Meningococcal fHBP polypeptides

Abstract

fHBP is a protein in Neisseria meningitidis. Three families of fHBP are known. To increase the ability of a fHBP protein to elicit antibodies that are cross-reactive between the families, fHBP is selected or engineered to have a sequence which can elicit broad-spectrum bactericidal anti-meningococcal antibodies after administration to a host animal.

Claims

1. An immunogenic composition comprising (a) an isolated polypeptide comprising an amino acid sequence (i) which has at least 94% sequence identity to SEQ ID NO: 76 and/or (ii) comprises a first fragment and a second fragment of SEQ ID NO: 76, wherein said first fragment includes at least 28 contiguous amino acids from within amino acids 89-154 of SEQ ID NO: 76, and said second fragment includes at least 28 contiguous amino acids from within amino acids 187-248 of SEQ ID NO: 76, and (b) an immunostimulatory amount of an aluminium salt adjuvant.

2. A plasmid comprising an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence (i) which has at least 94% sequence identity to SEQ ID NO: 76 and/or (ii) comprises a first fragment and a second fragment of SEQ ID NO: 76, wherein said first fragment includes at least 28 contiguous amino acids from within amino acids 89-154 of SEQ ID NO: 76, and said second fragment includes at least 28 contiguous amino acids from within amino acids 187-248 of SEQ ID NO: 76.

3. An isolated host cell transformed with the plasmid of claim 2.

4. The host cell of claim 3, wherein the cell is a meningococcal bacterium.

5. An immunogenic composition comprising: (a) isolated membrane vesicles, wherein the vesicles include a polypeptide comprising an amino acid sequence (i) which has at least 94% sequence identity to SEQ ID NO: 76 and/or (ii) comprises a first fragment and a second fragment of SEQ ID NO: 76, wherein said first fragment includes at least 28 contiguous amino acids from within amino acids 89-154 of SEQ ID NO: 76, and said second fragment includes at least 28 contiguous amino acids from within amino acids 187-248 of SEQ ID NO: 76, and (b) an immunostimulatory amount of an aluminium salt adjuvant.

6. The immunogenic composition of claim 1 or claim 5, further comprising a second polypeptide that, when administered to a mammal, elicits an antibody response that is bactericidal against meningococcus, provided that the second polypeptide is not a meningococcal factor H binding protein (fHBP).

7. The immunogenic composition of claim 1 or claim 5, further comprising a conjugated capsular saccharide from N. meningitidis serogroup A, C, W135 and/or Y.

8. The immunogenic composition of claim 1 or claim 5, further comprising a conjugated pneumococcal capsular saccharide.

9. A method for raising an antibody response against meningococcus in a mammal, comprising administering the immunogenic composition of claim 1.

10. A method for raising an antibody response against meningococcus in a mammal, comprising administering the immunogenic composition of claim 5.

11. The immunogenic composition of claim 1, wherein the aluminium salt adjuvant is aluminium hydroxide.

12. The immunogenic composition of claim 5, wherein the aluminium salt adjuvant is aluminium hydroxide.

Description

MODES FOR CARRYING OUT THE INVENTION

(1) With the wild-type MC58 sequence (SEQ ID NO: 1) as a baseline, 72 modified fHBP sequences have been prepared. These are shown in the sequence listing as SEQ ID NOs: 4 to 75.

(2) Polypeptides have been expressed in E. coli with a N-terminus methionine followed immediately by a SEQ ID NO amino acid sequence. The polypeptides have been combined with Freund's complete adjuvant, MF59 or an aluminium hydroxide adjuvant and then used to immunise mice. Antisera from the mice have been tested in a bactericidal assay against a panel of ten meningococcal strains. The panel included strains from each of the three fHBP families. Wild-type MC58 polypeptide was also prepared.

(3) Two of the polypeptides which were expressed and purified are SEQ ID NOs 79 (PATCH_9C) and 80 (PATCH_10A), comprising SEQ ID NOs: 20 and 23, respectively. All of the tested polypeptides elicited sera which displayed bactericidal activity (SBA titre128) against at least two strains in the panel (and usually more), but the 9C and 10A polypeptides were noteworthy because their sera showed good bactericidal activity across the whole panel, including usefully against the M1239 strain. SBA titres with 9C and 10A were as follows:

(4) TABLE-US-00003 SEQ ID NO: 1 SEQ ID NO: 79 SEQ ID NO: 80 Strain fHBP family FCA AlH MF59 AlH MF59 AlH MC58 1 >32768 32768 16384 >32768 2048 16384 NM008 1 4096 <16 1024 4096 64 128 M4030 1 2048 256 4096 32768 64 1024 GB185 1 2048 32 256 >8192 512 2048 NZ 1 4096 128 256 >8192 16 256 961-5945 2 128 <16 1024 4096 4096 2048 M3153 2 128 <16 1024 2048 4096 4096 C11 2 32 <16 64 512 512 128 M2552 2 <16 <16 4096 2048 >8192 2048 M1239 3 64 <16 <16 512 2048 1024

