AU2006200732B2 - Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins - Google Patents

Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins Download PDF

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AU2006200732B2
AU2006200732B2 AU2006200732A AU2006200732A AU2006200732B2 AU 2006200732 B2 AU2006200732 B2 AU 2006200732B2 AU 2006200732 A AU2006200732 A AU 2006200732A AU 2006200732 A AU2006200732 A AU 2006200732A AU 2006200732 B2 AU2006200732 B2 AU 2006200732B2
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AU2006200732A1 (en
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Marzia Giuliani
Mariagrazia Pizza
Rino Rappuoli
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GlaxoSmithKline Biologicals SA
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Novartis Vaccines and Diagnostics SRL
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    • Y—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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Description

AUSTRALIA
Patents Act 1990 CHIRON S.R.L.
COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins The following statement is a full description of this invention including the best method of performing it known to us:- S -1 A- )OUTER MEMBRANE VESICLE (OMV) VACCINE COMPRISING N. MENINGITIDIS SEROGROUP B OUTER x1 MEMBRANE PROTEINS All documents cited herein are hereby incorporated by reference in their entirety.
*i 1 TECHNICAL FIELD This invention relates to vaccines against Neisseria meningitidis, serogroup B (NmB).
Si BACKGROUND ART S Neisseria meningitidis is a non-motile, Gram-negative diplococcus human pathogen. It colonises the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman et al. (1996) JAMA 275(19):1499-1503; Schuchat et al (1997) N Engl J Med 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).
Based on the organism's capsular polysaccharide, 12 serogroups of N.meningitidis have been identified. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Following the success of the vaccination against H.influenzae, however, conjugate vaccines against serogroups A and C have been developed Serogroup B remains a problem, however, and it is currently responsible for approximately of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of a(2-8)linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if a response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero Outschoom (1994) Clin Microbiol Rev 7(4):559-575).
An efficacious outer-membrane vesicle (OMV) vaccine against serogroup B has been produced by the Norwegian National Institute of Public Health Bjune et al (1991) Lancet S 338(8775):1093-96]. Whilst this vaccine is safe and prevents NmB disease, its efficacy is c limited to the strain used to make the vaccine. Other vaccines based around outer-membrane preparations have also been reported. It is an object of the present invention to broaden the efficacy of these vaccines to other strains.
C DISCLOSURE OF THE INVENTION O Surprisingly, it has been found that the addition of further defined components to OMV CN vaccines significantly broadens their efficacy.
o Thus the present invention provides a composition comprising a NmB outer membrane preparation, and an immunogenic component selected from one or more of the following: a protein disclosed in W099/57280, or an immunogenic fragment thereof; a protein disclosed in W099/36544, or an immunogenic fragment thereof; a protein disclosed in W099/24578, or an immunogenic fragment thereof; a protein disclosed in WO00/66791, or an immunogenic fragment thereof; a protein disclosed in Tettelin et al. [Science (2000) 287:1809-1815], or an immunogenic fragment thereof; a protein disclosed in Parkhill et al. [Nature (2000) 404:502-506], or an immunogenic fragment thereof; a protein disclosed in W097/28273, or an immunogenic fragment thereof; a protein disclosed in W096/29412, or an immunogenic fragment thereof; a protein disclosed in W095/03413, or an immunogenic fragment thereof; a protein disclosed in W099/31132, or an immunogenic fragment thereof; a protein disclosed in W099/58683, or an immunogenic fragment thereof; a protein disclosed in W099/55873, or an immunogenic fragment thereof; and/or Neisseria meningitidis protein PorA, TbpA, TbpB, PilC, OpA, or If the composition comprises a protein disclosed in W099/24578, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ IDs 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72,74,76,78,80,82,84,86,88,90,92, 94, 96,98,100,102,104,106,108, 110, 112,114, 116, 118, 120,122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160., 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, (1 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, S244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266., 268, 270, 272, 274, 276, 278, 280, 282, 284, S286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, r~~370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, S 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, c 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, S 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, S 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602,.604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 614, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,7118, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822,1824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, as disclosed in W099/24578 (or a protein comprising an immunogenic fragment of one or more of these SEQ IDs, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 90%, 95%, 99% or more) to one of these SEQ IDs).
If -the composition comprises a protein disclosed in W099/36544, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ IDs 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, as disclosed in W099/36544 (or a protein comprising an immunogenic fragment of one or more of these SEQ W~s, or a protein comprising a sequence having sequence identity (preferably grea ter than 50% eg. 60%, 90%, 95%, 99% or more) to one of these SEQ IDS).
If the composition comprises a protein disclosed in Tettelin et a protein encoded by one of the genes disclosed therein), said protein preferably comprises an amidno acid sequence selected from the group consisting of NMBOOO1 to NMB2160 (or a protein comprising an immunogenic fragment of one or more of these 2160 genes, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 70%, 80%, S 95%, 99% or more) to one of these 2160 genes).
S If the composition comprises a protein disclosed in Parkhill et al., said protein preferably comprises an amino acid sequence selected from the group consisting of the 2121 coding N sequences disclosed therein (or a protein comprising an immunogenic fragment of one or more of these 2121 sequences, or a protein comprising a sequence having sequence identity o) (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to one of these 2121 sequences).
If the composition comprises a protein disclosed in W099/57280, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ IDs 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72,74,76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 3 18, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 8-30, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068,1070, 1072, 1074, 1076, 1078., 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102,1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136,1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170,1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204,1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238,1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272,1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306,1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340,1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358. 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374,1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408,1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456,.1.458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476,1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512,1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1612,1614, 1616, 1646, 1648, 1650, 1680, 1682, 1684, 1714, 1716, 1718, 1748, 1750, 1752, 1782,1784, 1786, 1816, 1818, 1820, 1850, 1852, 1854, 1884, 1886, 1888, 1918, 1920, 1922, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 160'8, 1610, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1818, 1880, 1882, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, r 2088,2090,2092,2094,2096,2098,2100,2102,2104,2106,2108,2110,2112,2114,2116,2118,2120, 2122,2124,2126,2128,2130,2132,2134,2136,2138,2140,2142,2144,2146,2148,2150,2152,2154, c 2156,2158,2160,2162,2164,2166,2168,2170,2172,2174,2176,2178,2180,2182,2184,2186,2188, 2190,2192,2194,2196,2198,2200,2202,2204,2206,2208,2210,2212,2214,2216,2218,2220,2222, 2224,2226,2228,2230,2232,2234,2236,2238,2240,2242,2244,2246,2248,2250,2252,2254,2256, C 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, S 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, C1 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, \O 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, C1 2394,2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428,2430,2432,2434,2436,2438,2440,2442,2444,2446,2448,2450,2452,2454,2456,2458,2460, 2462,2464,2466,2468,2470,2472,2474,2476,2478,2480,2482,2484,2486,2488,2490,2492,2494, 2496,2498,2500,2502,2504,2506,2508,2510,2512,2514,2516,2518,2520,2522,2524,2526,2528, 2530,2532,2534,2536,2538,2540,2542,2544,2546,2548,2550,2552,2554,2556,2558,2560,2562, 2564,2566,2568,2570,2572,2574,2576,2578,2580,2582,2584,2586,2588,2590,2592,2594,2596, 2598,2600,2602,2604,2606,2608,2610,2612,2614,2616,2618,2620,2622,2624,2626,2628,2630, 2632,2634,2636,2638,2640,2642,2644,2646,2648,2650,2652,2654,2656,2658,2660,2662,2664, 2666,2668,2670,2672,2674,2676,2678,2680,2682,2684,2686,2688,2690,2692,2694,2696,2698, 2700,2702,2704,2706,2708,2710,2712,2714,2716,2718,2720,2722,2724,2726,2728,2730,2732, 2734,2736,2738,2740,2742,2744,2746,2748,2750,2752,2754,2756,2758,2760,2762,2764,2766, 2768,2770,2772,271,2776,2778,2780,2782,2784,2786,2788,2790,2792,2794,2796,2798,2800, 2802;2804,2806,2808,2810,2812,2814,2816,2818,2820,2822,2824,2826,2828,2830,2832,2834, 2836,2838,2840,2842,2844,2846,2848,2850,2852,2854,2856,2858,2860,2862,2864,2866,2868, 2870,2872,2874,2876,2878,2880,2882,2884,2886,2888,2890,2892,2894,2896,2898,2900,2902, 2904,2906,2908,2910,2912,2914,2916,2918,2920,2922,2924,2926,2928,2930,2932,2934,2936, 2938,2940,2942,2944,2946,2948,2950,2952,2954,2956,2958,2960,2962,2964,2966,2968,2970, 2972,2974,2976,2978,2980,2982,2984,2986,2988,2990,2992,2994,2996,2998,3000,3002,3004, 3006, 3008, 3010, 3012, 3014, 3016, 3018 3020, as disclosed in W099/57280 (or a protein comprising an immunogenic fragment of one or more of these SEQ IDs, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 90%, 95%, 99% or more) to one of these SEQ IDs).
If the composition comprises a protein disclosed in W099/28273, said protein is preferably the protein disclosed in Figure 4 or Figure 13 of W097/28273.
SIf the composition comprises a protein disclosed in W096/29412, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ IDs 1-8 c disclosed in W096/29412 (or a protein comprising an immunogenic fragment of one or more of these SEQ IDs, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to one of these SEQ IDs).
c, r- If the composition comprises a protein disclosed in W095/03413, said protein preferably
O
o comprises an amino acid sequence selected from the group consisting of SEQ IDs 1-23 0 disclosed in W095/03413 (or a protein comprising an immunogenic fragment of one or more o of these SEQ IDs, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to one of these SEQ IDs).
If the composition comprises a protein disclosed in W099/31132, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ ID 2 disclosed in W099/31132 (or a protein comprising an immunogenic fragment of SEQ ID 2, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 90%, 95%, 99% or more) to SEQ ID 2).
If the composition comprises a protein disclosed in W099/58683, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ ID 2 or SEQ ID 4 disclosed in W099/58683 (or a protein comprising an immunogenic fragment of SEQ ID 2 or SEQ ID 4, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to SEQ ID 2 or SEQ ID 4).
If the composition comprises a protein disclosed in W099/55873, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ ID 2 or SEQ ID 4 disclosed in W099/55873 (or a protein comprising an immunogenic fragment of SEQ ID 2 or SEQ ID 4, or a protein comprising a sequence having sequence identity (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to SEQ ID 2 or SEQ ID 4).
Details of Opa and PorA can be found in Wiertz et al. [Infect. Immun. (1996) 61:298-304].
PilC is disclosed in Nassif et al. [PNAS USA (1994) 91:3769-73]. Omp85 is disclosed in Manning et al. [Microb. Pathog. (1998) 25:11-21]. TbpA and TbpB are disclosed in Ala'Aldeen Borriello [Vaccine (1996) 14:49-53] and also in Legrain et al, [Prorein Expr Purif(1995) 6:570-578].
Preferred proteins for component are: 00 C protein '919', typified by SEQ IDs 3069-3074 and 3207-3241 of W099/57280 (see also Figure 23 and Example 15 therein).
1* protein '235', typified by SEQ IDs 869-874 and 3149-3178 of WO99/57280 (see Salso Figure 20 and Example 12 therein).
protein '519', typified by SEQ IDs 3045-3056 and 3185-3206 of W099/57280 (see also Figure 22 and Example 14 therein).
protein '225', typified by SEQ IDs 793-804 and 3115-3148 of W099/57280 (see O also Figure 19 and Example 11 therein).
C protein 'ORF40', typified by example 1 (SEQ IDs 1-6) of WO99/36544 I 10 corresponding to SEQ IDs 15-20 disclosed in the sequence listing herein (see Salso Figure 1 ofWO00/66741, corresponding to SEQ IDs 21-41 disclosed in the sequence listing herein; see also W099/31132 and W099/58683, corresponding to SEQ IDs 42-44 disclosed in the sequence listing herein).
protein '287', typified by example 9 of WO99/57280 (see SEQ IDs 1199-1204, 3103-3108 and 3179-3184 therein).
protein 'ORFI', typified by example 77 (SEQ IDs 647-654) ofW099/24578 (see also W099/55873 and accession number AJ242535).
protein 'ORF4', typified by example 26 (SEQ IDs 215-226) of W099/24578 (see also Figure 2 of WO00/66741).
protein 'ORF46', typified by example 55 (SEQ IDs 457-466) of W099/24578 (see also Figure 12 ofWO00/66741).
Component of the composition is preferably a NmB protein. It is preferred that component includes a protein from a different NmB strain from that from which the OMV of component is derived i.e. the OMV in component is preferably supplemented by immunogenic component from a different NmB strain.
One or more of the components (or all of them) may be adsorbed on Al(OH) 3 The outer membrane preparation component The compositions of the invention include a NmB outer membrane preparation as component This is preferably in the form of outer membrane vesicles (OMVs).
The preparation of OMVs from NmB is well-known in the art. Methods for obtaining suitable preparations are disclosed in, for instance: Claassen et al. [Vaccine (1996) 00 8A ri 14:1001-1008]; Cartwright et al. [Vaccine (1999) 17;2612-2619]; Peeters et A.
[Vaccine (1996) 14:1009- 1015]; Fu et al. [Biotechnology NY (1995) 12:170-74]; Davies et al. [J.lmmunol.Meth. (1990) 134:215-225]; Saunders et al. [Infect. Immun. (1999) 67:113-119]; Draabick et al. [Vaccine (2000) 18:160-172]; Moreno et al. [Infect. Immun. (1985) 47:527-533]; Milagres et al. [Infect.
Immun. (1994) 62:4419-4424]; Naess et al. [Infect. Immun. (1998) 66:959-965]; Rosenqvist et al. [Dev.Biol.Stand. (1998) 92:323-333]; Haneberg et al. [Infect. Immunn. (1998) 66:1334-41]; C Andersen et al. [Vaccine (1997) 15:1225-34]; Bjune et al. [Lancet (1991) 338:1093-96] etc.
O
0 OMVs are preferably a deoxycholate extract from NmB obtained from NmB by ,O deoxycholate extraction). The preferred extraction protocol is that described by Fredriksen et o al. [Production, characterization and control of MenB-vaccine "Folkehelsa": an outer membrane vesicle vaccine against group B meningococcal disease (1991) NIPH Ann.
14(2):67-80].
A preferred strain from which to extract OMVs is the 44/76 strain (B:15:P1.7,16:P5.5:L3,7,9) of N.meningitidis.
Further details of the OMV component can be found in, for instance, Bjune et al. [Lancet (1991) 338(8775):1093-96], or Fredriksen et al. [Characterization of high molecular weight component in MenB-vaccine 'Folkehelsa', an outer membrane vesicle vaccine against group B meningococcal disease. Pages 818-824 of Pathobiology and immunobiology of Neisseriaceae (eds. Conde-Glez et al.) ISBN 968-6502-13-0].
The OMV component may be adsorbed to aluminium hydroxide adjuvant. A preferred protein:adjuvant ratio is 1:67 (wt/wt).
A typical dose of vaccine for a human contains 25lg protein, 2gpg LPS and 1.67mg AI(OH) 3 and can be injected in 0.5ml volumes into the deltoid muscle.
The OMV component as obtained by deoxycholate extraction) may be treated to remove certain components. For instance, pyrogens or toxic components may be removed LPS).
It is preferred that the OMV component should retain the 80kDa antigenic component described by Fredriksen et al. [pages 818-824 of Pathobiology and immunobiology of Neisseriaceae].
More preferably, the OMV component should retain a protein comprising one or more of the following amino acid sequences: SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQ ID 11, SEQ ID 13 [or a protein having sequence identity to SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, r' SEQ ID 11, or SEQ ID 13 depending on the particular SEQ ID, the degree of sequence D) identity is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more), which S includes mutants and allelic variants, or (ii) a protein comprising an immunogenic fragment of N SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQ ID 11, or SEQ ID 13 the fragment should comprise at least n consecutive amino acids from the sequence and, Cn depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more).] l^- 0 Combining components and (b) 'O Components and can be combined by simply mixing component with an outer- O membrane preparation by mixing ORF4 with Norwegian OMVs).
As an alternative, they can be combined by manipulating a bacterium such that it produces (preferably hyperproduces) component in its outer membrane an outer-membrane preparation from such a recombinant bacterium will comprise both component and component Suitable bacteria for manipulation in this way include Neisseria meningitidis (any serogroup or strain), Neisseria lactamica, Neisseria cinerea or any other non-typable Neisseria. Other Gram-negative bacteria can also be used, such as E.coli, Salmonella, Shigella, Bordetella, Yersinia, Helicobacter, etc. Transformation methods are well known in the art.
Multivalent vaccines Optionally, the composition of the invention may also comprise one or more of the following components: a protective antigen against Neisseria meningitidis serogroup A; a protective antigen against Neisseria meningitidis serogroup C; a protective antigen against Neisseria meningitidis serogroup Y; a protective antigen against Neisseria meningitidis serogroup W; a protective antigen against Haemophilus influenzae; a protective antigen against pneumococcus; a protective antigen against diphtheria; a protective antigen against tetanus; a protective antigen against whooping cough; -11a protective antigen against Helicobacter pylori; a protective antigen against polio; and/or a protective antigen against hepatitis B virus.
-1 Preferred examples of these optional components are: a polysaccharide antigen against Neisseria meningitidis serogroup A; 0 a polysaccharide antigen against Neisseria meningitidis serogroup C, such as that C described in Costantino et al. (1992) Vaccine 10:691-698; a polysaccharde antigen against Neisseria meningitidis serogroup Y; a polysaccharide antigen against Neisseria meningitidis serogroup W; a polysaccharide antigen against Haemophilus influenzae; a polysaccharide antigen against pneumococcus; a protective antigen against diphtheria, consisting of a diphtheria toxoid, such as the CRM197 mutant [eg. Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70].
a protective antigen against tetanus, consisting of a tetanus toxoid [eg. Wassilak Orenstein, Chapter 4 of Vaccines (eds. Plotkin Mortimer), 1988] a protective antigen against whooping cough, comprising pertussis holotoxin (PT) and filamentous haemagglutinin (FHA); optionally further comprising pertactin and/or agglutinogens 2 and 3 [eg. Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238].
a protective antigen against H.pylori, comprising one or more of CagA (eg.
W093/18150), VacA (eg. W093/18150), NAP (eg. W099/53310), HopX (eg.
W098/04702), HopY (eg. W098/04702), urease.
a protective antigen against hepatitis B virus, consisting of a HBV surface antigen and/or a HBV core antigen.
Where the composition comprises an antigen against diphtheria, it preferably also comprises antigens against tetanus and polio. Where the composition comprises an antigen against tetanus, it preferably also comprises antigens against diphtheria and polio. Where the composition comprises an antigen against polio, it preferably also comprises antigens against diphtheria and tetanus.
SPertussis toxin is a toxic protein and, when present in the composition, it is preferably detoxified. Detoxification may be by chemical and/or genetic means. A preferred detoxified C mutant is the 9K/129G double mutant [eg. Rappuoli (1997) Nature Medicine 3:374-376].
Where the composition includes a protein that exists in different nascent and mature forms, the N mature form of the protein is preferably used. For example, where NspA is included, r- (W096/29412; see also Martin et al. (1997) J. Exp. Med 185 1173-1183) the mature form of 0 the protein lacking the signal peptide is preferably used.
0 Where the composition includes a polysaccharide antigen, the polysaccharide is preferably conjugated to a carrier protein.
Therapy, prophylaxis, diagnosis The composition of the invention is preferably a vaccine. Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
The invention also provides the compositions of the invention for use as medicaments (preferably as vaccines) or as diagnostic reagents. It also provides the use of a composition according to the invention in the manufacture of: a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria. Said Neisserial bacteria may be any species or strain (such as N.gonorrhoeae) but are preferably N.meningitidis, especially serogroup B (NmB).
The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition according to the invention. The method is preferably immunisation.
Processes According to further aspects, the invention provides various processes.
A process for producing a composition of the invention is provided, comprising the step of extraction deoxycholate extraction) of OMVs from N.meningitidis.
-13- SEQUENCE LISTING The sequences in the sequence listing are:.
S' SEQ ID DESCRIPTION 1 N-terminal sequence of N.meningitidis serogroup B protein, 80-85kDa S2 Complete gene from N.meningitidis serogroup B 3 Encoded protein from SEQ ID 2 4 Signal peptide protein from SEQ ID 3 'A 5 Mature protein from SEQ ID 3 S 6 Complete gene from N.gonorrhoeae, homologous to SEQ ID 2 1 7 Encoded protein from SEQ ID 6 8 Signal peptide protein from SEQ ID 7 9 Mature protein from SEQ ID 7 Complete gene from N.meningitidis serogroup A, homologous to SEQ ID 2 11 Encoded protein from SEQ ID 12 Signal peptide protein from SEQ ID 11 13 Mature protein from SEQ ID 11 14 Protein '919' from nmb strain 2996 MODES FOR CARRYING OUT THE INVENTION A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.
General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D.N Glover ed. 1985); Oligonucleotide Synthesis Gait ed, 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. 1984); Transcription and Translation Hames S.J. Higgins eds. 1984); Animal Cell Culture (R.I.
Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 155; Gene Transfer Vectors for Mammalian Cells Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, and Handbook of Experimental Immunology, Volumes l-IV Weir and C. C. Blackwell eds 1986).
C Standard abbreviations for nucleotides and amino acids are used in this specification.
Proteins used with the invention can be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.).
Cn They are preferably prepared in substantially pure form (ie. substantially free from other Neisseria or host O cell proteins).
N Nucleic acid used with the invention can be prepared in many ways (eg. by chemical synthesis, from o genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single C stranded, double stranded, vectors, probes etc.). The term "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
Definitions A composition containing X is "substantially free of" Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.
The term "comprising" means "including" as well as "consisting" eg. a composition "comprising" X may consist exclusively of X or may include something additional to X, such as X+Y.
The term "heterologous" refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.
An "origin of replication" is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.
Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with S parameters gap open penalty=12 and gap extension penalty=l. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
C As used herein, an "allelic variant" of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant S typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5' or 3' untranslated regions of D the gene, such as in regulatory control regions (eg. see US patent 5,753,235).
O Expression systems The Neisserial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.
i. Mammalian Systems Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In Molecular Cloning: A Laboratory Manual, 2nd ed.].
Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad M LP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or -16downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al.
(1989) Molecular Biology of the Cell, 2nd Enhancer elements derived from viruses may be C particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long r terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] S and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are 0 regulatable and become active only in the presence of an inducer, such as a hormone or metal ion \0 [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].
0 A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule,.in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.
The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific posttranscriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic RNA. In Transcription and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.
Examples of transcription terminater/polyadenylation signals include those derived from (Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Laboratory Manual].
Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian -17cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the S replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian S cells for expression and in a prokaryotic host for cloning and amplification. Examples of such 3 mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and O pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].
The transformation procedure used depends upon the host to be transformed. Methods for introduction of 1 heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.
ii. B aculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculoviruslinsect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith").
-18- Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and C transcription termination sequence, are usually assembled into an intermediate transplacement construct C (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable
O
O maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it O0 to be maintained in a suitable host for cloning and amplification.
O Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373.
Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.
The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev.
Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.
Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream to 3') transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression," in: The Molecular Biology ofBaculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the plO protein, Vlak et al., (1988), J. Gen. Virol. 69:765.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human -19aointerferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, C1 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al.
(1988) DNA, 7:99, can also be used to provide for secretion in insects.
