGCCATCAAGATACTGATTGGATTCAGGAAGGAGATAGGCCGCATGCTGAACATCTTGAACGGGAGAAAAAGGTC ArgTrpGlyGlnLeuLysLysAsnLysAlaIleLysIleLeuIleGlyPheArgLysGluIleGlyArgMetLeuAsnIleLeuAsnGlyArgLysArgSer> 410420430440450460470480490500 TGCAGCCATGCTCCTCATGCTGCTGCCCACAGCCCTGGCGTTCCATTTGACCACACGAGGGGGAGAGCCACACATGATAGTTAGTAAGCAGGAAAGAGGA AlaAlaMetLeuLeuMetLeuLeuProThrAlaLeuAlaPheHisLeuThrThrArgGlyGlyGluProHisMetIleValSerLysGlnGluArgGly> 510520530540550560570580590600 AAGTCACTGTTGTTTAAGACCTCTGCAGGCATCAATATGTGCACTCTCATTGCGATGGATTTGGGAGAGTTATGCGAGGACACAATGACCTACAAATGCC LysSerLeuLeuPheLysThrSerAlaGlyIleAsnMetCysThrLeuIleAlaMetAspLeuGlyGluLeuCysGluAspThrMetThrTyrLysCys> 610620630640650660670680690700 CCCGGATCACTGAGGCGGAACCAGATGACGTTGACTGCTGGTGCAATGCCACAGACACATGGGTGACCTATGGGACGTGTTCTCAAACCGGCGAACACCG ProArgIleThrGluAlaGluProAspAspValAspCysTrpCysAsnAlaThrAspThrTrpValThrTyrGlyThrCysSerGlnThrGlyGluHisArg> 710720730740750760770780790800 ACGAGACAAACGTTCCGTGGCACTGGCCCCACACGTGGGACTTGGTCTAGAAACAAGAACCGAAACATGGATGTCCTCTGAAGGTGCCTGGAAACAAGTA ArgAspLysArgSerValAlaLeuAlaProHisValGlyLeuGlyLeuGluThrArgThrGluThrTrpMetSerSerGluGlyAlaTrpLysGlnVal> 810820830840850860870880890900 CAAAAAGTGGAGACTTGGGCTTTGAGACACCCAGGATTCACGGTGACAGCCCTTTTTTTAGCACATGCCATAGGAACATCCATTACTCAGAAAGGGATCA GlnLysValGluThrTrpAlaLeuArgHisProGlyPheThrValThrAlaLeuPheLeuAlaHisAlaIleGlyThrSerIleThrGlnLysGlyIle> 9109209309409509609709809901000 TTTTCATTCTGCTGATGCTAGTAACACCATCAATGGCCATGCGATGTGTGGGAATAGGCAACAGAGACTTCGTTGAAGGACTGTCAGGAGCAACGTGGGT IlePheIleLeuLeuMetLeuValThrProSerMetAlaMetArgCysValGlyIleGlyAsnArgAspPheValGluGlyLeuSerGlyAlaThrTrpVal> 1010102010301040105010601070108010901100 GGACGTGGTATTGGAGCATGGAAGCTGCGTCACCACCATGGCAAAAGATAAACCAACATTGGACATTGAACTCTTGAAGACGGAGGTCACAAACCCTGCC AspValValLeuGluHisGlySerCysValThrThrMetAlaLysAspLysProThrLeuAspIleGluLeuLeuLysThrGluValThrAsnProAla> 1110112011301140115011601170118011901200 GTCTTGCGCAAACTGTGCATTGAAGCTAAAATATCAAACACCACCACCGATTCAAGGTGTCCAACACAAGGAGAGGCTACACTGGTGGAAGAACAGGACT ValLeuArgLysLeuCysIleGluAlaLysIleSerAsnThrThrThrAspSerArgCysProThrGlnGlyGluAlaThrLeuValGluGluGlnAsp> 1210122012301240125012601270128012901300 CGAACTTTGTGTGTCGACGAACGTTTGTGGACAGAGGCTGGGGTAATGGCTGCGGACTATTTGGAAAAGGAAGCCTACTGACGTGTGCTAAGTTCAAGTG SerAsnPheValCysArgArgThrPheValAspArgGlyTrpGlyAsnGlyCysGlyLeuPheGlyLysGlySerLeuLeuThrCysAlaLysPheLysCys> 1310132013301340135013601370138013901400 TGTGACAAAACTAGAAGGAAAGATAGTTCAATATGAAAACTTAAAATATTCAGTGATAGTCACTGTCCACACTGGGGACCAGCACCAGGTGGGAAACGAG ValThrLysLeuGluGlyLysIleValGlnTyrGluAsnLeuLysTyrSerValIleValThrValHisThrGlyAspGlnHisGlnValGlyAsnGlu> 1410142014301440145014601470148014901500 ACTACAGAACATGGAACAATTGCAACCATAACACCTCAAGCTCCTACGTCGGAAATACAGCTGACTGACTACGGAGCCCTCACATTGGACTGCTCGCCTA