(5) Thus anti-fHBP antibodies elicited by the wild-type MC58 sequence are effective against strains that express a family I fHBP, but are relatively ineffective against strains in fHBP family 2 or 3. In contrast, sera elicited by the two modified sequences are effective against a panel of strains including all three fHBP families. SEQ ID NO: 79 (PATCH_9C) is particularly effective in this regard. In particular, in many cases the sera obtained against this polypeptide were at least as effective against the ten strains, as control sera raised against the strain's own fHBP (when using an aluminium hydroxide adjuvant). In all but one case there was no more than one dilution's decrease:

(6) TABLE-US-00004 Strain fHBP family Homologous fHBP SEQ ID NO: 79 MC58 1 32768 >32768 NM008 1 no data 4096 M4030 1 256 32768 GB185 1 2048 >8192 NZ 1 2048 >8192 961-5945 2 2048 4096 M3153 2 2048 2048 C11 2 1024 512 M2552 2 >8192 2048 M1239 3 1024 512

(7) Activity of the PATCH_9C mutant was confirmed after formulation with Freund's Complete Adjuvant (FCA) or a mixture of IC31 with alum:

(8) TABLE-US-00005 Strain fHBP family FCA adjuvant IC31 + Alum MC58 1 8192 >32768 NM008 1 1024 1024 M4030 1 8192 2048 GB185 1 8192 4096 NZ 1 2048 4096 961-5945 2 8192 4096 M3153 2 2048 4096 C11 2 2048 1024 M2552 2 2048 4096 M1239 3 512 1024

(9) SEQ ID NO: 77 is a fHBP sequence found in a wild-type strain (NL096). It is encoded by SEQ ID NO: 78. Although this is a natural sequence it does not fit well into the variants which have previously been reported. Instead, it appears to be an intermediate between families I and II. Sera raised using a G form of the NL096 sequence, using two different adjuvants (FCA or aluminium hydroxide), were tested against a panel of eight strains and bactericidal titres were as follows:

(10) TABLE-US-00006 Strain fHBP family FCA AlH NL096 8192 1024 MC58 1 32768 2048 NM008 1 4096 1024 GB185 1 16384 2048 NZ98/254 1 1024 256 961-5945 2 >32768 8192 M3153 2 >32768 8192 M1239 3 4096 256

(11) Thus the NL096 polypeptide elicits antibodies that display a broad spectrum of bactericidal activity. For all of the heterologous strains except NZ98/254 the titres obtained using FCA were at least as high as the titres obtained using IC31+alum with the PATCH_9C mutant.