A recombinant polypeptide or polyprotein may be expressed intracellularly or,if it is expressed with the r proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign O proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable C= translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may o be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
C Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.
After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome.
Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by- homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91.The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3' by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles.
These occlusion bodies, up to 15 .lm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or S absence (indicative of recombinant virus) of occlusion bodies. "Current Protocols in Microbiology" Vol.
C 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).
Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
Cn- For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa S californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (W O S 891046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) O Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).
O
O Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculoviruslexpression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.
The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, eg.
proteins, lipids and polysaccharides.
In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
iii. Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art.
Exemplary plant cellular genetic expression systems include those described in patents, such as: US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol.
Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J.
Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the -1 phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R.L.
Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,. Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and SHickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987) Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an S expression cassette comprising genetic regulatory elements designed for operation-in plants. The 0 expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165- 185.
Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5' untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette allow for easy insertion into a pre-existing vector.
A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the 1) desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the CI signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or S scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be
O
O secreted from the cells in which the protein is produced, this facilitates the isolation and purification of s. the recombinant protein.
O Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the "intron" region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.
The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982.
Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.
The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, I3 Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, r Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Penniselum, Ranunculus, Senecio, C- Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and C shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be Sinduced from the protoplast suspension. These embryos germinate as natural embryos to form plants.
SThe culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such O species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.
iv. Bacterial Systems Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.
In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
CI Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter C sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids 0 Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; US patent 4,738,921; EP-A-0036776 and C. EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128) and T5 (US patent 4,689,406] promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [US patent 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci.
82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E.
coli operator region (EPO-A-0 267 851).
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine- Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA [Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R.F.
Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual].
A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which C\ is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).
CM Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N- O terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5' end of C heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al.
(1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol.
135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.
ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) BiolTechnology 7:698].
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [US patent 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Nail. Acad. Sci. 82:7212). As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva etal. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].
Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by -26the DNA. Transcription termination sequences frequently include DNA sequences of about nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples A include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.
Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.
A plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and S amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about to about 150. A host containing a high copy number plasmid will preferably contain at least about and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A- 0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors.
Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Nail. Acad.
Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84104541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) -27- D Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ.
Microbiol. 54:655], Streptomyces lividans [US patent 4,745,056].
'l Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCI 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation ,n procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS j Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP- 7A A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al.
3 (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Nail. Acad. Sci. 69:2110; 3 Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) "An improved method for transformation of Escherichia coli with ColEl-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al.
(1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al.
(1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], (Barany et al.
(1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect.
Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].
v, Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression.
Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PH05 gene, -28- D encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al. (1983) Proc.
Natl. Acad. Sci. USA 80:1].
'A In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include T the ADH regulatory sequence linked to the GAP transcription activation region (US Patent Nos.
j 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the 7 regulatory sequences of either the ADH2, GAL4, GALIO, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556).
3 Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. lmmunol..96:119; Hollenberg et al. (1979) "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae," in: Plasmids of Medical, Environmental and Commercial Importance (eds. K.N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].
A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the Nterminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5' terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. W088/024066).
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vive or in vitro. The leader -29- .0 sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct 1( the secretion of the protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (US patent Oi 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide S for secretion in yeast (EP-A-0 060 057).
A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that S can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as ~l well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (US Patents 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.
Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.
Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCI/l [Brake et al. (1984) Proc. Nall. Acad. Sci USA 81:4642-4646], and YRpl7 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.
Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and _0 the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for A inclusion in the vector. See Orr.Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the nr vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to S allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRPI, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUPI allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].
Alternatively, some of the above described components can be put together into transformation vectors.
Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts:Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol.
6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.
202:302], Kluyveromyces fragilis [Das, et al. (1984) 1. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris (Cregg, et al.
(1985) Mol. Cell. Biol. 5:3376; US Patent Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae (Hinnen et al. (1978) Proc. Nail. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].
Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.
Transformation procedures usually vary with the yeast species to be transformed. See eg. (Kurtz et al.
(1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al.
(1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; -31- [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den r Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; (Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Ci Kunze et.al. (1985) J. Basic Microbiol. 25:141; US Patent Nos. 4,837,148 and 4,929,555; Pichia]; I [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; (Davidow et al.
r'n (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].
0 Antibodies 0 CN As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An "antibody combining site" is the three-dimensional bding space with 0) one antibody combining site. An "antibody combining site" is the three-dimensional binding space with San internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. "Antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishinglidentifying Neisserial proteins.
Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.
Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 gglinjection is typically sufficient.
Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25 0 C for one hour, followed by incubating at 4°C for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
Monoclonal antibodies are prepared using the standard method of Kohler Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above.
However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the -32- ,C plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective C1 medium (eg. hypoxanthine, aminopterin, thymidine medium, The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting cn hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in o vivo (as ascites in mice).
C If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional S techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly P and S '121), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 121 may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, M Abs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 25I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
Pharmaceutical Compositions Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.
The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels.
Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.
The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.
Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for -33- D a given situation can be determined by routine experimentation and is within the judgement of the clinician.
1 For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
1 A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as j antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical 1 carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, 7A slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.
Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Vaccines Vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does -34- .0 not itself induce the production of antibodies harmful to the individual receiving the composition.
j Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, 1 polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as n immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
K Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) 3 aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example MF597 M
(WO
90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model I IOY microfluidizer (Microfluidics, Newton, MA), SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and Ribi T M adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL CWS (DetoxrM); saponin adjuvants, such as Stimulon T M (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59 are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-Disoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl- L-alanyl-D-isoglutam inyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-hydrox yphosphoryloxy)ethylamine (MTP-PE), etc.
The immunogenic compositions (eg. the immunising antigen/immunogen/polypeptide/protein/ nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
,C Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or n suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may CN also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
C Immunogenic compositions used as vaccines comprise an immunologically effective amount of the r antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as O needed. By "immunologically effective amount", it is meant that the administration of that amount to an N individual, either in a single dose or as part of a series, is effective for treatment or prevention. This o amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermallyltranscutaneously (eg. W098/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].
Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in viva or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994)Nature Genetics 6:148-153.
C Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-XI, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses Seg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA O Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
C Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, O retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous
O
C Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US patent 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see W 096/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.
Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see W095/30763 and W092105266), and can be used to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.
Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.
Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) 1 Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No.
VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or isolated from known sources using commonly available techniques.
Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, W089/02468; W089/05349, W089/09271, W090/02806, W090/07936, W094/03622, W093/25698, W093/25234, W093/11230, W093/10218, W091/02805, W091/02825, W095/07994, US 5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res r 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Nail CK Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.
Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and W093/07283, W093/06223, and W093/07282. Exemplary known adenoviral gene therapy vectors 0 employable in this invention include those described in the above referenced documents and in C W094/12649, W093/03769, W093/19191, W094/28938, W095/11984, W095100655, W095127071, W095129993, W095/34671, W096/05320, W094/08026, W094/11506, W093/06223, W094/24299, 0 W095114102, W095124297, W095102697, W094/28152, W094/24299, W095/09241, W095/25807, W095105835, W094/18922 and W095109654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, W093/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleolides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary A.AV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector.
Construction of the Double-D ITR vector is disclosed in US Patent 5,478,745. Still other vectors are those disclosed in Carter US Patent 4,797,368 and Muzyczka US Patent 5,139,941, Chartejee US Patent 5,474,935, and Kotin W094/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in US 5,354,678, US 5,173,414, US 5,139,941, and US 5,252,479.
The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in US 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in W095/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in W090/09441 and W092107945, HSV Us3::pgC-lacZ CN described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
c n Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred 0 alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC C VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), C Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC r VR-532), and those described in US patents 5,091,309, 5,217,879, and W092/10578. More particularly, those alpha virus vectors described in US Serial No. 08/405,627, filed March 15, 1995,W094/21792, W092/10578, W095/07994, US 5,091,309 and US 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see USSN 08/679640).
DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See W095/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.
Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.
Standardization 1:115; rhinovirus, for example ATCC VR- 110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in US 4,603,112 and US 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in US 5,166,057 and in Enami (1990) Proc Nail Acad Sci 87:3802-3805; Enami Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah -39virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; r Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC C VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR.372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for cr example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and S ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Q Hamre (1966) Proc Soc Exp Biol Med 121:190.
O Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see US Serial No. 081366,787, filed December 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No.08/240,030, filed May 9, 1994, and US Serial No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in US Patent 5,149,655, ionizing radiation as described in US5,206,152 and in W092/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Nall Acad Sci 91:1581-1585.
Particle mediated gene transfer may be employed, for example see US Serial No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.
Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and US 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
Liposomes that can act as gene delivery vehicles are described in US 5,422,120, W095/13796, W094/23697, W091/14445 and EP-524,968. As described in USSN. 601023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, CN linked to cell targeting ligands such as asialoorosomucoid, insolin, galactose, lactose, or transferrin.
Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable
C]"
m for use includes mechanical delivery systems such as the approach described in Woffendin el al (1994) 0 Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of C] expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for O example, use of hand-held gene transfer particle gun, as described in US 5,149,655; use of ionizing radiation for activating transferred gene, as described in US 5,206,152 and W092/11033 Exemplary liposome and polycationic gene delivery vehicles are those described in US 5,422,120 and 4,762,915; in WO 95/13796; W094/23697; and W091/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem.176:420.
A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered directly to the subject; delivered ex vivo, to cells derived from the subject; or in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and -described in eg. W093/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.
Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, CI polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
jC Polvnucleotide and polvpetide pharmaceutical compositions O In addition to the pharmaceutically acceptable carriers and salts described above, the following additional C- agents can be used with polynucleotide and/or polypeptide compositions.
0O 0 A.Polypeptides
C
N
I One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.
B.Hormones. Vitamins, etc.
Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.
C.Polvalkylenes, Polvsaccharides, etc.
Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide) D.Lipids. and Liposomes The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.
Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta.
1097:1-17;Straubinger (1983) Meth. Enzymol. 101:512-527.
Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); -42- 0 mRNA (Malone (1989) Proc. Nall. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.
Cationic liposomes are readily available. For example, N[l-2,3-dioleyloxy)propyll-N,N,Ntriethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAPIDOPE (Boerhinger). Other cationic liposomes can be prepared S from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc.
S Natl. Acad. Sci. USA 75:4194-4198; W090111092 for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are well known in the art.
The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immuno. 101:512-527; Szoka (1978) Proc. Natl.
Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem.
Biophys. Res. Commun. 76:836; Fraley (1979). Proc. Natl. Acad. Sci. USA 76:3348); Enoch Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.
E.Lipoproteins In addition, lipoproteins can be included with the polynucleotidelpolypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.
Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, All, AIV; CI, CII, CIII.
-43- C A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons n comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL C1 comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.
The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Anno Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; 0 Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.
CI Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and 0 phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For.example, C chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.
Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA. Further description of lipoproteins can be found in Zuckermann et al.
W 098/06437.
F.Polvcationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.
Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.
The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X 174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid .0 condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, 1 Prot-1,Sp-l, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.
Organic polycationic agents include: spermine, spermidine, and purtrescine.
The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list 'l above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.
7- Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene.
S Lipofectin
T
and lipofectAMINE T M are monomers that form polycationic complexes when combined 0 with polynucleotides/polypeptides.
3 Immunodianostic Assays Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisserial proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.
Nucleic Acid Hybridisation "Hybridization" refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solhtion.
Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.
.0 "Stringency" refers to conditions in a hybridization reaction that favor association of very similar 0 sequences over sequences that differ. For example, the combination of temperature and salt concentration 1 should be chosen that is approximately 120 to 200°C below the calculated Tm of the hybrid under study.
The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.
S Variables to consider when performing, for example, a Southern blot are the complexity of the DNA K being blotted and the homology between the probe and the sequences being detected. The total S amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to li g for a plasmid or 7 phage digest to 10' 9 to 10' g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with I pg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10 8 cpmlpg. For a single-copy mammalian gene a conservative approach would start with 10 pg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10' cpmlp g, resulting in an exposure time of -24 hours.
Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm= 81 16.6(logloCi) C)]-0.6(%formamide) 600/n-1.5(% mismatch).
where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth Wahl (1984) Anal. Biochem. 138: 267-284).
In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.
o In general, convenient hybridization temperatures in the presence of 50% formamide are 42 0 C for a probe with is 95% to 100% homologous to the target fragment, 37*C for 90% to 95% homology, and N 32 0 C for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash cr" conditions which are nonstringent. If non-specific bands or high background are observed after 0 autoradiography, the filter can be washed at high stringency and reexposed. If the time required for 0 exposure makes this approach impractical, several hybridization andlor washing stringencies should be o tested in parallel.
N Identification of the meningococcal 80-85kDa protein It was observed that various outer membrane vesicle preparations from N.meningitidis serogroup B contained a component of approximately 80-85kDa. This protein was purified from SDS-PAGE gels and N-terminal sequenced (SEQ ID 1).
Antibodies raised against the SDS-PAGE purified protein cross-reacted with equivalent proteins in more than 50 N.meningitidis strains of diverse serogroups and serotypes.
Cross-reactivity with N.gonorrhoeae, N.polysaccharia and N.lactamica was also observed.
Post-immune sera from vaccinated patients also reacted with the protein.
The complete gene was cloned from serogroup B N.meningitidis (SEQ ID 2) and the encoded protein was inferred (SEQ ID By comparison with the N-terminal sequencing described above, a signal peptide (SEQ ID 4) and a mature sequence (SEQ ID 5) are inferred.
Identification of corresponding genes in N.meningitidis serogroup A and N.gonorrhoeae On the basis of the serogroup B N.meningitidis sequence, the corresponding genes from N.meningitidis serogroup A and N.gonorrhoeae were cloned and sequenced.
The complete gene from serogroup A N.meningitidis is shown in SEQ ID 6, with the encoded protein in SEQ ID 7. The signal peptide and mature sequence are SEQ IDs 8 and 9.
The complete gene from N.gonorrhoeae is shown in SEQ ID 10, with the encoded protein in SEQ ID 11. The signal peptide and mature sequence are SEQ IDs 12 and 13.
Sequence comparisons The protein sequences were compared and are highly homologous.
_0 The Mmeningitidis serogroup B sequence and the N.gonorrhoeae sequence show 95.4% Sidentity in 797 aa overlap: K110 20 30 .40 50 ~1 orf2l .pep MKLKQIASALMMLGISPLALADFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGSA Orf 2lng .pep MIKLKQIASALI.ThLGISPLAFAnFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGSA K110 20 30 40 50 80 90 100 110 120 orf21. pep I IKSLYATGPFDDVRVETADGQLLLTVIERPTIGSLNITGAKMLQNDAIKKNLESFGLiAQ orf 2 ng .pep I IKSLYATGFFDDVRVETADGQLLLTVIERPTIGSLNITGAKMLQNDAIKKNLESFGLAQ .80 90 100 110 120 130 140 150 160 170 180 Sorf 21.pep SQYFNQATLNQAVAGLKEEYLGRGKLNIQITPKVTKLARNRVDIDITIDEGKSAKITDIE orf2lng .pep SQYFNQATLNQAVAGLKEEYLGRGKLNIQITPKVTILARNRVDIDITIDEGKSAKITDIE 130 140 150 160 170 180 190 200 210 220 230 240 or 21. pep FEGNQVYSDRKLMRQMSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFRIL orE 2 ng .pep FEGNQVYSDRKLMRQ!4SL'PEGGIWTWLTRSDRFDRQKFAQDMEKVTDFYQNNGYFDFRIL 190 200 210 220 230 240 250 260 270 280 290 300 orf 21.pep DTDIQTNEDKTKQTIKITVHEGGRFRWGKVSIEGDTNEVPKAELEKLLTMPGKWERQQ orf 2lirg .pep DTDIQTNEDKTRQTIKITVHEGGRFRWGKVSIEGDTNEVPKAELEKLLTMPGKWYERQQ 250 260 270 280 290 300 310 320 330 340 350 360 orE 21. pep MTAVLGEIQNRMGSAGYAYSEISVQPLPNAETKTVDFVLHIEPGRKIYVNEIHITGNNKT orE 2 ng .pep MTAVLGEIQNRMGSAGYAYSEISVQPLPNAGTKTVDFVLHIEPGRKIYVNEIHITGNNKT 310 320 330 340 350 360 370 380 390 400 410 420 orf21. pep RDEVVRRELRQMESAPYDTSKLQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLNMSLTE orf 2 ng .pep RDEVVRRELRQMESAPYDTSKLQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLNMSLTE 370 380 390 400 410 420 430 440 450 460 470 480 or E2 1 pep RSTGSLDLSAGWVQDTGLVMSAGVSQDNLFGTGKSAALRASRSKTTLNGSL.SFTDPYFTA orf 2 ng .pep RSTGSLDLSAGWVQDTGLVMSAGVSQDNLFGTGKSAALRASRSKTThNGSLSFTDPYFTA 430 440 450 460 470 480 490 500 510 520 530 540 orf21. pep DGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRMSVPVTEYDRVNFGLVAEHLTVNT orE 2 ng .pep DGVSLGYDIYGKAFDPRKASTSVKQYKTTTAGGGVRMGI PVTEYDRVNFGIJAAEHLTVN4T 490 500 510 520 530 540 550 560 570 580 590 600 or 21. pep YNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSALWPTRGYLTGVNAEIA orf2 ing pep YNKAPKRYADFIRKYGKTDGADGSFKGLLYKGTVGWGRNKTDSASWPTRGYLTGVNAEIA 550 560 570 580 590 600 610 620 630 640 650 660 or f21. pep LPGSKLjQYYSATHNQ'NFPLSKTFTLMLGGEVGIAGGYGRTKEIPFFENFYGGGWiSVR III1111IIIIIIII1111IIIIIII1111 1111 1111 -48orf2lng .pep orf 21.pep orf2lng.pep orE 2l.pep Orf2lng .pep orE 2l.pep orf2lng .pep LPGSKLQYYSATHNQTWFFPLSKTFTLHMjGGEVGIAGGYGRTKEIPFFENFYGGGLGSVR 610 620 630 640 650 660 670 680 690 700 710 720
GYESGTLGPKVYDEYGEKISYGGNKKANVSAELLFPPGAKDARTVRLSLFADAGSVWDG
GYESGTLGPKVYDEYGEKISYGGNKKMA1VSAELLFPMPGAKDARTVRLSLFADAGSVWDG 670 680 690 700 710 720 730 740 750 760 770 780
KTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGGAVTWLSPLGPHKPSYAYPLKK
RTY- TAAENGNNKSVYSE-I4AHKSTFTNELRYSAGGAVTWLSPLGPMKFSYAYPLKK 730 740 750 760 770 790
KPEDEIQRFQFQLGTTF
ll11II1II III
KPEDEIQRFQFQLGTTFX
780 790 The NMmeningitidis serogroup A and B sequences show 99.9% identity in 797 aa overlap: or 21. pep orf2la .pep orf2l .pep orf2la .pep orf2l .pep orf2la .pep orf2l .pep orf2la .pep orf2l .pep orf2la.pep orf2l. pep orf2la.pep orf2l.pep orf2la .pep 20 30 40 50 MKLKQIASALMNLGI SPISALADFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGSA 14KLKQIASALMVLGISPLALADFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGSA 20 30 40 50 80 90 100 110 120 I IKSLYATGFFDDVRVETADGQLLLTVIERPTIGSLNITGAKMLQNDAIKKNLESFGLAQ I IKSILYATGFFDDVRVETADGQLLLTVIERPTISLNITGAKMLQNDAIKKNLESFGLAQ 80 90 100 110 120 130 140 150 160 170 180
SQYFNQATLNQAVAGLKEEYLGRGKLNIITPKVTKLANRVDIDITIDEGKSAKITDIE
SQYFNQATLNQAVAGLKEEYLGRGKLNIITPKVTKLARNRVDIDITIDEGKSAKITDIE
130 140 150 160 170 180 190 200 210 220 230 240
FEGNQVYSDRKLMRQMSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFRIL
FEGNQVYSDRKL14RQMSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFRIL 190 200 210 220 230 240 250 260 270 280 290 300
DTDIQTNEDKTKQTIKITVHEGGRRWGKVSIEGDTNEVPKAELEKLLTMKPGKKYERQQ
DTDIQTNEDKTKQTIKITVHEGGRRWGKVSIEGTNEPKAELEKLLTMPGKWYERQQ
250 260 270 280 290 300 310 320 330 340 350 360
MTAVLGEIQNRMGSAGYAYSEISVQPLPNAETKTVDFVLHIEPGRKIYVNEIHITGNNKT
MTAVLGEIQNIU4GSAGYAYSEISVQPLPNAETKTVDFVLHIEPGRKIYVNEIHITGNNKT 310 320 330 340 350 360 370 380 390 400 410 420
RDEVVRRELRQMESAPYDTSKLQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLNMSLTE
RDEVVRRELRQMESAPYDTSKLQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLNMSLTE
370 380 390 400 410 420 orfl.pep Orf2l.pep Qrf2la.pep orf2l.pep orf2la.pep 0 0 orf2l.pep 0 O orf2la.pep orf2la.pep orf2la.pep orf2l.pep orf2l.pep orf2l.pep orf2la.pep orf2l .ep orEla.ep 430 440 450 460 470 480
RSTSLDLSAGWVQDTGLVMSAGVSQDNLFGTGSAALRASRSKTTLNLSFTDPYFTA
III I I I I 1 1111*1111 1111 111111 1 111 1.11 1 1111 1I 1.1 1111 1111 111 11 I
RSTGSLDLSAGWVQDTGLVSAGVSQDNLFGTKSAALRASRSKTTLNGSLSFTDPYFTA
430 440 450 460 470 480 490 500 510 520 530 540
DGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRSVPVTEYDRVNFLVAEHLTNT
liii I 1111111 ill111llil11l1ll till111111 IIIIIIII11IIl lli1il
DGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRMSVPVTEYDRVNFGLVAEHLTVNT
490 500 510 520 530 540 550 560 570 580 590 600
YNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSALWPTRGYLTGVNAEIA
III I I I IIIIIIIIIIIIIII11111111111111 11111111111111111 I11111 I
YNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSALWPTRGYLTGVNAEIA
550 560 570 580 590 600 610 620 630 640 650 660
LPGSKLQYYSATHNQTWFFPLSKTFTLMLGGEVGIAGGYGRTKEIPFFENFYGGILGSVR
IIIIIIIIIIIIII11 I IIIIllIIIIIIIIII1111111I1111 1111111 11111111 I
LPGSKLQYYSATHNQTWFFPLSKTFTLMLGGEVGIAGGYGRTKEIPFFENFYGGGLGSVR
610 620 630 640 650 660 670 680 690 700 710 720
GYESGTLGPKVYDEYGEKISYGGNKKAVSAELLFPMPGAKDARTVRLSLFADAGSVWG
1111111111 11111111111111111 111111111 II II IIIIIIIIIIIII 111111 GYESGTLGPKVYDEYGEKISYGNKKANVSAELL FPGAKDA TVRLSLFAAGTD 670 680 690 700 710 720 730 740 750 760 770 780
KTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGGAVTWLSPLGPMKFSYAYPLKK
11111111111111 I I 111111111111 111111 111111111 1111111111111 I
KTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGGAVTWLSPLGPMKFSYAYPLKK
730 740 750 760 770 780 790
KPEDEIQRFQFQLGTTF
111111111 II IIII
KPEDEIQRFQFQLGTTFX
790 The high degree of conservation suggests that a single protein may be able to induce immune responses against a variety of Neisseriae species.
Vaccines The three proteins identified above were expressed and used for immunisation. Good immune responses were observed against the proteins.