ThrThrGluHisGlyThrIleAlaThrIleThrProGlnAlaProThrSerGluIleGlnLeuThrAspTyrGlyAlaLeuThrLeuAspCysSerPro> 1510152015301540155015601570158015901600 GAACAGGGCTGGACTTTAATGAGATGGTTCTATTGACAATGAAAGAAAAATCATGGCTTGTCCACAAACAATGGTTTCTAGACTTACCACTGCCTTGGAC ArgThrGlyLeuAspPheAsnGluMetValLeuLeuThrMetLysGluLysSerTrpLeuValHisLysGlnTrpPheLeuAspLeuProLeuProTrpThr> 1610162016301640165016601670168016901700 TTCAGGAGCTTCAACATCTCAAGAGACTTGGAACAGACAAGATTTGCTGGTCACATTCAAGACAGCTCATGCAAAGAAACAGGAAGTAGTCGTACTGGGA SerGlyAlaSerThrSerGlnGluThrTrpAsnArgGlnAspLeuLeuValThrPheLysThrAlaHisAlaLysLysGlnGluValValValLeuGly> 1710172017301740175017601770178017901800 TCACAGGAAGGAGCAATGCACACTGCGTTGACTGGGGCGACAGAAATCCAGACGTCAGGAACGACAACAATCTTTGCAGGACACCTGAAATGCAGACTAA SerGlnGluGlyAlaMetHisThrAlaLeuThrGlyAlaThrGluIleGlnThrSerGlyThrThrThrIlePheAlaGlyHisLeuLysCysArgLeu> 1810182018301840185018601870188018901900 AAATGGATAAACTGACTTTAAAAGGGATGTCATATGTAATGTGCACAGGCTCATTTAAGCTAGAGAAGGAAGTGGCTGAGACCCAGCATGGAACTGTTTT LysMetAspLysLeuThrLeuLysGlyMetSerTyrValMetCysThrGlySerPheLysLeuGluLysGluValAlaGluThrGlnHisGlyThrValLeu> 1910192019301940195019601970198019902000 AGTGCAGGTTAAATACGAAGGAACAGATGCGCCATGCAAGATCCCTTTTTCGGCCCAAGATGAGAAAGGAGTGACCCAGAATGGGAGATTGATAACAGCC ValGlnValLysTyrGluGlyThrAspAlaProCysLysIleProPheSerAlaGlnAspGluLysGlyValThrGlnAsnGlyArgLeuIleThrAla> 2010202020302040205020602070208020902100 AACCCCATAGTCACTGACAAAGAAAAACCAGTCAACATTGAGACAGAACCACCTTTTGGTGAGAGCTACATCGTGGTAGGGGCAGGTGAAAAAGCTTTGA AsnProIleValThrAspLysGluLysProValAsnIleGluThrGluProProPheGlyGluSerTyrIleValValGlyAlaGlyGluLysAlaLeu> 2110212021302140215021602170218021902200 AACTGAGCTGGTTCAAGAAAGGGAGCAGCATAGGGAAAATGTTCGAAGCAACTGCCCGAGGAGCGCGAAGGATGGCTATCCTGGGAGACACCGCATGGGA LysLeuSerTrpPheLysLysGlySerSerIleGlyLysMetPheGluAlaThrAlaArgGlyAlaArgArgMetAlaIleLeuGlyAspThrAlaTrpAsp> 2210222022302240225022602270228022902300 CTTTGGCTCTATAGGAGGAGTGTTCACATCAGTGGGAAAATTGGTACACCAGGTTTTTGGAGCCGCATATGGGGTTCTGTTCAGCGGTGTTTCTTGGACC PheGlySerIleGlyGlyValPheThrSerValGlyLysLeuValHisGlnValPheGlyAlaAlaTyrGlyValLeuPheSerGlyValSerTrpThr> 2310232023302340235023602370238023902400 ATGAAAATAGGAATAGGGATTCTGCTGACATGGCTAGGATTAAACTCGAGGAACACTTCAATGGCTATGACGTGCATAGCTGTTGGAGGAATCACTCTGT MetLysIleGlyIleGlyIleLeuLeuThrTrpLeuGlyLeuAsnSerArgAsnThrSerMetAlaMetThrCysIleAlaValGlyGlyIleThrLeu> 24102420 TTCTGGGCTTCACAGTTCAAGCA PheLeuGlyPheThrValGlnAla Bases 1 to 404 (PstI): DEN4 Bases 405 (PstI) to 2345 (XhoI): DEN1 Bases 2346 (XhoI) to 2423: DEN4 Bases 102 to 440: C protein ORF Bases 441 to 938: prM protein ORF Bases 939 to 2423: E protein ORF
(176) While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.