(12) It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(13) References

(14) [1] Jodar et al. (2002) Lancet 359(9316):1499-1508. [2] Pizza et al. (2000) Science 287:1816-1820. [3] WO99/57280. [4] Masignani et al. (2003) J Exp Med 197:789-799. [5] Welsch et al. (2004) J Immunol 172:5605-15. [6] Hou et al. (2005) J Infect Dis 192(4):580-90. [7] WO03/063766. [8] Fletcher et al. (2004) Infect Immun 72:2088-2100. [9] Zhu et al. (2005) Infect Immun 73(10):6838-45. [10] WO01/64920. [11] WO03/020756. [12] WO2004/048404. [13] WO2006/024954. [14] WO2007/060548. [15] Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453. [16] Rice et al. (2000) Trends Genet. 16:276-277. [17] Achtman (1995) Global epidemiology of meningococcal disease. Pages 159-175 of Meningococcal disease (ed. Cartwight). ISBN: 0-471-95259-1. [18] Caugant (1998) APMIS 106:505-525. [19] Maiden et al. (1998) Proc. Natl. Acad. Sci. USA 95:3140-3145. [20] WO01/30390. [21] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [22] WO03/009869. [23] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum. [24] WO00/23105. [25] WO90/14837. [26] WO90/14837. [27] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203. [28] Podda (2001) Vaccine 19: 2673-2680. [29] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). [30] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan. [31] U.S. Pat. No. 5,057,540. [32] WO96/33739. [33] EP-A-0109942. [34] WO96/11711. [35] WO00/07621. [36] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271. [37] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338. [38] Niikura et al. (2002) Virology 293:273-280. [39] Lenz et al. (2001) J Immunol 166:5346-5355. [40] Pinto et al. (2003) J Infect Dis 188:327-338. [41] Gerber et al. (2001) J Virol 75:4752-4760. [42] WO03/024480. [43] WO03/024481. [44] Gluck et al. (2002) Vaccine 20:B10-B16. [45] EP-A-0689454. [46] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278. [47] Evans et al. (2003) Expert Rev Vaccines 2:219-229. [48] Meraldi et al. (2003) Vaccine 21:2485-2491. [49] Pajak et al. (2003) Vaccine 21:836-842. [50] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400. [51] WO02/26757. [52] WO99/62923. [53] Krieg (2003) Nature Medicine 9:831-835. [54] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [55] WO98/40100. [56] U.S. Pat. No. 6,207,646. [57] U.S. Pat. No. 6,239,116. [58] U.S. Pat. No. 6,429,199. [59] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658. [60] Blackwell et al. (2003) J Immunol 170:4061-4068. [61] Krieg (2002) Trends Immunol 23:64-65. [62] WO01/95935. [63] Kandimalla et al. (2003) BBRC 306:948-953. [64] Bhagat et al. (2003) BBRC 300:853-861. [65] WO03/035836. [66] Schellack et al. (2006) Vaccine 24:5461-72. [67] WO95/17211. [68] WO98/42375. [69] Beignon et al. (2002) Infect Immun 70:3012-3019. [70] Pizza et al. (2001) Vaccine 19:2534-2541. [71] Pizza et al. (2000) Int J Med Microbiol 290:455-461. [72] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313. [73] Ryan et al. (1999) Infect Immun 67:6270-6280. [74] Partidos et al. (1999) Immunol Lett 67:209-216. [75] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293. [76] Pine et al. (2002) J Control Release 85:263-270. [77] Tebbey et al. (2000) Vaccine 18:2723-34. [78] Domenighini et al. (1995) Mol Microbiol 15:1165-1167. [79] WO99/40936. [80] WO99/44636. [81] Singh et all (2001) J Cont Release 70:267-276. [82] WO99/27960. [83] U.S. Pat. No. 6,090,406. [84] U.S. Pat. No. 5,916,588. [85] EP-A-0626169. [86] WO99/52549. [87] WO01/21207. [88] WO01/21152. [89] Andrianov et al. (1998) Biomaterials 19:109-115. [90] Payne et al. (1998) Adv Drug Delivery Review 31:185-196. [91] Stanley (2002) Clin Exp Dermatol 27:571-577. [92] Jones (2003) Curr Opin Investig Drugs 4:214-218. [93] WO99/11241. [94] WO94/00153. [95] WO98/57659. [96] European patent applications 0835318, 0735898 and 0761231. [97] WO99/24578. [98] WO99/36544. [99] Costantino et al. (1992) Vaccine 10:691-698. [100] Costantino et al. (1999) Vaccine 17:1251-1263. [101] WO03/007985. [102] Watson (2000) Pediatr Infect Dis J 19:331-332. [103] Rubin (2000) Pediatr Clin North Am 47:269-285, v. [104] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207. [105] Bell (2000) Pediatr Infect Dis J19:1187-1188. [106] Iwarson (1995) APMIS103:321-326. [107] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80. [108] Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0. [109] Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70. [110] Gustafsson et al. (1996) N Engl. J. Med. 334:349-355. [111] Rappuoli et al. (1991) TIBTECH 9:232-238. [112] Sutter et al. (2000) Pediatr Clin North Am 47:287-308. [113] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126. [114] McMichael (2000) Vaccine 19 Suppl 1:S101-107. [115] Schuchat (1999) Lancet 353(9146):51-6. [116] WO02/34771. [117] Dale (1999) Infect Dis Clin North Am 13:227-43, viii. [118] Ferretti et al. (2001) PNAS USA 98: 4658-4663. [119] Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages 1218-1219. [120] Jones (2001) Curr Opin Investig Drugs 2:47-49. [121] Ravenscroft et al. (1999) Vaccine 17:2802-2816. [122] WO03/080678. [123] Research Disclosure, 453077 (January 2002). [124] EP-A-0372501. [125] EP-A-0378881. [126] EP-A-0427347. [127] WO93/17712. [128] WO94/03208. [129] WO98/58668. [130] EP-A-0471177. [131] WO91/01146. [132] Falugi et al. (2001) Eur J Immunol 31:3816-3824. [133] Baraldo et al. (2004) Infect Immun 72(8):4884-7. [134] EP-A-0594610. [135] Ruan et al. (1990) J Immunol 145:3379-3384. [136] WO00/56360. [137] Kuo et al. (1995) Infect Immun 63:2706-13. [138] Michon et al. (1998) Vaccine. 16:1732-41. [139] WO02/091998. [140] WO01/72337. [141] WO00/61761. [142] WO00/33882 [143] Lees et al. (1996) Vaccine 14:190-198. [144] WO95/08348. [145] U.S. Pat. No. 4,882,317 [146] U.S. Pat. No. 4,695,624 [147] Porro et al. (1985) Mol Immunol 22:907-919.s [148] EP-A-0208375 [149] WO00/10599 [150] Geyer et al. Med. Microbiol. Immunol, 165: 171-288 (1979). [151] U.S. Pat. No. 4,057,685. [152] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700. [153] U.S. Pat. No. 4,459,286. [154] U.S. Pat. No. 4,965,338 [155] U.S. Pat. No. 4,663,160. [156] U.S. Pat. No. 4,761,283 [157] U.S. Pat. No. 4,356,170 [158] WO02/09643. [159] Katial et al. (2002) Infect Immun 70:702-707. [160] WO01/52885. [161] European patent 0301992. [162] Bjune et al. (1991) Lancet 338(8775):1093-1096. [163] Fukasawa et al. (1999) Vaccine 17:2951-2958. [164] WO02/09746. [165] Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333. [166] WO01/09350. [167] European patent 0449958. [168] EP-A-0996712. [169] EP-A-0680512. [170] WO02/062378. [171] WO99/59625. [172] U.S. Pat. No. 6,180,111. [173] WO01/34642. [174] WO03/051379. [175] U.S. Pat. No. 6,558,677. [176] WO2004/019977. [177] WO02/062380. [178] WO00/25811. [179] Peeters et al. (1996) Vaccine 14:1008-1015. [180] Vermont et al. (2003) Infect Immun 71:1650-1655. [181] WO2006/081259. [182] WO02/09643. [183] Katial et al. (2002) Infect. Immun. 70:702-707. [184] U.S. Pat. No. 6,180,111. [185] WO01/34642. [186] WO2004/019977. [187] European patent 0011243. [188] Fredriksen et al. (1991) NIPH Ann. 14(2):67-80. [189] WO01/91788. [190] WO2005/004908. [191] WO98/56901. [192] Claassen et al. (1996) 14(10):1001-8. [193] WO99/10497. [194] Steeghs et al. (2001) The EMBO Journal 20:6937-6945. [195] WO01/52885. [196] WO00/25811. [197] WO2004/015099. [198] WO01/09350. [199] WO02/09746. [200] WO02/062378. [201] WO2004/014417. [202] WO2004/046177. [203] WO2004/094596
Alternative Names for Sequences in the Sequence Listing