Combination vaccines In addition, the proteins were each combined with antigens against other pathogenic organisms the Chiron polysaccharide vaccine against serogroup C meningitis). and used for immunisation. Good immune responses were observed.
4 Further NmB components Whilst it is efficacious, the protection elicited by the Norwegian OMV vaccine is restricted to the strain used to make the vaccine. The clinical trials on the vaccine obtained only 57.2% efficacy after 29 months in teenagers, although IgG responses were observed in almost 100% CN of patients Rosenqvist et al. (1995) Infect. Immun. 63:4642-4652].
O Surprisingly, it has been found that the addition of further defined components to the
O
N Norwegian OMV vaccine significantly broadens its efficacy.
o The Norwegian vaccine does not elicit protection against NmB strain 2996. Defined proteins Sfrom strain 2996 were added to the Norwegian vaccine, and it was shown that the efficacy of the vaccine was increased by a surprising degree. Furthermore, the addition of a NmC polysaccharide conjugate antigen Costantino et al. (1992) Vaccine 10:691-698] gave excellent results.
The bactericidal activities of the combinations are shown in the following table: Norwegian NmB NmC Bactericidal activity Group OMV antigen' antigen against NmB strain 2996 1 <4 2 #1 512 3 #2 >2048 4 #3 1024 #3 256 6 #3 2048 7 #3 2048 SThree different NmB antigens were used: ORFI e.g. example 77 of W099/24578 (see also W099/55873) protein '287' e.g. Figure 21 of W099/57280 (also SEQ IDs 3103-3108) a mixture in AI(OH) 3 of #2 and protein '919' (SEQ ID 14 herein; see also W099/57280 Figure 23 and SEQ IDs 3069-3074 therein).
It can readily be seen that the inefficacy of the Norwegian OMV vaccine against strain 2996 (group 1) can be overcome by adding defined antigens from strain 2996. The results using NmB protein '287' are particularly good. The Norwegian vaccine can thus be improved without needing to prepare OMVs from a number of different strains.
This vaccine also offers protection against heterologous MenB strains. The same vaccines, prepared using 2996 strain proteins, was tested against five other strains. Titres were as follows: 2996 BZ133 BZ232 1000 MC58 NGH38 <4 1024 <4 >2048 >2048 32 512 512 <4 >2048 >2048 256 4096 4096 256 1024 >2048 1024 1024 2048 <4 2048 >2048 64 256 >32000 <4 >2048 >2048 128 2048 2048 4 <4 64 4 2048 >32000 4 128 1024 128 32768 4096 8192 16384 16384 8192 Controls: strains 2996, BZ133 1000 OMVs prepared from homologous strain; strain BZ232 OMVs prepared from 2996; MC58 NGH38 SEAM3 A second study supplemented 'Norwegian' OMVs with proteins from NmB strain 2996: protein 919, expressed in E.coli without any fusion partner ORF1, expressed in E.coli as a His-tagged fusion Protein 287, expressed in E.coli as a GST fusion A mixture of these three proteins, optionally with the NmC conjugate SThe preparations were adjuvanted with AI(OH) 3 and tested against the homologous strain using the bactericidal assay. Results were as follows: NmB NmC Antigen 2996 NGH38 394/98 C11 BZ133 OMVs <4 32 1024* <4 1024 +919 <4 <4 4 <4 512 +ORF1 512 256 2048 4096 512 +287 4096 1024 1024 512 4096 +mix 1024 64 4 64 2048 +mix 256 128 2048 64000 >32000 +NmC the antibodies were bacteriostatic, not bactericidal Further work with antigen 287 Combinations of Norwegian OMVs with antigen 287 were investigated further. 20pg antigen 287 was combined with Norwegian OMP vaccine (15ptg OMP A1(OH) 3 and used to immunise mice. The antibodies were tested in the bactericidal assay, and were effective against all strains tested. The results were as follows: NmB ___NmA NmC Antigen 2996 BZ133 BZ232 1000 MCS8 NGH38 NZ F6124 Cll OMVs <4 1024 <4 >2048 32768 32 <4 287 8000 4096 256 <4 512 2048 1024 1024 2048 OMV+287 4096 4096 256 1024 4096 1024 1024 ,1 In almost all cases, therefore, the combination of OMVs protein 287 surprisingly gives better results that the OMVs alone.
1 Recombinant OMVs 0 0 Ecoli were transformed to express ORF1, ORF40 and ORF46. OMVs prepared from the recombinant E.coli were able to induce bactericidal antibodies against N.meningitidis.
ORFI, ORF40 and ORF46 (strain 2996) were expressed as His-tagged fusions in E.coli and were prepared either as pure proteins or in the form of OMVs. Bactericidal titres against both preparations were tested using strain 2996 as challenge: Antigen: ORF1 ORF40 ORF46 Purified 64 2048 16000 OMV 1024 256 128000 Bactericidal titres using heterologous challenge strains were also measured. ORF46 gives a titre against strain MC58 of 4096 in pure form, but this rises to 32000 when in the form of OMVs. ORF1 gives a titre against NmA strain F6124 of 128 in pure form, but this rises to 512 when in the form of OMVs. ORF40 gives a titre against strain MC58 of 512 in pure form, but this doubles when in the form of OMVs.
These data show that NmB antigens retain immunogenicity when prepared in E.coli as OMVs and, furthermore, that immunogenicity can actually be enhanced.
It will be understood that this application describes the invention by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims (15)

1. A composition comprising N.meningitidis serogroup B outer membrane Spreparation, and Neisseria meningitides protein
2. The composition as claimed in claim 1 wherein component is selected from Cc one or more of the following: \O a protein comprising an amino acid sequence selected from SEQ IDs 16, 18 or 20, or a protein comprising an immunogenic fragment of one or more of SEQ IDs 16, S18 or 20, or a protein comprising a sequence having greater than 50% sequence identity to one or more of SEQ IDs 16, 18 or a protein comprising an amino acid sequence selected from SEQ IDs 21 to 41 or an immunogenic fragment thereof; a protein comprising an amino acid sequence selected from the group consisting of SEQ ID 42, a protein comprising an immunogenic fragment of SEQ ID 42, and a protein comprising a sequence having greater than 50% sequence identity to SEQ ID 42; and/or a protein comprising an amino acid sequence selected from the group consisting of SEQ ID 43 or SEQ ID 44, a protein comprising an immunogenic fragment of SEQ ID 43 or SEQ ID 44, and a protein comprising a sequence having greater than sequence identity to SEQ ID 43 or SEQ ID 44.
3. The composition as claimed in claim 2 wherein component is a protein comprising an amino acid sequence selected from SEQ IDs 16, 18 or 20, or a protein comprising an immunogenic fragment of one or more of SEQ IDs 16, 18 or 20, or a protein comprising a sequence having greater than 50% sequence identity to one or more of SEQ IDs 16, 18 or
4. The composition of any one of the preceding claims, wherein component (b) includes a protein from a N.meningitidis serogroup B strain. The composition of any one of the preceding claims, wherein component (b) includes a protein from a different NmB strain from that from which component is derived. 00 i't 0 C 6. The composition of any one of the preceding claims, wherein one or more of its components is adsorbed on AI(OH) 3
7. The composition of any one of the preceding claims, wherein component (a) comprises OMVs. J 8. The composition of claim 7, wherein the OMVs are a deoxycholate extract from O NmB.
9. The composition of claim 7, or claim 8 wherein the OMVs are from the 44/76 strain of Neisseria meningitidis. The composition of any one of the preceding claims, wherein component is adsorbed on A1(OH) 3
11. The composition of any one of the preceding claims further comprising one or more of the following components: a protective antigen against Neisseria meningitidis serogroup A; a protective antigen against Neisseria meningitidis serogroup C; a protective antigen against Neisseria meningitidis serogroup Y; a protective antigen against Neisseria meningitidis serogroup W; a protective antigen against Haemophilus influenzae; a protective antigen against pneumococcus; a protective antigen against diphtheria; a protective antigen against tetanus; a protective antigen against whooping cough; a protective antigen against Helicobacter pylori; a protective antigen against polio; and/or a protective antigen against hepatitis B virus.
12. The composition of any one of the preceding claims, wherein the composition is a vaccine.
13. The composition of any one of the preceding claims, for use as a medicament. 00 C 14. The use of N.meningitidis serogroup B outer membrane preparation, and (b) Neisseria meningitidis protein ORF40 according to any one of the preceding claims in j the manufacture of: a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria. A Neisseria meningitidis serogroup B outer membrane preparation comprising OMVs and protein 010
16. A bacterial outer membrane preparation as claimed in claim 1 wherein the bacterium has been manipulated to hyperproduce ORF40 in its outer membrane.
17. A bacterial outer membrane preparation comprising Neisseria meningitidis protein
18. A composition comprising OMVs and Neisseria meningitidis protein Dated this TWENTY SIXTH day of SEPTEMBER, 2008 Novartis Vaccines and Diagnostics S.r.l. Patent Attorneys for the Applicant: F B RICE CO SEQUENCE LISTING <110> CHIRON SPA <120> SUPPLEMENTED OMV VACCINE AGAINST MENINGOCOCCUS <130> P023785W0 <140> <1431> 2 001-01-17 <150> GB-0001067.8 <151> 2000-01-17 <150> GB-0005699.4 <151> 2000-03-09 <160> 14 <170> SeqjWin99, version 1.02 <210> <211> <212> <213> <400> Asp Phe I PRT Neisseria meningitidis Thr Ile Gin Asp Ile Arg Vai Glu Gly Leu Gin Arg Thr 5 10 <210> 2 <212> 2394 <212> DNA <213> Neisseria, meningitidis <400> 2 atgaaactga. gccgacttca gtattcaact atcatcaaaa gggcagctcc gcaaaaatgc tcgcaatact ctcgggcgcg cgcgtcgaca tttgaaggca ggcggcattt gatatggaaa gataccgaca gaaggcggac aaagccgaac atgaccgccg gaaatcagcg atcgaaccgg cgcgacgaag aagctgcaac gatgctgtcc cgttccaccg tccgcaggcg tccaggagca aacagattgc ccatccaaga acctgcccgt gcc tgtacgc tgctgaccgt tgcaaaacga ttaatcaggc gcaaactcaa tcgacatcac accaagtcta ggacatggct aagtaaccga tccaaaccaa gtttccgttg tggaaaaac t ttttgggtga tacagccgct gccggaaaat tcgtccgccg gttccaaaga cgcttgccgg gttccctgga tttcccaaga aaaccacgct ttccgcactg catccgcgtc caaagtcggc caccggtttc tatcgaacgc cgccattaag gacactcaat tatccaaatc gattgacgag ttccgaccgc gacacgaagc cttctaccaa cgaagacaaa gggcaaagtc gctgaccatg gattcagaac gccgaacgct ctacgtcaac tgaattacgc gcgcgtcgag cacgcccgac tttgagcgcg caacctgttc taacggctcg atgatgttgg gaaggcttgc gacacctaca tttgacgacg cccaccatcg aaaaacctcg caggcagtcg acgcccaaag ggcaaatccg aaactgatgc aaccaattca aataacggct accaagcaga. tccatcgaag aagcccggca cgoatgggct gaaaccaaaa, gaaatacaca, caaatggaat cttttgggct aaagtcgatt ggttgggttc ggtacgggca ctgtcgttta gcatatcgcc agcgtaccga acgacacaca tacgcgtcga gctcgctcaa aatcgttcgg ccggcctgaa taaccaaact ccaaaatcac ggcaaatgtc acgagcagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggcta ccgtcgattt tcaccggcaa ccgcacctta acttcgacaa tgaacatgag aagataccgg agtcggccgc ctgacccgta tttggcactt gccgagtacc cggcagtgcc aactgcggac catcaccggc gctggcgcag agaagaatac cgcccgcaac cgacatcgaa cctgaccgaa atttgcccaa. ccgtatcctc caccgtccac cgaagt cccc acgccagcag cgcatacagc cgtcctgcac caacaaaacc cgacacctcc tgtccagttt tctgaccgaa gttggtcatg actgcgcgcc cttcacggca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 gacggggtca accagcatca gttekccgaat tacaacaaag acagacggca accgacagcg ctgcctggca ctgagcaaaa agaaccaaag gga tacgaaa tacggcggca aaagacgcgc aaaacctacg gccggcaata gttacctggc aaaccggaag gcctgggcta aacaatataa acgaccgcgt cgcccaaaca gcttcaaagg cgttatggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgc t acaaaaaagc gcaccgtccg acgacaacag cccataaatc tctcgccttt acgaaatcca cgatgtttac aaccaccacg gaatttcggt ctatgccgac ctggctgtac gacgcgcggc atactactcc gatgctcggc ctttgaaaac cggtccgaaa caacgtctcc cctgagcctg cagttccgcg cacctttacc aggcccgatg acgettccaa ggaaaagcct gcaggcgcag ttggtggcag tttatcaaga aaaggtaccg tacc tgacgg gccacccaca ggcgaagtcg ttctacggcg gtctatgacg gccgagctgc tttgccgacg accggcggca aacgaattgc aaattcagc t ttccaactcg tcgacccgcg gcatccgcat aacacctgac aatacggcaa tcggctgggg gcgtgaacgc accaaacctg gcattgcggg gcggcctggg aatacggcga tcttcccgat caggcagcgt gggttcaaaa gctattccgc acgcctaccc gcacgacgtt caaagcatcg gagcgtgcct cgtcaacacc aaccgacggc gcgcaacaaa cgaaatcgcc gttcttcccc cggctacggc ttcggtgcgc aaaaatcagc gcccggcgcg gtgggacggc catttacggc cggcggcgcg gctgaagaaa ctaa 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2394 <210> 3 <211> 797 <212> PRT <213> Neisseria meningitidis <400> 3 Met Lys Leu Lys 1 Gin Ilie Ala Ser Ala Leu Met 5 10 Met Leu Gly Ile Ser Pro Leu Ala Leu Ala Asp Phe Leu Gin Arg Thr Giu Pro Ser Thr Ile 25 Thr Val 40 Gin Asp Ile Phe Asn Tyr Arg Vai Glu Gly Leu Pro Val Lys Vai Gly Asp Thr Tyr Asn Asp 55 Thr His Gly Ser Ala Ilie Ile Lys Ser Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Val Giu Thr Ala Gly Gin Leu Leu Leu Thr Val Ile Giu Arg 90 Pro Thr Ile Giy Ser Leu Asn Ilie Thr Leu Giu Ser 115 Gly Ala 100 Lys Met Leu Asn Asp Ala Ile Lys Lys Asn 110 Gin Ala Thr Phe Gly Leu Ala Ser Gin Tyr Phe Leu Asn 130 Gin Ala Val Ala Leu Lys Giu Giu Leu Gly Arg Gly Leu Asn Ile Gin Thr Pro Lys Val Thr Ile Asp Glu 170 Lys Leu Ala Arg Arg Vai Asp Ilie Asp Ilie 165 Gly Lys Ser Ala Lys Ile 175 Thr Asp Ile Giu Phe Glu Gly Asn 180 Val Tyr Ser Asp Arg Lys Leu 190 Met Arg Gin Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr 195 Arg Ser Asn Gln Phe Asn
210- Val Thr Asp Phe Tyr Gin 225 230 Asp Thr Asp Ile Gin Thr 245 Ile Thr Val His Glu Gly 260 Glu Gly Asp Thr Asn Glu 275 Thr Met Lys Pro Gly Lys 290 Leu Giy Giu Ile Gin Asn 305 310 Giu Ile Ser Vai Gin Pro 325 Phe Val Leu His Ile Glu 340 His Ile Thr Gly Asn Asn 355 Leu Arg Gin Met Giu Ser 370 Ser Lys Giu Arg Val Glu 385 390 Asp Ala Vai Pro Leu Ala 405 Ser Leu Thr Glu Arg Ser 420 Vai Gin Asp Thr Gly Leu 435 Leu Phe Giy Thr Giy Lys 450 Thr Thr Leu Asn Gly Ser 465 470 Asp Gly Val Ser Leu Gly 485 Arg Lys Aia Ser Thr Ser 500 200 Glu Gin I 215 Asn Asn C Asn Glu 2 Gly Arg i Val Pro 280 Trp Tyr 295 Arg Met Leu Pro Pro Gly Lys Thr 360 Ala Pro 375 Leu Leu Gly Thr Thr Gly Val Met 440 Ser Ala 455 Leu Ser Tyr Asp Ile Lys ys P ;iy sp I Phe 1 265 LrysZ lu ly Asn Arg i 345 Arg Tyr Gly Pro Ser 425 Ser Ala Phe Val Gin 505 'he ~yr jys !50 ~rg kla krg Ser Ala 330 Liys Asp Asp Tyr Asp 410 Leu Ala Leu Thr Tyr 490 Tyl 2 Ala Gin A 220 Phe Asp F 235 Thr Lys C Trp Giy I Glu Leu C Gin Gin b 300 Ala Giy 315 Glu Thr i Ile Tyr Glu Val Thr Ser 380 Phe Asp 395 Lys Val Asp Leu Gly Val Arg Ala 460 Asp Pro 475 Gly Lye Lys Thr ,sp 'he ln 11Ys flu 185 let Pyr Lys V'al Val 365 Lye Asn Asp Ser Ser 445 Ser Tyr Ala Thi Met Arg Thr Vai 270 Lys Thr Ala Thr Asn 350 Arg Leu Val Leu Ala 430 Gln Arg Phc Ph Thl Glu I Ile L 2 Ile I 255 Ser I Leu I Ala Tyr Val 335 Glu Arg I Gin Gin Asn 415 Gly Asp Ser Thr a Asp 495 r Ala 'ye leu jys le Jeu ral 3er ksp Ile 31u Arg Phe 400 Met Trp Asn Lys Ala 480 Pro Gly 510 Ala Gly Ile Arg Met Ser Val Pro Val Thr Giu Tyr Asp Arg Val Asn Phe Gly 530 Pro Lys 545 Thr Asp Gly Arg Thr Gly Tyr Ser 610 Phe Thr 625 Arg Thr Gly Ser Asp Glu Val Ser 690 Thr Val 705 Lys Thr Asn Ile Leu Arg Pro Met 770 Glu Ile 785 515 Leu His Gly Asn Val 595 Ala Leu Lys Val Tyr 675 Ala Arg Tyr Tyr Tyr 755 Lys Gin His Val Ala Glu
535. Tyr Ala Asp Phe 550 Ser Phe Lys Gly 565 Lys Thr Asp Ser 580 Asn Ala GJu Ile Thr His Asn Gin 615 Met Leu Gly Gly 630 Glu Ile Pro Phe 645 Arg Gly Tyr Glu 660 Gly Giu Lys Ile Glu Leu Leu Phe 695 Leu Ser Leu Phe 710 Asp Asp Asn Ser 725 Gly Ala Gly Asn 740 Ser Ala Giy Gly Phe Ser Tyr Ala 775 Arg Phe Gin Phe 520 Leu Ile Trp Ala Ala 600 Thr Glu Phe Ser Ser 680 Pro Ala Ser Thr Ala 760 Tyr Lys Leu Leu 585 Leu rrp Val Glu Gly 665 Tyr Met Asp Ser His 745 Val Lys Tyr 570 Trp Pro Phe Gly Asn 650 Thr Gly Pro Ala Ala 730 Lys Thr Thr Val Asn Thr Tyr( 555 Lys Pro Gly Phe Ile 635 Phe Leu Gly Gly Gly 715 Thr Ser Trp 540 3iy Gly Thr Ser Pro 620 Ala Tyr Gly Asn Ala 700 Ser Gly Thr Leu Lys 780 525 Tyr Lys Thr Arg Lys 605 Leu Gly Gly Pro Lys 685 Lys Val Gly Phe Ser 765 Lys Thr Vlal Gly 590 Leu Ser Gly Gly Lys 670 Lys Asp Trp Arg Thr 750 Pro Pro Asp Gly 575 Tyr Gin Lys Tyr Gly 655 Val Ala Ala Asp Vai 735 Asn Leu Glu ksn Lys Ala Gly 560 Trp Leu Tyr Thr Gly 640 Leu Tyr Asn Arg Gly 720 Gin Glu Gly Asp Pro Leu Lys Gin Leu Gly Thr Thr Phe 795 <210> 4 <211> 21 <212> PRT <213> Neisseria meningitidis <400> 4 Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met. Leu Gly Ile Ser 1 5 10 Pro Leu Ala Leu Ala' <210> <211> 776 <212> PRT <213> Neisseria menirgitidis <400> Asp Phe Thr Ile Gn Asp Ile Arg Val Giu Gly Leu Gin Arg Thr Glu 1 Pro Asn Phe Thr Lye Leu Ala Ile Ile 145 Glu Leu Asn Gin Ser Asp Phe Val Met Ala Gly Thr 130 Thr Gly Thr Glu Asn 210 Thr Thr I Asp Ile Leu Gin Leu 115 Pro Ile Asn Glu Gin 195 Asn Ja1 His Asp Glu Gin Ser 100 Lys Lys Asp Gin Gly 180 Lys Glj 5 Phe Gly Val Arg Asn Gin Glu Val Glu Val 165 Gly Phe Tyr Asn Tyr Leu 1 Ser Ala Ile 40 Arg Vai Glu 55 Pro Thr Ile 70 Asp Ala Ile Tyr Phe Asn I Glu Tyr Leu 120 Thr Lys Leu 135 Gly Lys Ser 150 Tyr Ser Asp Ile Trp Thr Ala Gln Asp 200 Phe Asp Phe 215 Thr Lys Gln 230 Trp Gly Lys 10 !ro 55 Ele hr ly Lys 3ml 105 Gly Ala Ala Arg Trp 185 Met Arg Thr Val Vai I Lys Ala Ser Lys 90 Ala Arg Arg Lye Lys 170 Leu Glu Ile Ile Ser 250 ys 3er ksp Leu 75 Asn Thr Gly ASn Ile 155 Leu Thr Lys Leu Lys 235 Ile Val Leu Gly Asn Leu Leu Lys Arg 140 Thr Met Arg Val Asp 220 Ile Glu Gly Tyr Gin Ile Glu Asn Leu 125 Val Asp Arg Ser Thr 205 Thr Thr Gly Asp Thr Ala Thr Leu Leu Thr Gly Ser Phe I Gin Aia 110 Asn Ile Asp Ile Ile Glu Gin Met 175 Asn Gin 190 Asp Phe Asp Ile Val His Asp Thr 255 [yr 3ly Leu Ala BO Gly Val Gin Asp Phe 160 Ser Phe Tyr Gin Glu 240 Asn Thr Asn Glu Asp Lys Gly Giy Arg Phe Arg 245 Glu Val Pro Lys Ala Glu Leu Giu Lys Leu Leu Thr Met Lys Pro Gly Lys Trp Ty'r 275 Asn Arg 290 Pro Leu 305 Glu Pro I Asn Lys Ser Ala Glu Leu 370 Ala Gly 385 Ser Thr Leu Val Lys Ser Ser Leu 450 Gly Tyr 465 Ser lie Ser Val Glu His Asp Phe 530 Lye Gly 545 Asp Ser Met Pro Gly Thr Pro 355 Leu Thr Gly Met Ala 435 Ser Asp Lys Pro Leu 515 Ile Tr Ali 260 Glu Gly Asn Arg Arg 340 Tyr Gly Pro Ser Ser 420 Ala Phe Val Gin Val 500 Thr Lys Leu Leu Ser Ala Lye 325 Asp Asp Tyr Asp Leu 405 Ala Leu Thr Tyr Tyr 485 Thr Val Lys Tyr Trp Ala Glu 310 lie Glu Thr Ph. Lys 390 Asp Gly Arg Asp Gly 470 Lys Glu Asn Tyr Lye 550 Pro Arg Gin Gin Met Gly Tyr 295 Thr Lye Tyr Val Val Val Ser Lys 360 Asp Asn 375 Vai Asp Leu Ser Val Ser Ala Ser 440 Pro Tyr 455 Lys Ala Thr Thr Tyr Asp Thr Tyr 520 Gly Lye 535 Gly Thr Thr Arg 265 [hr Ala kia Tyr rhr Val ksn Glu 330 Arg Arg 345 Leu Gin Val Gin Leu Asn Ala Gly 410 Gin Asp 425 Arg Ser Phe Thr Phe Asp Thr Ala 490 Arg Val 505 Asn Lys Thr Asp Val Gly Gly Tyr 570 Val Leu Giy C 285 Ser Giu lie 1 300 Asp Ph. Val I 315 Ile His lie Glu Leu Arg Arg Ser Lye 365 Ph. Asp Ala 380 Met Ser Leu 395 Trp Val Gin Asn Leu Ph. Lys Thr Thr 445 Ala Asp Gly 460 Pro Arg Lys 475 Gly Aia Gly Asn Ph. Gly Ala Pro Lys 525 Gly Thr Asp 540 Trp Gly Arg 555 Leu Thr Gly er eu [hr 31n 350 Glu Val Thr Asp Gly 430 Leu Val Ala Ii Leu 510 His Gly Asr Val 2 Val His Gly 335 Met Arg Pro Glu Thr 415 Thr Asn Ser Ser Arg 495 Val Tyr Ser Lys Asn 575 iu Ile Gln 3;l Ile 320 ksn Glu Vtal Leu Axg 400 Gly Gly Gly Leu Thr 480 Met Ala Ala Ph. Thr 560 Ala 565 Giu lie Ala Leu Pro Gly Ser Lys Leu Gin Tyr Tyr Sen Ala Thr His Asn Gin Thr 595 Trp Phe Phe Pro Leu Ser Lys Thr Phe Thr 600 605 Leu Met Leu LJys Glu Ile Gly Sly Glu Val Sly Ile Ala 610 615 Gly Gly Tyr Gly Arg Thr 620 Phe Phe Glu Asn Phe 630 Tyr Gly Gly Sly Leu 635 Gly Ser Val Arg Tyr Glu.Ser Gly Leu Gly Pro Lys Val .650 Tyr Asp Glu Tyr Gly Glu 655 Lys Ile Ser Leu Phe Pro 675 Gly Gly Asn Lys Ala Asn Val Ser Ala Glu Leu 670 Arg Leu Ser Met Pro Sly Ala Asp Ala Arg Thr Leu Phe 690 Ala Asp Ala Sly Val Trp Asp Gly Lys 700 Thr Tyr Asp Asp Asn Ser Ser Ser Ala 705 Sly Asn Thr His Lys 725 Sly Gly Arg Val Asi Ile Tyr Sly Ser Thr Phe Thr Asn 730 Siu Leu Arg Tyr Ser Ala 735 Sly sly Ala Val 740 Tyr Ala Tyr Pro
755. Thr Trp Lou Ser Leu Sly Pro Met Lys Phe Ser 750 Sin Arg Phe Leu Lys Lys Pro Glu-Asp Slu Ile 765 Gin Phe 770 Sin Lou Sly Thr Thr Phe 775 <210> 6 <211> 2379 <212> DNA <213> Neisseria gonorrhoeae <400> 6 atgaaactga aacagattgc gccgacttca ccatccaaga gtattcaact acctgcccgt atcatcaaaa gcctgtacgc gggcagcttc tgctgaccgt gccaaaatgc tgcaaaacga tcgcaatact ttaatcaggc ctcgggcgtg gcaaactcaa cgcgtcgaca tcgacatcac tttgaaggca accaagtcta ggcggcattt ggacatggct gacatggaaa aagtaaccga gataccgaca tccaaaccaa gaaggcggac gtttccgctg aaggccgaac tggaaaaac t atgaccgccg ttttgggtga ctccgcactg catccgtgtc caaagtcggc caccggtttc tatcgaacgc cgccatcaag gacac tcaac tatccaaatc gattgacgag ttccgaccgc gacacgaagc cttctaccag cgaagacaaa gggcaaagtg gctgaccatg gattcagaac atgatgttgg gaaggcttgc gacacctaca tttgacgacg cccaccatcg aaaaacctcg caggcagtcg acgcccaaag ggcaaatccg aaactgatgc gaccggttcg aacaacggct accaggcaga tcgattgaag aagcccggca cgcatgggct gcatatcgcc agcgtaccga acgacacaca tacgagtcga gctcgctcaa aatcgttcgg ccggcctgaa taaccaaact ccaaaatcac ggcagatgtc accgccagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggcta tttggcattt gccgagcacc cggcagtgcc aactgcggac catcaccggc gctggcgcag agaagaatac cgcccgcaac cgacatcgaa gctgaccgaa attcgcccaa ccgtatcctc caccgtccac cgaagtcccc acgccagcag cgcatacagc 120 180 240 300 360 420 480 540 600 660 720 780 840 *900 960 gaaatcagcg atcgaaccgg cgcgacgaag aagctgcaac gatgccgtcc cgc tccaccg tccgccggcg tcgcgaagca gacggggtca accagcgtca gttaccgaat tacaacaaag gcagacggca accgacagcg ctgcccggca ttaagcaaaa agaaccaaag ggctacgaaa tacggcggca aaagacgcac agaacctata aaatccacct cct ttgggtc atccaacgct tacagccgct gccggaaaat tcgtgcgccg gc tccaaaga cgcttgccgg gctcgctcga tatcgcagga aaaccacgct gcctgggcta aacaatataa acgaccgcgt cacccaaacg gcttcaaagg cgtcatggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgct acaaaaaagc gcaccgtccg ccgccgccga ttaccaacga cgatgaaatt tccaattcca gccgaacocc ctacgtcaac cgaattgcgc gcgcgtcgag tacgcccgac cttgagcgcg caacctgttc caacggctcg cgatatttac aaccaccacc caatttcggg ctatgccgac cctgctgtac gacgcgcggc atactactcc gatgctcggc ctttgaaaac cggcccgaaa caacgtctcc cctgagcctg aaacggtaac attgcgctat cagctacgcc gctcggcacg ggaaccaaaa ccgtcgattt gaaatccaca tcaccggcaa caaatggaat ccgcgcctta cttttgggct acttcgacaa aaagtcgatt tgaacatgag ggctgggttc aggataccgg ggtacgggca agtcggccgc ctgtcgttta ccgacccgta ggaaaagcct tcgacccgcg gccggcggcg gcgtaaggat ctggcggcgg aacacctgac tttatcagga aatacggcaa aaaggcaccg tcggctgggg tacctgaccg gcgtaaatgc gccacccaca accaaacctg ggcgaagtcg gcattgcggg ttctacggcg gcggcctggg gtgtatgacg aatacggcga gccgagctgc tcttcccgat tttgccgacg caggcagcgt aacaaatcgg tttactcgga tccgccggcg gcgcggttac tacccgctga agaaaaaacc acgttctaa cgtcctgcac caacaaaacc cgacacctcc cgtacagttt cctgaccgaa cttggtcatg cctgcgcgcc cttcacggca caaagcatcg gggtatcccc cgtcaacacc aaccgacggc gcgcaacaag cgaaatcgcc gttcttcccc cggc tacggc ttcggtgcgc aaaaatcagc gCccggtgcg gtgggacggc aaacgcgcat ctggCtctcg ggaagacgaa 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2379 <210> 7 <211> 792 <212> PRT <213> Neisseria gonorrhoeae <400> 7 Met Lys Leu Lys Gin Ilie Ala Ser Ala Leu Met 10 Met Leu Gly Ile Ser Pro Leu Ala Phe Ala Asp Phe Thr Gin Asp Ile Arg Pro Vai Lys Leu Gin Arg Thr Glu Pro Ser Val Phe Asn Tyr Val Gly Asp Thr Tyr Asn Asp Thr His Giy Ser Ile Ile Lys Ser Tyr Ala Thr Gly Phe Asp Asp Vai Val Glu Thr Ala Gly Gin Leu Leu Thr Val Ile Giu Pro Thr Ile Gly Ser Leu Asn Ile Thr Leu Glu Ser 115 Ala Lys Met Leu Gin Asn Asp Aia Ile 105. Lys Lys Asn 110 Phe Gly Leu Ala Ser Gin Tyr Phe Aen Gin Ala Thr Leu Asn 130 Lys Leu 145 Gin Ala Val Ala Gly Leu Lye Giu Giu Tyr 135 140 Leu Gly Arg Gly Asn Ile Gin Ile Thr Pro Lye Val Thr Lys Leu Ala Arg 150 155 Arg Val Asp Ile Asp Ile Thr Ile Asp Giu Gly Lys Ser Ala Lys Ile 165 170 175 Thr Asp Ile Giu Phe Giu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu 180 185 190 Met Arg Gin Met Ser Leu Thr Giu Gly Gly Ile Trp Thr Trp Leu Thr 195 200 205 Atg Ser Asp Arg Phe Asp Arg Gin Ly's Phe Ala Gin Asp Met Giu Lys 210 215 220 Val Thr Asp Phe Tyr Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu 225 230 235 240 Asp Thr Asp Ile Gin Thr Asn Giu Asp Lys Thr Arg Gin Thr Ile Lys 245 250 255 Ile Thr Val His Giu Gly Gly Axg Phe Arg Trp Gly Lys Val Ser Ile 260 265 270 Giu Gly Asp Thr Asn Glu Val Pro Lys Aia Giu Leu Giu Lys Leu Leu 275 280 285 Thr Met Lys Pro Gly Lys Trp Tyr Giu Arg Gin Gin Met Thz Ala Vai 290 295 300 Leu Gly Giu Ile Gin Asn Arg Met Giy Ser Ala Gly Tyr Ala Tyr Ser 305 310 315 .320 Giu Ilie Ser Vai Gin Pro Leu Pro Asn Ala Gly Thr Lys Thr Val Asp 325 330 335 Phe Val Leu His Ile Glu Pro Giy Arg Lys Ile Tyr Val Asn Giu Ile 340 345 350 His Ilie Thr Gly Asn Asn Lys Thr Arg Asp Giu Vai Val Arg Arg Giu 355 360 365 Leu Arg Gin Met Giu Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg 370 375 380 Ser Lys Giu Arg Val Giu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe 385 390 395 400 Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met 405 410 415 Ser Leu Thr Giu Arg Ser Thr Giy Ser Leu Asp Leu Ser Ala Gly Trp 420 425 430 Val Gin Asp Thr Gly Leu Val Met Ser Ala Giy Val Ser Gin Asp Asn 435 440 445 Leu Phe Gly Thr Giy Lys Ser Ala Aia Leu Arg Ala Ser Arg Ser Lys 450 455 460 Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala 465 470 475 480 Asp Gly Val Ser Lou Gly Tyr Asp 485 Arg Gly Phe Pro 545 Ala Gly Thr Phe 625 Arg Gly Asp Val Thr 705 Arg Giu Gly Tyr Lys Ala Gly Val 515 Gly Leu 530 Lys Arg Asp Gly Arg Asn Gly Val 595 Ser Ala 610 Thr Lou Thr Lys Ser Val Glu Tyr 675 Ser Ala 690 Val Arg Thr Tyr Asn Ala Gly Ala 755 Ala Tyr 770 Ser 500 Arg Ala Tyr Ser Lys 580 Asn Thr met Glu Arg 660 Gly Giu Lou Thr His 740 Val Pro Thr Met Ala Ala Phe 565 Thr Ala His Lou Ile 645 Gly Glu Leu Ser Ala 725 Lys Thr Lou Ser Val Gly Ile Glu His 535 Asp Phe 550 Lays Gly Asp Ser Glu Ile Asn Gin 615 Gly Gly 630 Pro Phe Tyr Glu Lys Ile Lou Phe 695 Lou Phe 710 Ala Glu Ser Thr Trp, Leu Lys Lys Lys Pro 520 Lou Ile Leu Ala Ala 600 Thr Glu Phe Ser Ser 680 Pro Ala Asn Phe Ser 760 Lys Ile Tyr Gly 490 Gin Tyr Lys 505 Vai Thr Glu Thr Val Aen Arg Lys Tyr 555 Leu Tyr Lays 570 Ser Trp Pro 585 Lou Pro Gly Trp Phe Phe V/al Gly Ile 635 Giu Asn Phe 650 Gly Thr Leu 665 Tyr Giy Gly Met Pro Gly Asp Ala Gly 715 Giy Asn Asn 730 Thr Asn Giu 745 Pro Lou Gly Pro Glu Asp Lays Thr Tyr Thr 540 Gly Gly Thr S er Pro 620 Ala Tyr Gly Asn Ala 700 Ser Lys Lou Pro Glu 780 Ala Phe Asp Pro 495 Thr Thr Ala Giy 510- Asp Arg Vai Asn 525 Tyr Asn Lys Ala Lys Thr Asp Gly 560 Thr Val Gly Trp, 575 Arg Giy Tyr Leu 590 Lys Lou Gin Tyr 605 Leu Ser Lys Thr Gly Giy Tyr Gly 640 Gly Gly Gly Leu 655 Pro Lys Val Tyr 670 Lys Lys Ala Asn 685 Lys Asp Ala Arg Val Trp Asp Gly 720 Ser Vai Tyr Ser 735 Arg Tyr.Ser Ala 750 Met Lays Phe Ser 765 Ile Gin Arg Phe Gin Phe 785 Gin Leu Gly Thr Thr Phe -11- <210> 8 <211> 21 <212> PRT <213> Neisseria gonorrhoeae <400> 8 Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met Leu Giy Ile Ser 1 5 1.0 -Pro Leu Ala Phe Ala <210> 9 <211> 771 <212> PRT <213> Neisseria gonorrhoeae <400> 9 Asp Phe Thr Ile Gin Asp Ile Arg Vai Glu Gly Leu Gin Arg 1 Pro Asn Phe Thr Lys Leu Ala Ile Ile 145 Giu Leu Asp Ser Asp Phe Val Met Ala Gly Thr 130 Thr Gly Thr Arg Thr Thr Asp Ilie Leu Gin Leu 115 Pro Ile Asn Giu Gin 195 5 Val Phe His Gly Asp Val Giu Arg Gin Asn Ser Gin 1.00 Lys Giu Lys Vai Asp Giu Gin Val 165 Gly Gly 180 Lys Phe Asn Ser Arg Pro 70 Asp Tyr Giu Thr Giy 150 Tyr Ile Ala Tyr Ala Val 55 Thr Ala Phe Tyr Lys 135 Lys Ser Trp Gin Asp 215 Leu Ile 40 Giu Ile Ile Asn Leu 120 Leu Ser Asp Thr Asp 200 10 Pro Vai 25 Ile Lys Thr Ala Gly Ser Lys Lys 90 Gin Ala 105 GJly Arg Ala Arg Ala Lys Arg Lys 170 Trp Leu 185 Met Giu Lys Ser Asp Leu 75 Asn Thr Giy Asn Ile 155 Leu Thr Val Leu Gly Asn Leu Leu Lys Arg 140 Thr Met Arg Giy. Tyr Gin Ile Giu Asn Leu 125 Val Asp Arg Ser Asp Ala Leu Thr Ser Gin 110 Asn Asp Ile Gin Asp 190 Thr Thr Thr Leu Gly Phe Ala Ile Ile Glu Met 175 Arg Giu Tyr Gly Leu Ala Giy Val Gin Asp Phe 160 Ser Phe Lys Val Thr Asp Phe Tyr 205 Gin Asn 210 Asn Gly Tyr Phe Phe Arg Ile Leu Asp 220 Thr Asp Ile Gin -12- Thr Asn Giu Asp Lys Thr Arg Gin Thr Ile Lys Ile Thr Val His Giu 225 230 235 240 Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile Glu Gly Asp Thr Asn 245 250 255 Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu Thr Met Lys Pro Gly 260 265 270 Lyb Trp Tyr Glu Arg Gin Gin Met Thr Ala Val Leu Gly Glu Ile Gin 275 .280 285 Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser Glu Ile Ser Val Gin 290 295 300 Pro Leu Pro Asn Aia Gly Thr Lys Thr Val Asp Phe Val Leu His Ile 305 310 315 320 Giu Pro Gly Arg Lys Ile Tyr Vai Asn Giu Ile His Ile Thr Gly Asn 325 330 335 Asn Lys Thr Arg Asp Giu Val Val Arg Arg Giu Leu Arg Gin Met Giu 340 345 350 Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg Ser Lys Glu Arg Val 355 360 365 Glu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe Asp Ala Vai Pro Leu 370 375 380 Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met Ser Leu Thr Giu Arg 385 390 395 400 Ser Thr Gly Ser Leu Asp Leu Ser Ala Giy Trp Val Gin Asp Thr Gly 405 410 415 Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn Leu Phe Gly Thr Gly 420 425 430 Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys Thr Thr Leu Asn Giy 435 440 445 Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala Asp Gly Val Ser Leu 450 455 460 Gly Tyr Asp Ile Tyr Gly Lys Ala Phe Asp Pro Arg Lys Ala Ser Thr 465 470 475 480 Ser Val Lys Gin Tyr Lys Thr Thr Thr Ala Gly Gly Gly Val Arg Met 485 490 495 Giy Ilie Pro Vai Thr Giu Tyr Asp Arg Val Asn Phe Gly Leu Ala Ala 500 505 510 Giu His Leu Thr Vai Asn Thr Tyr Asn Lys Ala Pro Lys Arg Tyr Ala 515 520 525 Asp Phe Ile Arg Lys Tyr 530 Gly Lys Thr Asp (fly 535 Ala Asp Gly Ser Phe 540 -13- Lys 545 Asp Glu Asn Gly Pro 625 TIyr Lys Leu Leu Ala 705 Ser Trp, Lys Thr Gly Ser Ile Gin Gl y 610 Phe Glu Ile Phe Phe 690 Giu- Thr Leu Lys Thr 770 Leu Ala Ala Thr 595 Glu Ph~e Ser Ser Pro 675 Ala Asn Phe Ser Lys 755 Phe Tyr Trp 565 Pro Phe Gly Asn Thr 645 Gly Pro Ala Asn Asn 725 Leu Thr Arg Lys Leu 600 Gly Gly Pro Lys Lys 680 Val Ser Arg Met Val Gly Trp 555 Gly Tyr Leu 570 Leu Gin Tyr 585 Ser Lys Thr Gly Tyr Gly Gly Gly Leu 635 Lys Val Tyr 650 Lys Ala Asn 665 Asp Ala Arg Trp Asp Gly Val Tyr Ser 715 Tyr Ser Ala 730 Lys Phe Ser 745 Gly Arg Asn Lys Thr Tyr Phe Arg 620 Gly Asp Val Thr Arg 700 Giu GJly Tyr Gly Ser Thr 605 Thr Ser Glu Ser Val 685 Thr Asn Gly Ala Val Ala 590 Leu Lys Val Tyr Ala 670 Arg Tyr Ala Ala Tyr 750 Asn 575 Thr Met Glu Arg Gly 655 Giu Leu Thr His Val 735 Pro Thr 560 Ala His Leu Ile Giy 640 Giu Leu Ser Ala Lys 720 Thr Leu Glu Asp Glu Ile Gin Arg Phe Gin Phe Gin Leu Giy <210> <211> 2394 <212> DNA <213> Neisseria meningitidis <400> atgaaactgA aacagattgc ttccgcactg atggtcttgg gcatatcgcc tttggcactt gccgacttca ccatccaaga catccgcgtc gaaggcttgc agcgtaccga gccgagtacc gtattcaact acctgcccgt caaagtcggc gacacctaca acgacacaca cggcagtgcc atcatcaaaa gcctgtacgc caccggtttc tttgacgacg tacgcgtcga aactgcggac gggcagctcc tgctgaccgt tatcgaacgc cccaccatcg gctcgctcaa catcaccggc gcaaaaatgc tgcaaaacga cgccattaag aaaaacctcg aatcgttcgg gctggcgcag tcgcaatact ttaatcaggc gacactcaat caggcagtcg ccggcctgaa agaagaatac ctcgggcgcg gcaaactcaa tatccaaatc acgcccaaag taaccaaact cgcccgcaac 120 180 240 300 360 420 480 -14- cgcgtcgaca tttgaaggca ggcggcattt gacatggaaa gataccgaca gaaggcggac aaagccgaac atga~CCgccg gaaatcagcg atcgaaccgg cgcgacgaag aagc tgcaac gatgccgtcc cgttccaccg tccgcaggcg tcacgaagca gacggggtca accagcatca gttaccgaat tacaacaaag acagacggca accgacagcg ctgcccggca ttaagcaaaa agaaccaaag ggatacgaaa tacggcggca aaagacgcgc aaaacctacg gccggcaata gttacctggc aaaccggaag tcgacatcac accaagtcia ggacatggct aagtaaccga tccaaaccaa gtttccgttg tggaaaaact ttttgggtga tacagccgct gccggaaaat tcgtgcgccg gctccaaaga cgcttgccgg gctcgctcga tttcccaaga aaaccacgct gcctgggcta aacaatataa acgaccgcgt cgcccaaaca gcttcaaagg cgttatggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgct acaaaaaagc gcaccgtccg acgacaacag cccataaatc tctcgccttt acgaaatcca gattgacgag ttccgaccgc gacacgaagc cttctaccag cgaagacaaa gggcaaagtc gctgaccatg gattcagaac gccaaacgcc ctacgtcaac cgaattgcgc gcgcgtcgag cacacccgac cttgagcgcg caacc tgttc caacggctcg cgatgtttac aaccaccacg gaatttcggt ctatgccgac ctggctgtac gacgcgcggc atactactcc gatgctcggc ctttgaaaac cggtccgaaa caacgtctcc cc tgagcctg cagttccgcg cacctttacc aggcccgatg acgcttccaa ggcaaatccg aaactgatgc aaccaattca aacaacggc t accaagcaga tccatcgaag aagcccggca cgcatgggct gaaaccaaaa gaaatccaca, caaatggaat cttttgggct aaagtcgatt ggctgggtac ggtacgggca ctgtcgttta ggaAaagcct gcaggcgcag ttggtggcag tttatcaaga aaaggtaccg tacctgacgg gccacccaca ggcgaagtcg ttctacggcg gtgtatgacg gccgagctgc tttgccgacg accggcggca aacgaattgc aaat tcagct ttccaactcg ccaaaatcac ggcagatgtc acgagcagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggcta ccgtcgattt tcaccggcaa ccgcgcctta acttcgacaa. tgaacatgag aggataccgg agtcggccgc ccgacccgta tcgacccgcg gcatccgcat aacacctgac aatacggcaa tcggctgggg gcgtgaacgc accaaacctg gcattgcggg gcggcctggg aatacggcga tcttcccgat caggcagcgt gggt tcaaaa gctattccgc acgcctaccc gcacgacgtt cgacatcgaa gctgaccgaa atttgcccaa. ccgcatcctc caccgtccac cgaagtcccc acqccagcag cgcatacagc cgtcctgcac caacaaaacc cgacacctcc cgtacagttt cctgaccgaa cctggtcatg cctgcgcgcc cttcacggca caaagcatcg gagcgtgcct cgtcaacacc aaccgacggc gcgcaacaaa cgaaatcgcc gttcttcccc cggctacggc ttcggtgcgc aaaaatcagc gcccggcgcg gtzgggacggc catttacggc cggcggcgcg gctgaagaaa c taa 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2394 <210> 11 <211> 797 <212> PRT <213> Neisseria ieningitidis <400> 11 Met Lys Leu 1 Pro Lou Ala Leu Gin Arg Lys Gin Ile Ala Ser Ala Leu Met Val Leu Gly Ile Ser Ala Asp Phe Thr Gin Asp Ile Arg Val Glu Gly Pro Val Lys Thr Glu Pro Ser Thr 40 Asp Thr 55 Val Phe Asn His Gly Ser Tyr Lou Val Gly Asp Thr Tyr Asn Ala Ile Ile Lys Ser Val Glu Thr Ala Asv Leu Tyr Ala Thr Gly Phe Leu Thr Phe Asp Asp Val Giy Gin Leu Leu Val Ilie Giu Arg Pro Thr Ile Gly Ser Lou 90 Met Laeu Gin Asn Asp Ala Ile Lys Lys Asn Asn Ile Thr Gly Ala Lys 100 Leu Giu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr 115 120 125 Leu Aen Gin Ala Val Ala Gly Leu Lys Giu Giu Tyr Leu Gly Arg Gly 130 135 140 Lys Leu Asn Ile Gin Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn 145 150 155 160 Arg Val Asp Ile Asp Ile Thr Ile Asp Giu Gly Lys Ser Ala Lys Ile 165 170 175 Thr Asp Ile Giu Phe Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu 180 185 190 Met Arg Gin Met Ser Leu Thr Giu Giy Gly Ile Trp Thr Trp Leu Thr 195 200 205 Arg Ser Aen Gin Phe Asn Glu Gin Lys Phe Ala Gin Asp Met Giu Lys 210 215 220 Vai Thr Asp Phe Tyr Gin Asn Asn Giy Tyr Phe Asp Phe Arg Ile Leu 225. 