(15) TABLE-US-00007 SEQ ID NO: Description 1 fHBP, strain MC58 - family I 2 fHBP, strain 961-5945 & 2996 - family II 3 fHBP, strain M1239 -family III 4 LOOP2 5 PATCH_1 6 PATCH_2 7 PATCH_2S 8 PATCH_2T 9 PATCH_2FAT 10 PATCH_3 11 PATCH_5 12 PATCH_5bis 13 PATCH_5tris 14 PATCH_5tetra 15 PATCH_5penta 16 PATCH_8 17 PATCH_8B 18 PATCH_9 19 PATCH_9B 20 PATCH_9C 21 PATCH_9D 22 PATCH_9E 23 PATCH_10A 24 PATCH_10B 25 PATCH_10C 26 PATCH_10D 27 PATCH_10E 28 PATCH_10F 29 PATCH_10G 30 PATCH_10H 31 PATCH_11 32 PATCH_11B 33 PATCH_11C 34 PATCH_11D 35 PATCH_11E 36 PATCH_11F 37 PATCH_11G 38 PATCH_11H 39 PATCH_11I 40 PATCH_11L 41 PATCH_12 42 PATCH_12B 43 PATCH_12C 44 PATCH_12D 45 PATCH_12E 46 PATCH_12F 47 PATCH_12G 48 PATCH_12H 49 PATCH_12I 50 PATCH_12L 51 PATCH_12M 52 PATCH_12N 53 PATCH_13 54 PATCH_13B 55 PATCH_13C 56 PATCH_14 57 PATCH_14B 58 PATCH_14C 59 PATCH_14D 60 PATCH_15A 61 PATCH_15B 62 PATCH_16A 63 PATCH_16B 64 PATCH_16C 65 PATCH_16D 66 PATCH_16E 67 PATCH_16F 68 PATCH_16G 69 PATCH_17A 70 PATCH_17B 71 PATCH_17C 72 PATCH_18A 73 PATCH_18B 74 PATCH_18C 75 PATCH_18D 76 NL096 77 & 78 NL096_FULL 79 Met-PATCH_9C 80 Met-PATCH_10A 81 IC31 82 IC31 83 aa 27-274 of SEQ 1