230 235 240 Asp Thr Asp Ile Gin Thr Asn Giu Asp Lys Thr Lys Gin Thr Ile Lys 245 250 255 Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Giy Lys Val Ser Ile 260 265 270 Giu Gly Asp Thr Asn Giu Val Pro Lye Ala Giu Leu Giu Lys Leu Leu 275 280 285 Thr Met Lys Pro Gly Lye Trp Tyr Giu Arg Gin Gin Met Thr Aia Val 290 295 300 Leu Gly Giu Ile Gin Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser 305 310 315 320 Giu Ile Ser Val Gin Pro Leu Pro Asn Ala Giu Thr Lys Thr Val Asp 325 330 335 Phe Val Leu His Ile Giu Pro Gly Arg Lye Ile Tyr Val Asn Giu Ile 340 345 350 His Ile Thr Gly Asn Aen Lye Thr Arg Asp Glu Val Val Arg Arg Giu 355 360 365 Leu Aig Gin Met Glu Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg 370 375 380 Ser Lys Giu Arg VJal Glu Leu Leu Gly Tyr Phe Asp Asn Vai Gin Phe 385 390 395 400 Asp Ala Val Pro Leu Ala Giy Thr Pro Asp Lys Val Asp Leu Asn Met 405 410 415 Ser Leu Thr Giu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp 420 425 430 Val Leu Thr 465 Asp Arg Ala Phe Pro 545 Thr Cly Thr Tyr Phe 625 Arg ly Asp Val Thr 705 Lys Asp Thr 435 Gly Thr Leu Asn Val Ser Ala Ser 500 Ile Arg 515 Leu Val His Tyr Gly Ser Asn Lys 580 Val Asn 595 Ala Thr Leu Met Lys Glu Val Arg 660 Tyr Gly 675 Ala Glu Arg Leu Tyr Asp Leu Lys Ser 470 Gly Ser Ser Glu Asp 550 Lys Asp Glu Asn Gly 630 Pro Tyr Lys Leu Leu 710 Asn Met 440 Ala Ser Asp Lys Pro 520 Leu Ile Trp Ala Ala 600 Thr Glu Phe Ser Ser 680 Pro Ala Ser Gly Val Arg Ala 460 Asp Pro 475 Gly Lys Lys Thr Glu Tyr Asn Thr 540 Tyr Gly 555 Lys Gly Pro Thr Gly Ser Phe Pro 620 Ile Ala 635 Phe Tyr Leu Gly Gly Asn Gly Ala 700 Gly Ser 715 Thr Gly Ser 445 Ser Tyr Ala Thr Asp 525 Tyr Lys Thr Arg LyS 605 Leu Gly Gly Pro LyS 685 Lys Val Gly Gin Asp Arg Ser Phe Thr Phe Asp 495 Thr Ala 510 Arg Val Asn Lys Thr Asp Val Gly 575 Gly Tyr 590 Leu Gin Ser Lys Gly Tyr Gly Gly 655 Lys Val 670 Lys Ala Asp Ala Trp Asp Arg Val 735 Asn Lys Ala 480 Pro Gly Asn Ala Gly 560 Trp Leu Tyr Thr Gly 640 Leu Tyr Asn Arg Gly 720 Gin Asn Ile Tyr Gly 740 Ala Gly Asn Thr His Lys Ser Thr Phe 745 Thr Asn Glu 750 I -17- Leu Arg Tyr Ser Ala Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly 755 760 765 Pro Net Lys Phe Ser Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp 770 775 780 Glu Ilie Gin Arg Phe Gin Phe Gin Leu Gly Thr Thr Phe 785 790 795 <210> 12 <211> 21 <212> PRT <213> Neisseria meningitidis <400> 12 Met Lys Leu Lys Gin Ile Aia Ser Ala Leu Met Val Leu Giy Ile Ser 1 5 10 Pro Leti Ala Leu Ala <216> 13 <211> 776 <212> PRT <213> Neisseria meningitidis <400> 13 Asp Phe Thr Ile Gin Asp Ile Arg Vai Giu Giy Leu Gin Arg Thr Giu 1 5 10 Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys Val Giy Asp Thr Tyr 25 Asn Asp Thr His Giy Ser Aia Ilie Ile Lys Ser Leu Tyr Ala Thr Giy 40 Phe Phe Asp Asp Vai Arg Val Glu Thr Ala Asp Giy Gin Leu Leu Leu 55 Thr Val Ilie Glu Arg Pro Thr Ile Giy Ser Leu Asn Ile Thr Gly Ala 70 75 Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn Leu Giu Ser Phe Gly 90 Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr Leu Asn Gin Ala Val 100 105 110 Ala Gly Leu Lys Giu Giu Ty'r Leu Gly Arg Gly Lye Leu Asn Ile Gin 115 120 125 Ile Thr Pro Lye Val Thr Lye Leu Ala Arg Aen Arg Val Asp Ile Asp 130 135 140 Ile Thr Ilie Asp Giu Gly Lye Ser Ala Lys Ile Thr Asp Ile Glu Phe 145 150 155 160 Giu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu Met Arg Gin Met Ser 165 170 175 Leu Thr Giu Gly Gly Ile 180 Trp, Thr Asn Giu Gin Lys Phe Ala Gin Thr 225 Gly Glu Lys Asn Pro 305 Glu Asn Ser Glu Ala 385 Ser Leu Lys Ser Gly 465 195 Aen Giu Arg Pro Tyr 275 Met Pro Gly Thr Pro 355 Leu Thr dly Met Ala 435 Ser Asp Tyr Phe Lys Thr 230 Arg Trp 245 Ala Giu Arg Gin Ser Ala Ala Glu 310. Lys Ile 325 Asp Giu Asp Thr Tyr Phe Asp Lys 390 Leu Asp 405 Ala Gly Leu Arg Thr Asp Tyr Gly 470 Gin Asp 215 Lys Gly Leu Gin Gly 295 Thr Tyr Val Ser Asp 375 Val Leu Val Ala Pro 455 Lys Trp Leu Thr 185 Met Glu Lye Arg Ile Leu Thr Ile Lys 235 Val Ser Ile 250 Lys Leu Leu 265 Thr Ala Val Ala Tyr Ser Thr Val Asp 315 Asn Giu Ile 3*3 0 Arg Arg Giu 345 Leu Gin Arg Val Gin Phe Leu Asn Met 395 Ala Gly Trp, 410 Gin Asp Asn 425 Arg Ser Lys Phe Thr Ala Phe Asp Pro 475 Arg Val Asp 220 Ile Giu Thr Leu Giu 300 Phe His Leu Ser Asp 380 Ser Val Leu Thr Asp 460 Arg Ser Thr 205 *Thr Thr Gly Met Gly 285 Ile Val Ile Arg Lys 365 Ala Leu Gin Phe Thr 445 Gly Lys Asn 190 Asp Asp Val Asp Lys 270 Giu Ser Leu Thr Gin 350 Giu Val Thr Asp Gly 430 Leu Val Ala Gin Phe Phe Tyr Ile Gin His Giu 240 Thr Asn 255 Pro Gly Ile Gin Val Gin His Ile 320 Gly Aen 335 Met Glu Arg Val Pro Leu Glu Arg 400 Thr Gly 415 Thr Gly Asn Gly Ser Leu Ser Thr 480 Ser Ile Lys Gin Lys Thr Thr Thr Ala Gly Ala Gly Ile Arg Met 490 1. 495 -19- Ser Val Pro Vai Thr Giu Tyr Asp Arg Val Asn Phe Gly Leu Val Ala 500 505 510 Glu His Leu Thr Val Asn Thr Tyr Asn Lys-Ala Pro Lys His Tyr Ala 515 520 525 Asp Phe Ile Lys Lys Tyr Gly Lys Thr Asp Gly Thr Asp Gly Ser Phe 530 535 540 Lys Gly Trp Leu Tyr Lys Gly Thr Val Gly Trp Gly Arg Asn Lys Thr 545 550 555 .560 Asp'Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu Thr Giy Val Asn Ala 565 570 575 Glu Ilie Ala Leu Pro Gly Ser Lys Leu Gin Tyr Tyr Ser Ala Thr His 580 585 590 Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr Phe Thr Leu Met Leu 595 600 605 Gly Gly Giu Vai Giy Ile Ala Gly Gly Tyr Giy Arg Thr Lye Glu Ile 610 615 620 Pro Phe Phe Giu Asn Phe Tyr Gly Gly Gly Leu Gly Ser Val Arg Gly 625 630 635 640 Tyr Giu Ser Giy Thr Leu Gly Pro Lys Val Tyr Asp Giu Tyr Giy Giu 645 650 655 Lys Ile Ser Tyr Gly Giy Asn Lys Lys Ala Asn Val Ser Ala Glu Leu 660 665 670 Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg Thx Val Arg Leu Ser 675 680 685 Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly Lye Thr Tyr Asp Asp 690 695 700 Asn Ser Ser Ser Ala Thr Gly Gly Arg Val Gin Asn Ile Tyr Gly Ala 705 710 715 720 Gly Asn Thr His Lye Ser Thr Phe Thr Aen Giu Leu Arg Tyr Ser Ala 725 730 735 Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly Pro Met Lye Phe Ser 740 745 750 Tyr Ala Tyr Pro Leu Lys Lye Lys Pro Giu Asp Giu Ile Gin Arg Phe 755 760 765 Gin Phe Gin Leu Gly Thr Thr Phe '770 775 <210> 14 .c211> 441 <212> PRT <213> Neisseria meningitidis <400> 14 Met Lys Lys Tyr Leu Phe Arg Ala Ala Ile Asp Pro Pro Leu Trp Gin Gly Leu 145 Gly Ser Thr Pro Arg 225 Asp Phe Lys Ser Thr 305 Ala Ala Ser Val Gly Thr Leu Ser Ser Phe Asp Val 100 Lys Gin 115 Gly Ser Gly Asp Pro Asp Lys Ala 180 Asp Asn 195 Thr Ala Leu Pro Lye Ala Met His 260 Ilie Arg 275 Gly Arg Cys Ile Thr Leu Arg Cys Phe Leu Asp Asp 165 Leu Thr Arg Tyr Pro 245 Ile Ile Tyr Gin Asn Vai Pro 70 Leu Ala Phe Ala Arg 150 Phe Val Giy Thr His 230 Ile Gin Gly Met Ser Gly Gly His Giy Gin Giu Gly 135 Arg Ile Arg Giy Thr 215 Thr Leu Gly Tyr Ala 295 LYS Pro 40 Gly Trp Cys Ala Arg 120 Thr Thr Ser Ile Thr 200 Ala Arg Gly Ser Ala 280 Asp Ser 25 Asp Giy Ala Ala Phe 105 Tyr Val Ala Vai Arg 185 His Ile Asn Tyr Giy 265 Asp Lys Leu Ile Arg Gly Ala Asn 90 Gin Phe Thr Gin Pro 170 Gin Thr Lye G3,n Ala 250 Arg LYS Gili Gin Pro Ala Gin 75 Leu Thr Thr Gly Ala 155 Leu Thr Ala Gly Ile 235 Giu Leu Asn 'Tyr Arg 315 Gly Thr Val Val Asp Lye Pro Pro Tyr 140 Axg Pro Gly Asp Arg 220 Asn Asp Lys Giu Leu 300 Ile Ala Phe Pro Gly Ile Tyr Thr Phe Ala Asn Arg Vai His 110 Trp, Gin 125 Tyr Giu Phe Pro Ala Gly Lys Asn 190 Leu Ser 205 Phe Glu Giy Gly Pro Vai Thr Pro 270 His Pro 285 Lys Leu klia Gln Pro V1al L~ys Gin Ser Val Pro Ile Leu 175 Ser Arg Gly Ala Glu 255 Ser Tyr Gly Ala Pro Asp Val Ser Gly Phe Ala Val Tyr 160 Arg Gly Phe Ser Leu 240 Leu Gly Val Gin Ser Met Gin Giy Ile Lye Ala Tyr Met Gin Asn Pro Gin Arg 320 -21- 00 Leu Ala Glu Val Leu Ala Gly Ser 340 Leu Met Gly Glu 355 Gly Ala Pro Leu Leu 325 Ser Gly Gin Asn Pro Tyr Ile Phe Phe Arg Glu 335 Asn Asp Gly Pro 345 Val1 Gly Ala Leu Tyr Ala Gly Asp Arg His Tyr 365 Thr Giy Thr Pro 350 Ile Thr Leu Arg Lys Ala Phe Val 370 Leu Asn Ala 375 Ala Ala His Pro Arg Leu Ile Gin Asp Thr 385 Ala Gly 395 Giy Aia Ile Lys Giy 400 Val Arg Val Asp 405 Gin Phe Trp Giy Asp Glu Ala Gly Glu 415 Leu Pro Leu Ala Giy Asn Gly Met 435 Lys Thr Thr Gl y 425 Pro Val Trp Gin Pro Giu Tyr <210> <211> 736 <212> DNA <213> Neisseria rueningitidis (W09936544-0001) <400> acactgttgt atatttagac cacgggagaa aggagtacta aaacggcaca aactgaaaaa aggcttgaat cggtattggt caacgacaac cgctggctgg cgtccgcact tgtggaaagc tattaaagaa ttgcaacggt cccgtacaac aaagaaaaag acagccagag aacttcacct ttatcgttta tttgcgaaag tcgactttga gttaccgatg aacattaaag tacgacacag aaagacaacg aaagac tcaggcaagt gcactgttgc tagaagaaaa aaatcaccyt actcgctgaa gcgcaaacgg aaacggctgg ccgatacgct acgagaaaaa gcgttaaacc tcgagttctt gcaagaaaac gctaaccaat cgtgttgata ttcagattgg caaagccggc aaaagacctc caataaagtc sacgaacggc gctgaatacc acgtgcggca cggtacaaca gagcgcagat cgaagttaaa gaagagcaag aagaagattt gtcaattccg gcagtatatt gacaacctga acagatctga aacatcacaa gacaccacgg ggagcgacca agcgttaaag gcttccgata acgaaaacaa atcggtgcga ataaagaagg tcaacgagaa aaatcaaaca ccagtgttgg gcgacaccaa ttcatctgaa caaacgtaac acgtattaaa acgttgattt cgactgttaa agacttctgt 120 180 240 300 360 420 480 540 600 660 720 736 <210> <211> <212> 22 <213> Neisseria meningitidis (W09936544-0002) 00 <220> <221> <222> <223> misc feature (70) Xaa can be any naturally occurring amino acid <400> 16 Thr 1 Leu Leu Phe Ala Thr Val Gin Ala Ser Ala Asn Gin Glu Giu Gin Giu Glu Asp Ile Val Asn Tyr Leu Asp Pro Val1Gin Arg Thr Val Ala Val Leu Lys Val Giu Ser Asp Lys Giu Gly 40 Thr Gly Giu Lys Giu Giu Asn Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Val Leu Thr Ala Asn Arg Giu Ile Thr Gly Thr Asn Phe Lys Ala Gly Asp As n Leu Lys Ile Lys Thr Tyr Ser Leu Lys Asp Leu Thr Asp Leu Thr Ser Val Val Asn Ile 115 Thr Glu Lys Leu Se r 105 Phe Ser Ala Asn Gly Asn Lys 110 Lys Giu Thr Thr Ser Asp Thr Gly Leu Asn Phe Ala 125 Ala Gly 130 Thr Asn Gly Asp Thr 135 Thr Val His Leu Gly Ile Gly Ser Thr 145 Leu Thr Asp Thr Leu Asn Thr Gly Ala 155 Thr Thr Asn Val Asn Asp Asn Val Thr 165 Asp Asp Glu Lys Arg Ala Ala Ser Val Lys 175 Asp Val Leu Thr Ala Ser 195 Ala Gly Trp Asn Ile 185 Lys Gly Val Lys Pro Gly Thr 190 Thr Val Giu Asp Asn Val Asp Val Arg Thr Tyr Asp 205 Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys 23 00 210 215 220 Asp Asn Gly Lys Lys Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val 225 230 235 240 ile Lys Glu Lys Asp 245 <210> 17 <211> 1776 <212> DNA <213> Neisseria meningitidis (W09936544-0003) <400> 17 atgaacaaaa gagctcacac acactgttgt tatttagacc acgggagaaa gqagtactaa aacggcacaa act gaa a aat ggcttgaatt ggtattggtt aacgacaacg gctggctgga gtccgcactt gtggaaagca attaaagaaa acagacgaag ggttggagaa accgttacat agtaaagatg aacgtcaatc tcgggcaaag aacattaatg acttcgatga tataccgcat gcaaccacac ttgcaacggt ccgtacaacg aagaaaaagt cagccagaga acttcaccta tatcgtttag ttgcgaaaga cgactttgac ttaccgatga acattaaagg acgacacagt aagacaacgg aagacggtaa gcgaaggctt tgaaaacaac caggcacaaa atcaaggcaa agctgcaaaa tcatcagcgg ccggcaacaa ccccgcagtt catttggaat caaacgcgcc tcaggcaagt cactgttgcc agaagaaaat aatcaccctc ctcgctgaaa cgcaaacggc aacggctggg cgatacgctg cgagaaaaaa cgttaaaccc cgagttcttg caagaaaacc gttggttact agtgactgca aaccgctaat tgtaaccttt catcactgtt cagcggttgg caatgtttcg catcgagatt ttccagcgtt agtgccctca tccgcaaccg gctaacaatg gtgttgatag tcagattggg aaagccggcg aaagacctca aataaagtca acgaacggcg ctgaataccg cgtgcggcaa ggtacaacag agcgcagata gaagttaaaa ggtaaagaca aaagaagtga ggtcaaacag gctagtggta atgtatgatg aatttggatt ccgagcaagg acccgcaacg tcgctcggcg atgcctgggt tgaagaccgc aagagcaaga tcaattccga cagtatattt acaacctgaa cagatctgac acatcacaag acaccacggt gagcgaccac gcgttaaaga cttccgataa cgaaaacaac tcggtgcgaa aaggcgagaa ttgatgcagt gtcaagctga aaggtacaac taaatgtcgg ccaaagcggt gaaagatgga gtaaaaatat cgggggcgga cgtcgtatcc cgtattggcg agaagattta taaagaaggc caacgagaaa aatcaaacaa cagtgttgga cgacaccaaa tcatctgaac aaacgtaacc cgtattaaac cgttgatttc gactgttaat gacttctgtt tggttcttct aaacaaggct caagtttgaa tgcgactgta cgatgcccta tgcaggttct tgaaaccgtc cgacatcgcc tgcgcccact 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 -24 00 ttgagcgtgg attaccaatg ggcgtggcgc atcgcccaag atggcgatcg atttccgacg ttcggtgctt atggggacgc tcgccccggg aaaacttgaa cgattgcaac gcggcggcac gcggaaattg ccgcatctgt attgaatgtc cgttaaagag caaccgcatc cgcaggtctg ttatcgcggc gattatcaaa cggttatcag ggcagcaaga ggggatgtta gacaatgtgg gttcaggcgt gaagccggtt ggcacggctt tggtaa aggacaacaa caaacgtcgc acggcaacgc atttgcccgg acgccatcgg ccggcaattc acccgtccgc acaacttaaa gcgtgcgggc caagagtatg ctactccagt gcgcggccat 1440 1500 1560 1620 1680 1740 1776 <210> 18 <211> 591 <212> PRT <213> Neisseria meningitidis (W09936544-0004) <400> 18 Met Asn Lys Ile Tyr 1 5 Arg Ile Ile Trp As n 10 Ser Ala Leu Asn Ala Trp Val Val Val Thr Vai Lys Ser Glu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Ser Ala Asn Asn Glu Gin Glu Glu Asp Leu Tyr Leu Asp Pro Val Gin Arg Thr Val Thr Gly Glu Lys Glu Ala Val Leu Ile Val Ser Asp Lys Glu Lys Val Glu Glu Asn 90 Ser Asp Trp Ala Val Tyr Phe Asn Glu Gly Asp Asn 115 Lys 100 Gly Val Leu Thr Arg Giu Ile Thr 110 Thr Tyr Ser Leu Lys Ile Lys Gin 120 Asn Gly Thr Asn Leu Lys 130 Lys Asp Leu Thr Leu Thr Ser Val Gly 140 Thr Glu Lys Leu Phe Ser Ala Asn Gly 150 Asn Lys Val Asn Ile 155 Thr Ser Asp Thr Lys 160 25 Gly Leu Asn Phe Ala Lys Glu Thr Ala 00 Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Asn 180 Gly Ile Gly Ser Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Giu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Lys Lys 210 Arg Ala Ala Ser Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Asp Asn Val Asp Val Arg Thr Tyr Thr Val Glu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Giu Ser Lys Asp 265 Asn Gly Lys Lys Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Glu Asn Gly Ser Thr Asp Giu Gly Giu 305 Gly Leu Val Thr Lys Glu Val Ile Asp 315 Ala Vai Asn Lys Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 Thr Val 370 Met Tyr Asp Val Asn Ser Gly Trp 390 Asn 375 Val Gly Asp Ala Asn Val Asn Gin Leu 385 Gin Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser Gly Lys Val Ile 405 Ser Gly Asn Val Pro Ser Lys Gly Lys Met 415 -26- Asp Glu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Thr 460 Leu Ser Val Asp Gly 465 Ile Asp Ala Leu Asn Thr Asn Val Ala 485 Val 470 Gly Ser Lys Lys Asn Lys Pro Val Arg 480 Pro Gly Val Lys Glu 490 Gly Asp Val Thr Asn Val 495 Ala Gin Leu Val Asp Gly 515 Lys 500 Gly Val Ala Gin Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Gly Leu 530 Val Gin Ala Tyr Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Gly 550 Glu Ala Gly Tyr Ala 555 Ile Gly Tyr Ser Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Gly His 580 Phe Gly Ala Ser Ala 585 Ser Val Gly Tyr Gin Trp 590 <210> 19 <211> 1779 <212> DNA <213> Neisseria meningitidis (W09936544-0005) <220> <221> misc feature <222> <223> n is a, c, g, or t <220> <221> misc feature <222> (420)..(420) <223> n is a, c, g, or t <220> <221> misc feature 27 00 <222> (608)..(608) <223> n is a, c, g, or t <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> misc feature (682)..(684) n is a, C, g, or t misc feature (763)..(763) n is a, C, g, misc feature (774)..(774) n is a, c. g, or t or t misc feature (1473)..(1473) n is a, c, g, or t misc feature (1492)..(1492) n is a, c, g, or t misc feature (1560)..(1560) n is a, c. g, or t <400> 19. atgaacaaaa gagctcacac acactgttgt gaatccgtac ttggaaacga gtagttaccc aatgccagta actgaaaaat ggcttgaatt ggtatcggtt ggtaaccnaa tggaatatta gtccgcactt gtggaaagca attaaagaaa tataccgcat gcaaccacac ttgcaacggt aacgctctgt tatcattatc tcaaagccgg gcttcaccta tatcgtttgg tcgcgaaaga cgactttgac gtacacatta agggtgttaa acgacacagt aagacaacgg aagacggtaa catttggaat agtgccctca atgcctgngt cgccgtatcc caaacgcgcc tcaggcgaat cgtagggagc aatgactaac cgacaacctg ctcgctgaaa cgcaaacggc aacggctqggg cgatacgctt cactcgtgca annnggctca cgagttcttg caagagaacc gttggttact tccgcaaccg gctaccgatg attcaagcca gacagcaagg aaaatcaaac aaagacctca aagaaagtca acgaacggcg gcgggttctt gcaagtatta acaactggtc agcgcagata gaagttaaaa ggtaaaggca tgaagaccgc aagatgaaga gtatggaagg aatttgtaga aaaacaccaa caggcctgat acatcataag acaccacggt ctgcttctca aggatgtgtt aatcagaaaa cgnaaacaac tcggtqcgaa aaggcgagaa cgtattggcg agaagagtta cagcggcgaa cccatacata tgaaaacacc caatgttgan cgacaccaaa tcatctgaac cgttgatgcg gaatgcgggt tgtcgatttc gacngttaat gacttctgtt tggttcttct -28- 00 acagacgaag ggttggagaa accgttacat agtaaagatg aacgtcaatc tcgggcaaag aacattaatg acttcgatgg ttaagcgtgg cgcattacca aaaggcgtgg ggcatcgccc atgatggcga agtatttccg catttcggtg gcgaaggctt tgaaaacaac caggcacaaa atcaaggcaa agctgcaaaa tcatcagcgg ccggcaacaa cgccgcagtt atgacgaggg atgtcgcccc cgcaaaactt aagcgattgc tcggcggcgg acggcggaaa cttccgcatc agtgactgca aaccgctaat tgtaaccttt catcactgtt cagcggttgg caatgtttcg catcgagatt ttccagcgtt cgcgttgaat gggcgttaaa gaacaaccgc aaccgcaggt cacttatcgc ttggattatc tgtcggttat aaagaagtga ggtcaaacag gctagtggta atgtatgatg aatttggatt ccgagcaagg agccgcaacg tcgctcggcg gtcggcagca ganggggatg atcgacaatg ctggttcagg ggcgaagccg aaaggcacgg cagtggtaa ttgatgcagt gtcaagctga aaggtacaac taaatgtcgg ccaaagcggt gaaagatgga gtaaaaatat cgggggcaga aggatgccaa ttacaaacgt tggacgqcaa cgtatctgcc gttacgccat cttccggcaa aa a ca aggc t caagtttgaa tgcgactgta cgatgcccta tgcaggttct tgaaaccgtc cgacatcgcc tgcgcccact caaacccgtc cncacaactt cgcgcgtgcn cggcaagagt cggctactcc ttcgcgcgqc 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> <211> 592 <212> PRT <213> Neisseria meningitidis (W09936544-0006) <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> misc feature Xaa can be any misc feature (140)..(140) Xaa can be any misc feature (203)..(203) Xaa can be any misc feature (228)..(228) Xaa can be any misc feature (255)..(255) Xaa can be any naturally occurring amino acid naturally occurring amino acid naturally occurring amino acid naturally occurring amino acid naturally occurring amino acid -29- <220> <221> misc feature <222> (491)..(491) <223> Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (498)..(498) <223> Xaa can be any naturally occurring amino acid <400> Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Xaa Val Ala Val Thr Val Lys Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Glu Ser Val Gin Arg Ser Val Val Gly Ile Gin Ala Ser Met 75 Glu Gly Ser Gly Glu Leu Glu Thr Ile Ser Leu Ser Met Thr Asp Ser Lys Glu Phe Val Asp Pro Tyr Lys Gin Asn 115 Val Val Thr Leu Lys 105 Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Glu Asn Asn Ala Ser Ser Phe 125 Leu Lys 130 Lys Asp Leu Thr Gly 135 Leu Ile Asn Val Thr Glu Lys Leu Ser 145 Phe Gly Ala Asn Lys Lys Val Asn Ile 155 Ile Ser Asp Thr Lys 160 Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Ala Gly 190 Ser Ser Ala 195 Ser His Val Asp Ala Gly Asn Xaa Ser 200 Thr His Tyr Thr 205 Arg Ala 210 Ala Ser Ile Lys Asp 215 Val Leu Asn Ala Gly Trp Asn Ile 220 Giu Asn Val Asp Lys Phe 240 Gly 225 Val Lys Xaa Gly Ser 230 Thr Thr Gly Gin Ser 235 Val Arg Thr Tyr Asp 245 Thr Val Giu Phe Leu 250 Ser Ala Asp Thr Xaa Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Glu Ser Lys Asn Gly Lys Arg Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Val1 280 Ile Lys Glu Lys Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Giu Gly Gly Leu Val Thr Ala 310 Lys Glu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Giu 340 Thr Val Thr Ser Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val 360 Ser Lys Asp Asp Thr Vai 370 Met Tyr Asp Vai Val Gly Asp Ala Leu 380 Asn Val Asn Gin Gin Asn Ser Gly T rp 390 Asn Leu Asp Ser Ala Val Ala Gly Ser Gly Lys Val Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Val1 420 Asn Ile Asn Ala Asn Asn Ile Glu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Ala Ser Val 450 rVlSer Leu Gly Ala Gly Ala Asp Ala Pro 450 455 Thr Leu Ser Val Asp 460 -31 Giu Gly Ala Leu Asn 470 Val Gly Ser Lys Ala Asn Lys Pro Arg Ile Thr Asn Ala Pro Gly Val Lys 490 Xaa Gly Asp Val Thr Asn 495 Val Xaa Gin Asn Val Asp 515 Leu 500 Lys Gly Val Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Gly Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala Gly 530 Leu Val Gin Ala Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly 545 Gly Gly Thr Tyr Arg 550 Gly Giu Ala Gly Tyr 555 Ala Ile Gly Tyr Ser Ile Ser Asp Asn Ser Arg Gly 580 Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 His Phe Gly Ala Ser Ala Ser Val Gly 585 Tyr Gin Trp 590 <210> 21 <21i> 591 <212> PRT <213> Neisseria meningitidis (W00066741-000l) <400> 21 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Lys Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ser Ala Asn Asn Glu Glu 55 Gin Glu Giu Asp Leu Tyr Leu Asp Pro Gin Arg Thr Val Val Leu Ile Val Asn Ser Asp Lys Giu Thr Gly Glu Lys Glu Lys Val Glu Glu Aso 90 Ser Asp Trp Ala Val Tyr 32 Phe Asn Glu Gly Asp Asn 115 Lys 100 Gly Val Leu Thr Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Leu Lys Ile Lys Gin 120 Asn Gly Thr Asn Leu Lys 130 Lys Asp Leu Thr Leu Thr Ser Val Giy 140 Thr Giu Lys Leu Phe Ser Ala Asn Gly 150 Asn Lys Val Asn Thr Ser Asp Thr Gly Leu Asn Phe Lys Glu Thr Ala Gi y 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Asn 180 Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Giu Thr Thr Asn Vai Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Ala Ala Ser Val1 215 Lys Asp Vai Leu Ala Gly Trp Asn Ile 225 Lys Gly Val Lys Gly Thr Thr Ala Ser 235 Asp Asn Val Asp Phe 240 Val Arg Thr Tyr Asp 245 Thr Val Giu Phe Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Val Glu Ser Lys Asp 265 Asn Gly Lys Lys Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Thr Asp Glu Gly Glu 305 Gly Leu Val Thr Ala 310 Lys Giu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Asn 330 Gly Gin Thr Giy Gin Ala 335 Asp Lys Phe Giu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser -33- 00 Gly Lys Gly 355 Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 Gly Asn Ile Thr Val 370 Met Tyr Asp Val Asn 375 Val Gly Asp Ala Asn Val Asn Gin Gin Asn Ser Gly Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser 400 Ser Giy Lys Val Ile 405 Ser Gly Asn Val Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Giy Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Ser Vai 450 Ser Leu Gly Ala Giy 455 Aia Asp Ala Pro Leu Ser Val Asp Gly 465 Asp Ala Leu Asn Val1 470 Gly Ser Lys Lys Asp 475 Asn Lys Pro Val Arg 480 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Glu 490 Giy Asp Val Thr Asn Val 495 Aia Gin Leu Val Asp Gly 515 Lys 500 Gly Val Ala Gin Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Gly Leu 530 Val Gin Ala Tyr Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Gly 550 Glu Ala Gly Tyr Ile Gly Tyr Ser Ser 560 Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Gly His 580 Phe Gly Ala Ser Ala 585 Ser Vai Gly Tyr Gin Trp 34 <210> 22 <211> 591 <212> ERT <213> Neisseria meningitidis (W00066741-0002) <400> 22 Asn Lys Ile Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Lys Ser Glu Leu Thr Thr Ala Val Leu Arg Ala His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Leu Leu Phe Ala Ser Ala Asn Asn Giu Gin Giu Glu Asp Leu Tyr Leu Asp Pro Gin Arg Thr Val Ala Val Leu Ile Val Ser Asp Lys Giu Gly Thr Gly Glu Lys Glu Lys Val Glu Giu Asn 90 Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Asp Asn 115 Gly Val Leu Thr Ala 105 Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Leu Lys Ile Lys Asn Gly Thr Asn Phe 125 Leu Lys 130 Lys Asp Leu Thr Asp 135 Leu Thr Ser Val Gly 140 Thr Giu Lys Leu Ser 145 Phe Ser Ala Asn Asn Lys Val Asn Ile 155 Thr Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Giu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Giu Thr Thr Asn Val Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Ala Ala Ser Val 215 Lys Asp Val Leu Asn Ala Gly Trp Asn 220 35 Lys Gly Val Lys 00 Pro 230 Gly Thr Thr Ala Ser 235 Asp Asn Vai Asp Phe 240 Val Arg Thr Tyr Thr Vai Glu Phe Leu Ser Ala Asp Thr 250 Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Glu Ser Lys Asn Gly Lys Lys Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Glu Lys Val Thr 290 Gly Lys Asp Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Giu Gly Gly Leu Val Thr Ala 310 Lys Giu Val Ile Ala Val Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Giy 355 Giu 340 Thr Val Thr Ser Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val 360 Ser Lys Asp Asp Thr Val 370 Met Tyr Asp Val Val Gly Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Gly Trp 390 Asn Leu Asp Ser Ala Val Ala Giy Ser Gly Lys Val Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asn Gly Lys 435 Val1 420 Asn Ile Asn Ala Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Ser Val 450 Gly Asp 465 Ser Leu Gly Ala Ala Asp Ala Pro Thr 460 Leu Ser Val Asp Ala Leu Asn Val Gly Ser Lys Lys AspAs Ly PrVaAg 470 47548 Asn Lys Pro Val Arg 480 36 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Gly Asp Val Thr Asn Val 495 Ala Gin Leu Gly Val Ala Gin Asn Leu Asn Asn Arg 505 Ile Asp Asn 510 Ala Thr Ala Vai Asp Gly 515 Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Ile 525 Giy Leu 530 Val Gin Ala Tyr Leu 535 Pro Gly Lys Ser Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Glu Ala Gly Tyr Ala 555 Ile Gly Tyr Ser Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile Gly Thr Ala Ser Gly Asn 575 Ser Arg Giy Phe Gly Ala Ser Ala 585 Ser Val Gly Tyr Gin Trp 590 <210> <211> <212> <213> 23 591 PRT Neisseria meningitidis (WO006674i-0003) <400> 23 Asn Lys Ile Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Lys Ser Glu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Ser Ala Asn Asn Glu Gin Giu Glu Asp Leu Tyr Leu Asp Pro Val Gin Arg Thr Val Ala Val Leu Ile Val Ser Asp Lys Giu Thr Gly Glu Lys Lys Val Giu Giu Asn Ser Asp Trp Ala Val Tyr Phe Asn Giu Lys 100 Gly Val Leu Thr Arg Giu Ile Thr Leu Lys Ala 110 37 Gly Asp Asn 115 Leu Lys Ile Lys Gin 120 Asn Gly Thr Asn Thr Tyr Ser Leu Lys 130 Lys Asp Leu Thr Leu Thr Ser Val Gly 140 Thr Giu Lys Leu Phe Ser Ala Asn Gly 150 Asn Lys Vai Asn Thr Ser Asp Thr Gly Leu Asn Phe Lys Giu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Asn 180 Gly Ile Gly Ser Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Giu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Ala Ala Ser Val1 215 Lys Asp Val Leu Asn Ala Gly Trp Asn 220 Asp Asn Val Asp Phe Ile 225 Lys Gly Val Lys Gly Thr Thr Ala Ser 235 Val Arg Thr Tyr Asp 245 Thr Val Giu Phe Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Val Giu Ser Lys Asp 265 Asn Gly Lys Lys Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Thr Asp Glu Gly Giu 305 Gly Leu Val Thr Lys Glu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys She Thr Val Thr Ser Gi y 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Lys Gly 355 Thr Thr Ala Thr Val Ser Lys Asp Asp 360 Gin Gly Asn Ile 365 -38- 00 Thr Val 370 Met Tyr Asp Val Asn 375 Val Gly Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Gly T rp 390 Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser Gly Lys Vai Ile 405 Ser Gly Asn Val Pro Ser Lys Giy Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Asn Ile Asn Ala Giy 425 Asn Asn Ile Giu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Ser Val 450 Ser Leu Giy Aia Ala Asp Ala Pro Thr Leu Ser Val Asp 460 Asn Lys Pro Vai Arg Gly 465 Asp Ala Leu Asn Val 470 Gly Ser Lys Lys Ile Thr Asn Val Pro Giy Vai Lys Giu 490 Gly Asp Val Thr Asn Vai 495 Ala Gin Leu Val Asp Gly 515 Lys 500 Gly Vai Ala Gin Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Ile 525 Gly Leu 530 Vai Gin Ala Tyr Pro Giy Lys Ser Met Met Ala Ile Gly 540 Ile Gly Tyr Ser Ser Giy 545 Gly Thr Tyr Arg Gly 550 Giu Ala Gly Tyr Ile Ser Asp Gly Ser Arg Gly His 580 Asn Trp le Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Phe Gly Ala Ser Ser Val Gly Tyr Gin Trp 590 <210> 24 <211> 591 <212> PRT <213> Neisseria meningitidis (W00066741-0004) -39- <400> 24 Met 1 Asn Lys Ile Arg Ile Ile Trp Asn 10 Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Giu Ser Giu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Vai Leu Ala Thr Leu Leu Phe Ala Ser Ala Asn Asn Giu Gin Giu Giu Asp Leu Tyr Leu Asp Pro Ser Asp Lys Giu Gly Gin Arg Thr Val Ala Val Leu Ile Val Thr Giy Giu Lys Giu Lys Val Giu Giu Asn 90 Ser Asp Trp Ala Val Tyr Phe Asn Glu Gly Asp Asn 115 Gly Val Leu Thr Ala 105 Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Leu Lys Ile Lys Asn Gly Thr Asn Phe 125 Leu Lys 130 Lys Asp Leu Thr Asp 135 Leu Thr Ser Val Thr Giu Lys Leu Ser 145 Phe Ser Ala Asn Asn Lys Val Asn Ile 155 Thr Ser Asp Thr Lys 160 Gly Leu Asn Phe Ala 165 Lys Giu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Asn 180 Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Glu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Lys Lys 210 Arg Ala Ala Ser Val 215 Lys Asp Val Leu Asn Ala Gly Trp 220 Asp Asn Vai Asp Asn Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Ser 235 Phe 240 Val Arg Thr Tyr Asp Thr Val Giu Phe Leu Ser Ala Asp Thr Lys Thr 250 00 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Giu Ser Lys Asn Gly Lys Lys 255 Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Val Ile Lys Glu Lys 280 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Ser 300 Thr Asp Giu Gly Gly Leu Val Thr Ala 310 Lys Giu Val Ile Ala Val Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Glu 340 Thr Val Thr Ser Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val 360 Ser Lys Asp Asp Thr Vai 370 Met Tyr Asp Val Vai Gly Asp Ala Leu 380 Asn Val Asn Gin Gin Asn Ser Gly T rp 390 Asn Leu Asp Ser Ala Val Ala Giy Ser Gly Lys Val Ser Gly Asn Val Ser 410 Pro Ser Lys Giy Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Val 420 Asn Ile Asn Ala Asn Asn Ile Giu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Ser Vai 450 Ser Leu Giy Ala Ala Asp Ala Pro Thr 460 Leu Ser Vai Asp Gly 465 Asp Ala Leu Asn Val1 470 Gly Ser Lys Lys Asn Lys Pro Val Ile Thr Asn Val Pro Gly Val Lys Glu 490 Gly Asp Vai Thr Asn Val 495 -41- Ala Gin Leu Lys Gly Val Ala Gin 00 Asn 505 Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Val Asp Gly 515 Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Gly Leu 530 Val Gin Ala Tyr Pro Gly Lys Ser Met 540 Met Ala Ile Gly Giy 545 Gly Thr Tyr Arg Gly 550 Glu Ala Gly Tyr Ile Gly Tyr Ser Ile Ser Asp Gly Giy 565 Asn Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Giy His 580 Phe Giy Ala Ser Ala 585 Ser Vai Giy Tyr Gin Trp 590 <210> <2ii> 591 <2i2> PRT <2i3> Neisseria meningitidis (WO006674i-0005) <400> Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Vai Val Thr Val Lys Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Aia Thr Vai Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Ala Asn Asn Glu Glu Gin Giu Glu Asp Tyr Leu Asp Pro Val Gin Arg Thr Val Val Leu Ile Val Asn 75 Ser Asp Lys Giu Gly Thr Gly Giu Lys Giu Lys Val Giu Giu Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Val Leu Thr Ala 105 Arg Glu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Gly Asp Asn Leu Lys Ile Lys Gln 120 Asn Gly Thr Asn Leu Lys 130 Lys Asp Leu Thr Asp 135 Leu Thr Ser Val Thr Glu Lys Leu Ser 145 Phe Ser Ala Asn Asn Lys Val Asn Ile 155 Thr Ser Asp Thr Lys 160 Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Glu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Lys Lys 210 Arg Ala Ala Ser Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Asp Asn Val Asp Phe 240 Val Arg Thr Tyr Thr Val Glu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Glu Ser Lys Asp 265 Asn Gly Lys Lys Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Glu Gly Glu 305 Gly Leu Val Thr Lys Glu Val Ile Asp 315 Ala Val Asn Lys Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 Thr Val 370 Met Tyr Asp Val Asn Val Gly Asp Ala 375 Asn Val Asn Gin -43- Leu 385 Gin Asn Ser Gly Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser 400 Ser Gly Lys Val Ile 405 Ser Gly Asn Val Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Leu Ser Val Asp Gly 465 Asp Ala Leu Asn Val 470 Gly Ser Lys Lys Asp 475 Asn Lys Pro Val Arg 480 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Glu 490 Gly Asp Val Thr Asn Val 495 Ala Gin Leu Val Asp Gly 515 Lys 500 Gly Val Ala Gin Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Asn Ala Arg Ala Gly 520 Ile Ala Gin Ala Gly Leu 530 Val Gin Ala Tyr Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Gly 550 Glu Ala Gly Tyr Ile Gly Tyr Ser Ile Ser Asp Gly Asn Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Gly His 580 Phe Gly Ala Ser Ser Val Gly Tyr Gin Trp 590 <210> 26 <211> 594 <212> PRT <213> Neisseria meningitidis (W00066741-0006) <400> 26 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 -44 Val Ala Val Thr Val Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gln Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gln Arg Thr Ala Pro Val Leu Phe His Ala Asp Glu Gly Thr Gly Glu Lys Glu Val Thr Glu Asp Ser Asn Trp Val Tyr Phe Asp Lys Lys Gly Val Leu Lys Ile Lys 115 Ala Gly Thr Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asp Glu 120 Asn Thr Asn Ala Tyr Ser 130 Leu Lys Lys Asp Thr Asp Leu Thr Ser 140 Val Glu Thr Glu Leu Ser Phe Gly Ala 150 Asn Gly Lys Lys Asn Ile Thr Ser Asp 160 Thr Lys Gly Leu Phe Ala Lys Glu Thr 170 Ala Gly Thr Asn Gly Asp 175 Thr Thr Val Leu Asn Thr 195 His 180 Leu Asn Gly Ile Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Asn 200 Val Thr Asn Asp Asp Glu 210 Lys Lys Arg Ala Ser Val Lys Asp Val 220 Leu Asn Ala Gly Trp Asn Ile Lys Gly 225 Asp Phe Vai Arg Thr 245 Val1 230 Lys Pro Gly Thr Ala Ser Asp Asn Tyr Asp Thr Val Glu 250 Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr 00 Glu Val Lys 275 Ile Gly Ala Lys Thr 280 Ser Val Ile Lys Giu 285 Lys Asp Gly Lys Leu 290 Val Thr Gly Lys Asp 295 Lys Gly Glu Asn Gi y 300 Ser Ser Thr Asp Glu 305 Gly Glu Gly Leu Thr Ala Lys Giu Val1 315 Ile Asp Ala Val Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Thr 330 Ala Asn Gly Gin Thr Giy 335 Gin Ala Asp Ala Ser Gly 355 Phe Giu Thr Val Thr 345 Ser Giy Thr Lys Val Thr Phe 350 Asp Gin Gly Asn Gly Thr Thr Thr Vai Ser Lys Asp 365 Asn Ile 370 Thr Val Lys Tyr Asp 375 Vai Asn Vai Gly Asp Ala Leu Asn Val 380 Ser Lys Ala Val Ala As n 385 Gin Leu Gin Asn Gly Trp Asn Leu Asp 395 Gly Ser Ser Gly Lys 405 Val Ile Ser Gly Val Ser Pro Ser Lys Giy 415 Lys Met Asp Thr Arg Asn 435 Glu 420 Thr Val Aso Ile Asn 425 Ala Gly Asn Asn Ile Giu Ile 430 Thr Pro Gin Gly Lys Asn Ile Ile Ala Thr Ser Met 445 Phe Ser 450 Ser Vai Ser Leu Gly 455 Ala Gly Ala Asp Ala Pro Thr Leu Ser 460 Lys Asp Ala Asn Lys Vali 465 Asp Asp Giu Giy Leu Asn Val Gly Ser 475 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly 490 Val Lys Giu Gly Asp Val 495 Thr Asn Val Gin Leu Lys Gly Val 505 Ala Gin Asn Leu Asn Asn His 510 00 Ile Asp Asn 515 Val Asp Gly Asn Arg Ala Gly Ile Ala 525 Gin Ala Ile Ala Thr 530 Ala Gly Leu Val Gin 535 Ala Tyr Leu Pro Gi y 540 Lys Ser Met Met Ile Gly Gly Gly Thr 550 Tyr Arg Gly Giu Gly Tyr Ala Ile Gly 560 Tyr Ser Ser Ile Asp Gly Gly Asn T rp 570 Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Ser 580 Arg Gly His Phe Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 27 <211> 594 <212> PRT <213> Neisseria meningitidis (W00066741-0007) <400> 27 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Pro Val Leu Phe His Ala Asp Ser 75 Glu Gly Thr Gly Glu Lys Giu Val Thr Glu Asp Ser Asn Trp Val Tyr Phe Asp Lys Lys Gly Val Leu Lys Ile Lys 115 Thr 100 Ala Gly Thr Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 Gin Asn Thr Asp Giu Asn Thr Asn Ala Ser 125 Ser Phe Thr 00 Tyr Ser 130 Leu Lys Lys Asp Leu 135 Thr Asp Leu Thr Val Glu Thr Glu Lys 145 Leu Ser Phe Gly Asn Gly Lys Lys Val1 155 Asn T1R Thr S -r Thr Lys Gly Leu As n 165 Phe Ala Lys Giu Ala Gly Thr Asn Gly Asp 175 Thr Thr Val Leu Asn Thr 195 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Val Thr Asn Asp As n 205 Asp Glu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val 220 Leu Asn Ala Gly T rp 225 Asn Ile Lys Gly Lys Pro Gly Thr Thr 235 Ala Ser Asp Asn Val1 240 Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Glu Val Lys 275 Val Asn Val Glu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Gly Ala Lys Ser Val Ile Lys Glu 285 Lys Leu 290 Val Thr Gly Lys Asp 295 Lys Gly Glu Asn Ser Ser Thr Asp Glu 305 Gly Glu Gly Leu Thr Ala Lys Glu Val 315 Ile Asp Ala Val Asn 320 Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Ala Ser Gly 355 Phe Glu Thr Val Thr 345 Ser Gly Thr Lys Val Thr Phe 350 Asp Gin Gly Asn Gly Thr Thr Thr Val Ser Lys Asp 365 Asn Ile 370 Thr Val Lys Tyr Asp 375 Val Asn Val Gly Ala Leu Asn Val Gin Leu Gin Asn Ser 390 Gly Trp Asn Lou Ser Lys Ala Val Ala 400 Gly Ser Ser Gly Val Ile Ser Gly Asn 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Glu 420 Thr Val Asn Ile Ala Gly Asn Asn Ile Glu Ile 430 Thr Pro Gin Gly Lys Asn Ile Asp 440 Ile Ala Thr Ser Phe Ser 450 Ser Val Ser Lou Ala Gly Ala Asp Ala 460 Pro Thr Lou Ser Val1 465 Asp Asp Glu Gly Ala 470 Lou Asn Val Gly Lys Asp Ala Asn Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly 490 Val Lys Glu Gly Asp Val 495 Thr Asn Val Ile Asp Asn 515 Gin Lou Lys Gly Val1 505 Ala Gin Asn Lou Asn Asn His 510 Gin Ala Ile Vai Asp Giy Asn Arg Aia Gly Ile Al a 525 Ala Thr 530 Aia Gly Lou Vai Gin 535 Aia Tyr Lou Pro Lys Ser Met Met Ile Gly Gly Gly Tyr Arq Gly Giu Aia 555 Gly Tyr Ala le Gly 560 Tyr Ser Ser Ile Ser 565 Asp Gly Giy Asn T rp 570 Ile Ile Lys Gly Thr Ala 575 Sor Gly Asn Ser 580 Arg Gly His Phe Gly 585 Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 28 <211> 594 <212> PRT <213> Noisseria meningitidis (W0006674i-0008) <400> 28 -49- Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Pro Val Leu Phe His Ala Asp Ser 75 Glu Gly Thr Gly Glu Lys Glu Val Thr Glu Asp Ser Asn Trp Val Tyr Phe Asp Lys Lys Gly Val Leu Lys Ile Lys 115 Ala Gly Thr Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asp Asn Thr Asn Ala Ser 125 Tyr Ser 130 Leu Lys Lys Asp Leu 135 Thr Asp Leu Thr Ser 140 Val Glu Thr Glu Leu Ser Phe Gly Asn Gly Lys Lys Asn Ile Thr Ser Asp 160 Thr Lys Gly Leu Asn 165 Phe Ala Lys Glu Ala Gly Thr Asn Gly Asp 175 Thr Thr Val His 180 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Leu Asn Thr 195 Gly Ala Thr Thr Asn Val Thr Asn Asp 200 Asp Glu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val 220 Leu Asn Ala Gly Asn Ile Lys Gly Val Lys Pro Gly Thr 230 Ala Ser Asp Asn Val 240 Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Giu Val Lys 275 Val Asn Val Giu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Giy Ala Lys Ser Val Ile Lys Giu 285 Lys Leu 290 Val Thr Gly Lys Asp 295 Lys Giy Giu Asn Ser Ser Thr Asp Giu 305 Gly Glu Gly Leu Val1 310 Thr Ala Lys Giu Val1 315 Ile Asp Ala Val Asn 320 Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Ala Ser Gly 355 Lys 340 Phe Giu Thr Val Thr 345 Ser Gly Thr Lys Val Thr Phe 350 Asp Gin Gly Asn Gly Thr Thr Ala 360 Thr Vai Ser Lys Asn Ile 370 Thr Val Lys Tyr Asp 375 Vai Asn Val Gly Asp 380 Ala Leu Asn Val Gin Leu Gin Asn Ser 390 Gly Trp Asn Leu Ser Lys Ala Val Gly Ser Ser Gly Val Ile Ser Gly Asn 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Giu 420 Thr Val Asn Ile Ala Gly Asn Asn Ile Glu Ile 430 Thr Pro Gin Gly Lys Asn Ile Asp 440 Ile Ala Thr Ser Phe Ser 450 Ser Val Ser Leu Ala Gly Ala Asp Ala 460 Pro Thr Leu Ser Asp Asp Giu Gly Ala 470 Leu Asn Val Gly Lys Asp Ala Asn Pro Val Arg Ile Asn Val Ala Pro Gly 490 Val Lys Giu Gly Asp Vai 495 Thr Asn Val Ala Gin Leu Lys Gly Val Ala Gin Asn Leu Asn Asn His1 -51 OO oO e, a, Cu Cqj Ile Asp Asn 515 Val Asp Gly Asn Ala Arg Ala Gly Ile 520 Gin Ala Ile Ala Thr 530 Ala Gly Leu Val Gin 535 Ala Tyr Leu Pro Gly 540 Lys Ser Met Met Ile Gly Gly Gly Thr 550 Tyr Arg Gly Glu Gly Tyr Ala Ile Tyr Ser Ser Ile Asp Gly Gly Asn Trp 570 Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Ser 580 Arg Gly His Phe Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 29 <211> 594 <212> PRT <213> Neisseria meningitidis (WO0066741-0009) <400> 29 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Val Val Leu Phe Arg Ser Asp Lys 75 Glu Gly Thr Gly Glu Lys Glu Gly Thr Glu Asp Ser Asn Trp Val Tyr Phe Asp Glu Lys Arg Val Leu Lys 100 Ala Gly Ala Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 Lys Ile Lys Gin Asn Thr Asn Glu Asn Thr Asn Asp Ser Ser Phe Thr 52 115 125 00 Tyr Ser 130 Leu Lys Lys Asp Leu 135 Thr Asp Leu Thr Ser 140 Val Glu Thr Glu Lys 145 Leu Ser Phe Gly Asn Gly Asn Lys Val Asn Ile Thr Ser 155 Thr Lys Gly Leu Asn 165 Phe Ala Lys Giu Ala Gly Thr Asn Gly Asp 175 Pro Thr Val Leu Asn Thr 195 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Val Thr Asn Asp Asn 205 Asp Glu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Leu Asn Ala Gly T rp 225 Asn Ile Lys Gly Lys Pro Gly Thr Thr 235 Ala Ser Asp Asn Val 240 Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Giu Val Lys 275 Val Asn Val Giu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Gly Ala Lys Ser Val Ile Lys Glu 285 Lys Leu 290 Val Thr Gly Lys Gi y 295 Lys Asp Glu Asn Gly Ser Ser Thr 300 Ile Asp Ala Val Asp Glu 305 Gly Glu Gly Leu Val1 310 Thr Ala Lys Glu Val1 315 As n 320 Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Phe Glu Thr Val Thr 345 Ser Gly Thr Asn Val Thr Phe 350 Asp Gin Gly Ala Ser Gly 355 Lys Gly Thr Thr Thr Val Ser Lys Asp 365 -53- 0O e, a, Cu U9 Asn Ile 370 Thr Val Lys Tyr Asp 375 Val Asn Val Gly Ala Leu Asn Val Asn 385 Gin Leu Gin Asn Gly Trp Asn Leu Asp 395 Ser Lys Ala Val Gly Ser Ser Gly Lys 405 Val Ile Ser Gly Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Thr Val Asn Ile Asn 425 Ala Gly Asn Asn Ile Glu Ile 430 Ala Pro Gin Gly Lys Asn Ile Ile Ala Thr Ser Met 445 Phe Ser 450 Ser Val Ser Leu Gly 455 Ala Gly Ala Asp Ala Pro Thr Leu Ser 460 Lys Asp Thr Asn Lys Val 465 Asp Asp Glu Gly Leu Asn Val Gly Ser 475 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Val Lys Glu Gly Asp Val 495 Thr Asn Val Ile Asp Asn 515 Gin Leu Lys Gly Val 505 Ala Gin Asn Leu Asn Asn Arg 510 Gin Ala Ile Val Asp Gly Asn Arg Ala Gly Ile Ala 525 Ala Thr 530 Ala Gly Leu Val Gin 535 Ala Tyr Leu Pro Lys Ser Met Met Ala 545 Ile Gly Gly Asp Tyr Arg Gly Glu Ala 555 Gly Tyr Ala Ile Gly 560 Tyr Ser Ser Ile Ser 565 Asp Gly Gly Asn Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Arg Gly His Phe Gly 585 Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> <211> <212> 594 PRT 54 00 <213> Neisseria meningitidis (W00066741-00i0) <400> Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Ala Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Val Val Leu Phe Arg Ser Asp Lys Glu Gly Thr Gly Lys Giu Gly Thr Giu Asp Ser Asn Trp Val Tyr Phe Asp Giu Lys Arg Val Leu Lys Ile Lys 115 Ala Gly Ala Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asn Asn Thr Asn Asp Se r 125 Tyr Ser 130 Leu Lys Lys Asp Leu 135 Thr Asp Leu Thr Val Giu Thr Giu Lys 145 Leu Ser Phe Gly Asn Gly Asn Lys Val 155 Asn Ile Thr Ser Thr Lys Gly Leu Asn 165 Phe Ala Lys Giu Ala Gly Thr Asn Gly Asp 175 Pro Thr Val Leu Asn Thr 195 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Val Thr Asn Asp Asn 205 Asp Giu 210 Trp Asn 225 Lys Lys Arg Ala Ile Lys Gly Val 230 Al a 215 Lys Ser Val Lys Asp Pro Gly Thr Thr 235 Val Leu Asn Ala Gly 220 Ala Ser Asp Asn Val 240 Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Glu Val Lys 275 Val Asn Val Glu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Gly Ala Lys Ser Val Ile Lys Glu 285 Lys Leu 290 Val Thr Gly Lys Gly 295 Lys Asp Glu Asn Ser Ser Thr Asp Glu 305 Gly Glu Gly Leu Val 310 Thr Ala Lys Glu Val 315 Ile Asp Ala Val Asn 320 Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Thr 330 Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Ala Ser Gly 355 Lys 340 Phe Glu Thr Val Ser Gly Thr Asn Val Thr Phe 350 Asp Gin Gly Lys Gly Thr Thr Ala 360 Thr Val Ser Lys Asn Ile 370 Thr Val Lys Tyr Val Asn Val Gly Asp 380 Ala Leu Asn Val Asn 385 Gin Leu Gin Asn Ser 390 Gly Trp Asn Leu Ser Lys Ala Val Gly Ser Ser Gly Val Ile Ser Gly Asn 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Glu 420 Thr Val Asn Ile Asn 425 Ala Gly Asn Asn Ile Glu Ile 430 Gly Lys Asn Ile Ile Ala Thr Ser Met Ala Pro Gin 445 Phe Ser 450 Ser Val Ser Leu Gly 455 Ala Gly Ala Asp Pro Thr Leu Ser Val 465 Asp Asp Glu Gly Leu Asn Val Gly Ser 475 Lys Asp Thr Asn Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly 490 Val Lys Glu Gly Asp Val 495 56 Thr Asn Val Ala 500 Gin Leu Lys Giy Ala Gin Asn Leu Asn Asn Arg 510 Ile Asp Asn 515 Val Asp Gly Asn Ala 520 Arg Ala Gly Ile Ala Gin Ala Ile 525 Ala Thr 530 Ala Gly Leu Val Gin 535 Ala Tyr Leu Pro Giy 540 Lys Ser Met Met Ala 545 Ile Gly Gly Asp Thr 550 Tyr Arg Giy Glu Ala 555 Gly Tyr Ala Ile Tyr Ser Ser Ile Ser 565 Asp Gly Gly Asn Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Arg Gly His Phe Gi y 585 Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 31 <211> 598 <212> PRT <213> Neisseria meninqitidis (W00066741-00ii) <400> 31 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Val Val Thr Vai Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Val Val Leu Phe Arg Ser Asp Lys Glu Gly Thr Gly Lys Glu Gly Thr Glu Asp Ser Asn Trp Val Tyr Phe Asp Giu Lys Arg Val Leu Ala Gly Ala Ile Thr 105 Leu Lys Ala Gly Asp Asn Leu 110 57 Lys Ile Lys 115 Gin Asn Thr Asn Asn Thr Asn Glu Asn 125 Thr Asn Asp Ser Ser 130 Phe Thr Tyr Ser Leu 135 Lys Lys Asp Leu Asp Leu Thr Ser Val1 145 Glu Thr Giu Lys Ser Phe Gly Ala Asn 155 Gly Asn Lys Val Ile Thr Ser Asp Thr 165 Lys Gly Leu Asn Ala Lys Giu Thr Ala Gly 175 Thr Asn Gly Thr Asp Thr 195 Pro Thr Val His Leu 185 Asn Gly Ile Gly Ser Thr Leu 190 Thr Asn Asp Leu Leu Asn Thr Ala Thr Thr Asn Val1 205 Asn Val 210 Thr Asp Asp Glu Lys 215 Lys Arg Ala Ala Val Lys Asp Val Leu 225 Asn Ala Gly Trp Ile Lys Gly Val Lys 235 Pro Gly Thr Thr Ser Asp Asn Val Asp 245 Phe Val Arg Thr Asp Thr Val Glu Phe Leu 255 Ser Ala Asp Gly Lys Lys 275 Thr 260 Lys Thr Thr Thr Val 265 Asn Val Giu Ser Lys Asp Asn 270 Val Ile Lys Thr Glu Val Lys Gly Ala Lys Thr Ser 285 Glu Lys 290 Asp Gly Lys Leu Val 295 Thr Gly Lys Giy Asp Glu Asn Gly Ser 305 Ser Thr Asp Glu Gly 310 Giu Gly Leu Val Thr 315 Ala Lys Glu Val Asp Ala Vai Asn Lys 325 Ala Giy Trp Arg Lys Thr Thr Thr Ala Asn 335 Gly Gin Thr Gly 340 Gin Ala Asp Lys Phe 345 Glu Thr Val Thr Ser Gly Thr 350 Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 58 00 Asp Asp 370 Gin Gly Asn Ile Thr 375 Val Lys Tyr Asp Val 380 Asn Val Gly Asp Ala 385 Leu Asn Val Asn Leu Gin Asn Ser Gi y 395 Trp Asn Leu Asp Lys Ala Val Ala Gly 405 Ser Ser Gly Lys Val1 410 Ile Ser Gly Asn Val Ser 415 Pro Ser Lys Asn Ile Glu 435 Lys Met Asp Glu Thr 425 Val Asn Ile Asn Ala Gly Asn 430 Ala Thr Ser Ile Thr Arg Asn Lys Asn Ile Asp Ile 445 Met Thr 450 Pro Gin Phe Ser Ser 455 Val Ser Leu Gly Gly Ala Asp Ala Pro 465 Thr Leu Ser Val Asp Giu Gly Ala Leu 475 Asn Val Gly Ser Asp Ala Asn Lys Pro 485 Val Arg Ile Thr Val Ala Pro Gly Val Lys 495 Glu Gly Asp Leu Asn Asn 515 Thr Asn Val Ala Gin 505 Leu Lys Gly Val Ala Gin Asn 510 Ala Gly Ile Arg Ile Asp Asn Asp Gly Asn Ala Arg 525 Ala Gin 530 Ala Ile Ala Thr Ala 535 Gly Leu Ala Gin Tyr Leu Pro Gly Lys 545 Ser Met Met Ala Gly Gly Giy Thr Tyr 555 Arg Gly Giu Ala Tyr Ala Ile Gly Tyr 565 Ser Ser Ile Ser Thr Gly Asn Trp Val Ile 575 Lys Gly Thr Ser Val Gly 595 Ser Gly Asn Ser Arg 585 Gly His Phe Gly Ala Ser Ala 590 Tyr Gin Trp -59- <210> 32 <211> 599 <212> PRT <213> Neisseria meningitidis (W00066741-0012) <400> 32 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Lys Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Glu Asp Giu Glu Giu Glu Giu Pro Val Val Arg Ser Ala Leu Val Gin Phe Met Ile Asp Lys Glu Gly Asn Glu Asn Giu Ser Thr Gly Asn Ile Giy Ser Ile Tyr Tyr Asp Asn His Asn Thr Leu Lys Ile 115 His Gly Ala Thr Val 105 Thr Leu Lys Ala Gly Asp Asn 110 Asn Thr Asn Lys Gin Asn Thr Lys Asn Thr Asn Giu 125 Asp Ser 130 Ser Phe Thr Tyr Ser 135 Leu Lys Lys Asp Thr Asp Leu Thr Ser 145 Val Glu Thr Glu Leu Ser Phe Gly Ala 155 Asn Gly Asn Lys Val1 160 Asn Ile Thr Ser Asp 165 Thr Lys Gly Leu Phe Ala Lys Glu Thr Ala 175 Gly Thr Asn Leu Thr Asp 195 Gly 180 Asp Thr Thr Val His 185 Leu Asn Gly Ile Gly Ser Thr 190 Val Thr Asn Thr Leu Leu Asn Thr 200 Gly Ala Thr Thr Asn 205 Asp Asn 210 Vai Thr Asp Asp Lys 215 Lys Lys Arg Ala Ala 220 Ser Val Lys Asp Leu Asn Ala Gly 00 qj c/^ Trp 230 Asn Ile Lys Gly Lys Pro Gly Thr Thr 240 Ala Ser Asp Asn Val 245 Asp Phe Val His Thr 250 Tyr Asp Thr Val Glu Phe 255 Leu Ser Ala Asn Gly Lys 275 Asp 260 Thr Lys Thr Thr Thr 265 Val Asn Val Glu Ser Lys Asp 270 Ser Val Ile Arg Thr Glu Val Lys 280 Ile Gly Ala Lys Lys Glu 290 Lys Asp Gly Lys Leu 295 Val Thr Gly Lys Gly 300 Lys Gly Glu Asn Ser Ser Thr Asp Glu 310 Gly Glu Gly Leu Thr Ala Lys Glu Val 320 Ile Asp Ala Val Asn 325 Lys Ala Gly Trp Arg 330 Met Lys Thr Thr Thr Ala 335 Asn Gly Gin Thr Asn Val 355 Thr 340 Gly Gin Ala Asp Phe Glu Thr Val Thr Ser Gly 350 Thr Val Ser Thr Phe Ala Ser Gly 360 Lys Gly Thr Thr Lys Asp 370 Asp Gin Gly Asn Thr Val Lys Tyr Asp 380 Val Asn Val Gly Ala Leu Asn Val Asn 390 Gin Leu Gin Asn Gly Trp Asn Leu Asp 400 Ser Lys Ala Val Ala 405 Gly Ser Ser Gly Lys 410 Val Ile Ser Gly Asn Val 415 Ser Pro Ser Asn Asn Ile 435 Lys 420 Gly Lys Met Asp Glu 425 Thr Val Asn Ile Asn Ala Gly 430 Ile Ala Thr Glu Ile Thr Arg Asn 440 Gly Lys Asn Ile Ser Met 450 Thr Pro Gin Phe Ser 455 Ser Val Ser Leu Gly 460 Ala Gly Ala Asp Pro Thr Leu Ser Val Asp Asp Lys Gly Ala 470 475 Leu Asn Val Gly Ser 480 -61 Lys Asp Ala Asn Lys 485 Pro Vai Arg Ile Thr Asn Val Ala Pro 490 Gly Val 495 Lys Glu Gly Asn Leu Asn 515 Asp 500 Val Thr Asn Val Ala 505 Gin Leu Lys Gly Val Ala Gin 510 Arg Ala Gly Asn Arg Ile Asp Val Asp Gly Asn Ala 525 Ile Ala 530 Gin Ala Ile Ala Thr 535 Aia Gly Leu Val Ala Tyr Leu Pro Gly 545 Lys Ser Met Met Ile Gly Gly Giy Thr 555 Tyr Arg Gly Giu Ala 560 Gly Tyr Aia Ile Giy 565 Tyr Ser Ser Ile Asp Gly Gly Asn Trp Ile 575 Ile Lys Gly Ala Ser Val 595 Ala Ser Gly Asn Ser 585 Arg Gly His Phe Gly Ala Ser 590 Gly Tyr Gin Trp <210> 33 <211> 592 <212> PRT <213> Neisseria meningitidis (W0006674i-00i3) <400> 33 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Asn Ser Ala Leu Asn 10 Ala Trp Vai Ala Val Thr Val Lys Ser Giu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala 40 Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Giu Giu Giu Giu Giu Giu Ser Val Gin Arg Ser Val Val Gly Ser Ile Gin Ala Ser Giu Gly Ser Gly Leu Giu Thr Ile Ser Leu Ser Met Thr Asn 90 Asp Ser Lys Giu Phe Val 62 Asp Pro Tyr Lys Gin Asn 115 Ile 100 Val Val Thr Leu Lys 105 Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Glu Asn Thr 120 Asn Ala Ser Ser Leu Lys 130 Lys Asp Leu Thr Leu Ile Asn Val Glu Thr Glu Lys Leu 140 Ile Ser Asp Thr Lys Ser Phe Gly Ala Asn 145 Gly Leu Asn Phe Ala 165 Gly 150 Lys Lys Val Asn Lys Giu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Ser Ser Ala 195 Asn 180 Gly Ile Gly Ser Leu Thr Asp Thr Leu Ala Gly 190 His Tyr Thr Ser His Val Asp Ala 200 Gly Asn Gin Ser Arg Ala 210 Ala Ser Ile Lys Val Leu Asn Ala Gly 220 Trp Asn Ile Lys Gly 225 Vai Lys Thr Gly Ser 230 Thr Thr Gly Gin Ser 235 Glu Asn Vai Asp Val Arg Thr Tyr Thr Val Giu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Giu Ser Lys Asp 265 Asn Gly Lys Arg Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Glu Gly Glu 305 Gly Leu Val Thr Al a 310 Lys Giu Val Ile Ala Vai Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe 34035 Thr Val Thr Ser Thr Asn Val Thr Phe Ala Ser 350 63 Gly Lys Gly 355 Thr Thr Ala Thr Val1 360 Ser Lys Asp Asp Gly Asn Ile Thr Val 370 Met Tyr Asp Val As n 375 Val Gly Asp Ala Leu Asn Vai Asn Gin 380 Ala Vai Ala Gly Ser Gin Asn Ser Gly Trp 390 Asn Leu Asp Ser Ser Gly Lys Val Ile 405 Ser Gly Asn Vai Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Giy Lys 435 Val1 420 Asn Ile Asn Ala Giy 425 Asn Asn Ile Giu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Aia 440 Thr Ser Met Ala Ser Vai 450 Ser Leu Gly Ala Ala Asp Ala Pro Thr Leu Ser Val Asp 460 Ala Asn Lys Pro Val Asp 465 Giu Gly Ala Leu Asn 470 Val Gly Ser Lys Arg Ile Thr Asn Ala Pro Gly Val Lys 490 Giu Gly Asp Val Thr Asn 495 Val Ala Gin Asn Val Asp 515 Leu 500 Lys Gly Val Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Gly Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala Gly 530 Leu Val Gin Ala Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly Gly Thr Tyr Arg 550 Gly Giu Aia Gly Ala Ile Gly Tyr Ser 560 Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg His Phe Gly Ala Ser 585 Ala Ser Val Gly Tyr Gin Trp 590 <210> 34 64 00 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0014) <400> 3 4 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Lys Ser Glu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Al a Ala Asn Ala Thr Asp Giu Asp Glu Giu Glu Giu Glu Ser Val Gin Arg Leu Ser Val Val Gly Glu Thr Ile Ser Ile Gin Ala Ser Met Giu Gly Ser Gly Leu Ser Met Thr Asp Ser Lys Glu Phe Val Asp Pro Tyr Lys Gin Asn 115 Vai Vai Thr Leu Lys 105 Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Glu Asn Asn Ala Ser Ser Phe 125 Leu Lys 130 Lys Asp Leu Thr Gly 135 Leu Ile Asn Val Thr Giu Lys Leu Ser 145 Phe Gly Ala Asn Lys Lys Val Asn Ile 155 Ile Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Gi y 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Ser Ser Ala 195 Gly Ile Giy Ser Thr 185 Leu Thr Asp Thr Leu Ala Gly 190 His Tyr Thr Ser His Val Asp Gly Asn Gin Ser Thr 205 Arg Ala 210 Ala Ser le Lys Asp 215 Val Leu Asn Ala Gly 220 Trp Asn Ile Lys Gly Val Lys Thr Gly Ser Thr Thr Gly Gin Ser Glu Asn Vai Asp Phe 65 230 240 00 Val Arg Thr Tyr Asp 245 Thr Val Glu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Glu Ser Lys Asp 265 Asn Gly Lys Arg Thr Glu Vai 270 Gly Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Giu Lys Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Giu Gly Gly Leu Val Thr Lys Glu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Asn Gly Gin Thr Gly 330 Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Vai Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Vai 360 Ser Lys Asp Asp Thr Val 370 Met Tyr Asp Val As n 375 Val Giy Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Gly Asn Leu Asp Ser Ala Val Ala Gly Ser 400 Ser Gly Lys Val Ile 405 Ser Gly Asn Val Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Ala Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Thr Leu Ser Val Asp 460 Ala Asn Lys Pro Val 480 Asp Glu Gly Ala Leu 465 Asn Val Gly Ser Lys Asp 470 475 66 Arg Ile Thr Asn 00 Val1 485 Ala Pro Gly Val Giu Gly Asp Val Thr Asn 495 Val Ala Gin Asn Val Asp 515 Lys Giy Vai Ala Gin 505 Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Gly Asn Ala Arg Gly Ile Ala Gin Ala 525 Ala Gly 530 Leu Val Gin Ala Tyr 535 Leu Pro Gly Lys Met Met Ala Ile Giy 545 Gly Gly Thr Tyr Gly Giu Ala Gly Tyr 555 Aia Ile Gly Tyr Ser 560 Ser Ile Ser Asp Gly 565 Giy Asn Trp Ile Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg His Phe Giy Ala Ser 585 Ala Ser Val Gly Tyr Gin Trp 590 <210> <211> 592 <2i2> PRT <2i3> Neisseria meningitidis (W0006674i-O0iS) <400> Asn Lys Ile Tyr Ar Ile Ile Trp Asn 10 Ser Ala Leu Asn Aia Trp is Val Ala Val Ser Giu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Asn Ala Thr Asp Giu Giu Giu Giu Giu Leu Giu Ser Val Gin Ser Val Val Gly Ser Ile Gin Ala Ser Giu Gly Ser Gly Giu Leu Giu Thr Ile Ser Leu Ser Met Thr Asn 90 Asp Ser Lys Giu Phe Val Asp Pro Tyr Ile 100 Val Val Thr Leu Ala Gly Asp Asn Leu Lys Ile 110 67 Lys Gin Asn 115 Thr Asn Glu Asn Asn Ala Ser Ser Phe 125 Thr Tyr Ser Le-u Lys 130 Lys Asp Leu Thr Gi y 135 Leu Ile Asn Val Thr Glu Lys Leu Se r 145 Phe Giy Ala Asn Lys Lys Val Asn Ile 155 Ile Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Ser Ser Ala 195 Gly Ile Gly Ser Thr 185 Leu Thr Asp Thr Leu Ala Gly 190 His Tyr Thr Ser His Vai Asp Gly Asn Gin Ser Thr 205 Arg Ala 210 Ala Ser Ile Lys Asp 215 Val Leu Asn Ala Trp Asn Ile Lys Gly 225 Val Lys Thr Giy Thr Thr Gly Gin Se r 235 Glu Asn Vai Asp Phe 240 Val Arg Thr Tyr Asp 245 Thr Val Glu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Giy 275 Asn 260 Val Giu Ser Lys Asp 265 Asn Gly Lys Arg Thr Giu Val 270 Giy Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Glu Lys Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser 300 Thr Asp Glu Gly Gly Leu Val Thr Ala 310 Lys Giu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Glu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Lys Gly 355 Thr Thr Ala Thr Gly Lys Gly Val Ser Lys Asp AspGiGyAs le 355 36036 Gln Gly Asn Ile 365 68 Thr Val 370 Met Tyr Asp Val Val Gly Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Gly T rp 390 Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser Gly Lys Val Ser Gly Asn Val Se r 410 Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asn Gly Lys 435 Val 420 Asn Ile Asn Ala Asn Asn Ile Giu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Ala Ser Val 450 Ser Leu Gly Ala Ala Asp Ala Pro Thr 460 Leu Ser Vai Asp Asp 465 Giu Gly Ala Leu Asn 470 Val Giy Ser Lys Aia Asn Lys Pro Arg Ile Thr Asn Vai 485 Ala Pro Gly Vai Lys 490 Giu Gly Asp Val Thr Asn 495 Val Ala Gin Asn Val Asp 515 Leu 500 Lys Gly Val Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Giy Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala Gly 530 Leu Val Gin Ala Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly Gly Thr Tyr Arg 550 Gly Giu Ala Gly Ala Ile Gly Tyr Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg His Phe Giy Ala Ser 585 Ala Ser Val Gly Tyr Gin Trp 590 <210> 36 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0016) <400> 36 69 Met 1 Asn Lys Ile Arg Ile Ile Trp Asn 10 Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Lys Ser Glu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Asn Ala Thr Asp Giu Giu Glu Glu Giu Leu Giu Ser Val Gin Ser Val Val Gly Ser Ile Gin Ala Ser Giu Gly Ser Gly Leu Glu Thr Ile Leu Ser Met Thr Asn Asp Ser Lys Giu Phe Val Asp Pro Tyr Lys Gin Asn 115 Ile 100 Val Val Thr Leu Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Giu Asn Thr 120 Asn Ala Ser Ser Leu Lys 130 Lys Asp Leu Thr Leu Ile Asn Val Giu 140 Thr Giu Lys Leu Phe Gly Ala Asn Gly 150 Lys Lys Val Asn Ile Ser Asp Thr Gly Leu Asn Phe Lys Giu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Ser Ser Ala 195 Asn 180 Gly Ile Gly Ser Leu Thr Asp Thr Leu Ala Gly 190 His Tyr Thr Ser His Vai Asp Ala 200 Giy Asn Gin Ser Arg Ala 210 Ala Ser Ile Lys Val Leu Asn Ala Gly Trp Asn Ile 220 Giu Asn Vai Asp Lys Phe 240 Val Lys Thr Gly Ser 230 Thr Thr Gly Gin Val Arg Thr Tyr Asp 245 AThr Val Giu Phe Leu Ser Ala Asp Thr 245 250 Lys Thr 255 70 Thr Thr Val Lys Ile Gly 275 As n 260 Val Giu Ser Lys Asp 265 Asn Gly Lys Arg Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Val1 280 Ile Lys Glu Lys Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser Thr Asp Glu Gly 300 Giu 305 Gly Leu Val Thr Lys Giu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 Thr Val 370 Met Tyr Asp Val Asn 375 Vai Gly Asp Ala Asn Val Asn Gin Leu 385 Gin Asn Ser Giy Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser Gly Lys Val Ile 405 Ser Giy Asn Val Pro Ser Lys Giy Lys Met 415 Asp Glu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Ala Pro 445 Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Leu Ser Val Asp Asp 465 Giu Gly Ala Leu Val Gly Ser Lys Asp 475 Ala Asn Lys Pro Arg Ile Thr Asn Ala Pro Gly Val Giu Gly Asp Val Thr Asn 495 Val Ala Gin Leu Lys Gly Val Ala Gin Asn Leu Asn Asn Arg Ile Asp -71- 00 Asn Val Asp Gly Asn Ala Arg 515 Gly Ile Ala Gin Ala 525 Ile Ala Thr Ala Gly 530 Leu Val Gin Ala Tyr 535 Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly 545 Gly Gly Thr Tyr Arg 550 Gly Giu Ala Giy Tyr 555 Ala Ile Gly Tyr Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg Gly 580 His Phe Gly Ala Ser Ala Ser Val Gly 585 Tyr Gin Trp 590 <210> 37 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0017) <400> 37 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Lys Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Ala Thr Asp Giu Asp Giu Giu Giu Giu Giu Ser Val Gin Arg Leu Ser Val Val Gly Giu Thr Ile Ser Ile Gin Ala Ser Met Giu Gly Ser Gly Leu Ser Met Thr Asp Ser Lys Glu Phe Val Asp Pro Tyr Lys Gin Asn 115 Val Val Thr Leu Lys 105 Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Giu Asn Asn Ala Ser Ser Phe 125 Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Giu Thr Glu Lys Leu 72 00 Ser Phe Gly Ala Asn 145 Gly Leu Asn Phe Ala 165 Gly 150 Lys Lys Val Asn Ile Ser Asp Thr Lys Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 Asn 180 Gly Ile Gly Ser Leu Thr Asp Met Leu Leu Asn 190 Asp Asp Glu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Ala Ala Ser Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Asp Asn Val Asp Val Arg Thr Tyr Thr Val Glu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 Asn 260 Val Glu Ser Lys Asn Gly Lys Lys Thr Glu Val 270 Gly Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Glu Lys Val Thr 290 Gly Lys Gly Lys Giu Asn Gly Ser Ser 300 Thr Asp Glu Gly Glu 305 Gly Leu Val Thr Ala 310 Lys Glu Val Ile Ala Val Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gln Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Giu 340 Thr Val Thr Ser Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val 360 Ser Lys Asp Asp Thr Val 370 rVlMet Tyr Asp Val Asn Val Gly Asp AlaLe 370 37538 Leu 380 Asn Val Asn Gin 73 00 Leu 385 Gin Asn Ser Gly I rp 390 Asn Leu Asp Ser Ala Val Ala Gly Ser Gly Lys Val Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Val1 420 Asn Ile Asn Ala Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Ser Val 450 Ser Leu Gly Ala Ala Asp Ala Pro Thr 460 Leu Ser Val Asp Lys Gly Ala Leu Asn 470 Val Gly Ser Lys Ala Asn Lys Pro Arg Ile Thr Asn Ala Pro Gly Vai Lys 490 Glu Gly Asp Val Thr Asn 495 Val Ala Gin Asn Val Asp 515 Leu 500 Lys Giy Val Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Gly Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala Gly 530 Leu Val Gin Ala Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly Gly Thr Tyr Arg 550 Gly Glu Ala Gly Ala Ile Gly Tyr Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg Gly 580 His Phe Gly Ala Ala Ser Val Gly Tyr Gin Trp 590 <210> 38 <211> 589 <212> PRT <213> Neisseria meningitidis (W00066741-0018) <400> 38 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 74 00 Val Val Val Thr Val Ala Giu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Vai Leu Thr Leu Leu Ser Ala Ala Asn Ala Thr Asp Thr Asp Giu Asp Glu Giu Giu Ser Val Val Arg Ser Ala Leu Val Leu 70 Gin Phe Met Ile Asp Lys Giu Giy Asn Glu Ile Glu Ser Thr Gly Asp Ile Gly Ser Ile Tyr Tyr Asp Asp His Asn Thr Leu Lys Ile 115 His Gly Ala Thr Val 105 Thr Leu Lys Ala Gly Asp Asn 110 Leu Lys Lys Lys Gin Ser Gly Asp Phe Thr Tyr Ser 125 Giu Leu 130 Lys Asp Leu Thr Val Giu Thr Giu Lys Leu Ser Phe Giy 140 Thr Lys Giy Leu Asn Ala 145 Asn Giy Asn Lys Val 150 Asn Ile Thr Ser Phe Ala Lys Glu Ala Gly Thr Asn Gly 170 Asp Pro Thr Val His Leu 175 Asn Gly Ile Ser His Val 195 Gly 180 Ser Thr Leu Thr Thr Leu Ala Gly Ser Ser Ala 190 Arg Ala Ala Asp Ala Giy Asn Gin 200 Ser Thr His Tyr Thr 205 Ser Ile 210 Lys Asp Val Leu Ala Gly Trp Asn Ile 220 Lys Gly Val Lys Thr 225 Gly Ser Thr Thr Gly 230 Gin Ser Giu Asn Asp Phe Val Arg Tyr Asp Thr Val Phe Leu Ser Ala Asp 250 Thr Lys Thr Thr Thr Vai 255 Asn Val Giu Ser 260 Lys Asp Asn Gly Lys 265 Arg Thr Giu Val Lys Ile Gly 270 75 Ala Lys Thr 275 Ser Val Ile Lys Gin 280 Lys Asp Gly Lys Leu 285 Val Thr Gly Lys Gly 290 Lys Gly Gin Asn Gly 295 Ser Ser Thr Asp Glu 300 Gly Giu Gly Leu Thr Ala Lys Giu Ile Asp Ala Val Asn 315 Lys Ala Gly Trp Arg 320 Met Lys Thr Thr Thr 325 Ala Asn Gly Gin Thr 330 Giy Gin Ala Asp Lys Phe 335 Gin Thr Vai Thr Thr Ala 355 Ser Gly Thr Lys Thr Phe Ala Ser Gly Asn Gly 350 Thr Val Lys Thr Val Ser Lys Asp Gin Gly Asn Tyr Asp 370 Val Asn Val Gly Asp 375 Ala Len Asn Val Asn Gin Len Gin 380 Gly Ser Ser Giy Asn Ser 385 Gly Trp Asn Len Ser Lys Ala Val Ala 395 Lys 400 Val Ile Ser Gly Asn 405 Val Ser Pro Ser Gly Lys Met Asp Gin Thr 415 Val Asn Ile Asn Ile Asp 435 Ala Gly Asn Asn Ile 425 Gin Ile Thr Arg Asn Gly Lys 430 Ser Val Ser Ile Ala Thr Ser Thr Pro Gin Phe Ser 445 Len Gly 450 Ala Gly Ala Asp Ala 455 Pro Thr Len Ser Val Asp Asp Gin Gly 460 Pro Val Arg Ile Thr Ala 465 Len Asn Val Gly Lys Asp Ala Asn Lys 475 Asn Val Ala Pro Gly 485 Val Lys Gin Gly Val Thr Asn Val Ala Gin 495 Len Lys Gly Ala Gin Asn Len Asn 505 Asn Arg Ile Asp Asn Val Asp 510 Gly Asn Aia 515 Arg Ala Gly Ile Ala Gin Ala Ile AlaThAlGyLe 52052 Thr Ala Gly Leu 525 76 Ala Gin Ala 530 Tyr Leu Pro Gly 535 Lys Ser Met Met Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala 545 550 Gly Tyr Ala Ile Gly 555 Tyr Ser Ser Ile Ser 560 Asp Thr Gly Asn Trp 565 Vai Ile Lys Gly Ala Ser Gly Asn Ser Arg 575 Gly His Phe Thr Ser Aia Ser Vali 585 Giy Tyr Gin Trp <210> 39 <211> 589 <212> PRT <2i3> Neisseria meningitidis (WO006674i-0019) <400> 39 Asn Lys Ile Tyr Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Vai Vai Val Thr Val Ala Ser Giu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala Thr Leu Leu Ser Ala Asn Ala Thr Asp Thr Glu Asp Giu Glu Leu Giu Ser Val Val Ser Ala Leu Val Leu Gin Phe Met Ile Lys Giu Gly Asn Gly Giu Ile Glu Ser Gly Asp Ile Gly Ser Ile Tyr Tyr Asp Asp His Asn Thr Leu Lys Ile 115 Leu 100 His Gly Ala Thr Val1 105 Thr Leu Lys Ala Gly Asp Asn 110 Leu Lys Lys Lys Gin Ser Gly Lys 120 Asp Phe Thr Tyr Giu Leu 130 Lys Asp Leu Thr Ser 135 Val Giu Thr Giu Lys 140 Leu Ser Phe Gly Asn Gly Asn Lys Val Asn Ile Thr Ser AspThLy GlLeAs 150 15516 Thr Lys Gly Leu Asn 160 77 Phe Ala Lys Glu Thr 165 Ala Gly Thr Asn Gly 170 Asp Pro Thr Val His Leu 175 Asn Gly Ile Ser His Val 195 Gly 180 Ser Thr Leu Thr Asp 185 Thr Leu Ala Gly Ser Ser Ala 190 Arg Ala Ala Asp Ala Gly Asn Gin 200 Ser Thr His Tyr Ser Ile 210 Lys Asp Val Leu Ala Gly Trp Asn Ile 220 Lys Gly Vai Lys Gly Ser Thr Thr Gly 230 Gin Ser Giu Asn Asp Phe Val Arg Thr 240 Tyr Asp Thr Val Giu 245 Phe Leu Ser Ala Asp 250 Thr Lys Thr Thr Thr Val 255 Asn Val Giu Ala Lys Thr 275 Ser 260 Lys Asp Asn Giy Arg Thr Glu Val Lys Ile Gly 270 Val Thr Gly Ser Vai Ile Lys Giu 280 Lys Asp Gly Lys Lys Gly 290 Lys Gly Giu Asn Gly 295 Ser Ser Thr Asp Gly Giu Gly Leu Thr Ala Lys Glu Ile Asp Ala Val Asn 315 Lys Ala Gly Trp Arg 320 Met Lys Thr Thr Thr 325 Ala Asn Gly Gin Thr Gly Gin Ala Asp Lys Phe 330 335 Giu Thr Val Thr Thr Ala 355 Ser Gly Thr Lys Val 345 Thr Phe Ala Ser Gly Asn Gly 350 Thr Val Lys Thr Val Ser Lys Asp Gin Gly Asn Ile 365 Tyr Asp 370 Val Asn Val Gly Asp 375 Ala Leu Asn Val Gin Leu Gin Asn Ser 385 Gly Trp Asn Leu Ser Lys Ala Val Ala 395 Gly Ser Ser Gly Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Giu Thr -78- 00 Val Asn Ile Asn Ile Asp 435 Asn 420 Ala Gly Asn Asn Giu Ile Thr Arg Asn Gly Lys 430 Ser Val Ser Ile Ala Thr Ser Met 440 Thr Pro Gin Phe Leu Giy 450 Ala Gly Ala Asp Pro Thr Leu Ser Val Asp Asp Giu Gly 460 Pro Val Arg Ile Thr Leu Asn Val Gly Ser 470 Lys Asp Ala Asn Asn Val Ala Pro Val Lys Giu Gly Asp 490 Val Thr Asn Val Ala Gin 495 Leu Lys Gly Gly Asn Ala 515 Val 500 Ala Gin Asn Leu Asn Arg Ile Asp Asn Val Asp 510 Ala Gly Leu Arg Ala Gly Ile Ala 520 Gin Ala Ile Ala Ala Gin 530 Ala Tyr Leu Pro Gly 535 Lys Ser Met Met Ala 540 Ile Gly Gly Gly Tyr Arg Gly Giu Ala 550 Gly Tyr Ala Ile Gly 555 Tyr Ser Ser Ile Ser 560 Asp Thr Giy Asn T rp 565 Val Ile Lys Gly Thr 570 Ala Ser Gly Asn Ser Arg 575 Giy His Phe Thr Ser Ala Ser Val 585 Gly Tyr Gin Trp <210> <211> <212> 595 PRT <213> Neisseria meningitidis (WG006674i-0020) <400> Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 Val Val Val Ser Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 25 Thr Val Giu Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gin 79 00 Ala Asn Ala Thr Asp Thr Asp Giu Asp Asp Glu Giu Pro Val Val Arg Ser Ala Leu Val Gin Phe Met Ile Asp 75 Lys Glu Gly Asn Gly Giu Ile Giu Ser Thr Giy Asp Ile Giy Ser Ile Tyr Tyr Asp Asp His Asn Thr Leui Lys Ile 115 His Giy Ala Thr Val1 105 Thr Leu Lys Ala Gly Asp Asn 110 Ser Ser Phe Lys Gin Asn Thr Giu Asn Thr Asn Thr Tyr 130 Ser Leu Lys Lys Asp 135 Leu Thr Asp Leu Thr 140 Ser Val Gly Thr Giu 145 Giu Leu Ser Phe Ala Asn Gly Asn Lys 155 Val Asn Ile Thr Ser 160 Asp Thr Lys Gly Leu 165 Asn Phe Ala Lys Lys 170 Thr Ala Gly Thr Asn Gly 175 Asp Thr Thr Leu Ala Gly 195 His Leu Asn Gly Ile 185 Gly Ser Thr Leu Thr Asp Thr 190 Gin Ser Thr Ser Ser Ala Ser Val Asp Ala Gly Asn 205 His Tyr 210 Thr Arg Ala Ala Ser 215 Ile Lys Asp Val Asn Ala Gly Trp Asn 225 Ile Lys Gly Val Thr Gly Ser Thr Thr 235 Gly Gin Ser Giu Asn 240 Val Asp Phe Val Arg 245 Thr Tyr Asp Thr Glu Phe Leu Ser Ala Asp 255 Thr Lys Thr Thr Giu Val 275 Thr Val Asn Val Giu 265 Ser Lys Asp Asn Gly Lys Arg 270 Glu Lys Asp Lys Ile Gly Ala Lys 280 Thr Ser Val Ile Lys 285 Gly Lys 290 Leu Val Thr Gly Gly Lys Gly Glu Asn Gly Ser Ser Thr 300 Val Ile Asp Ala Val Asp 305 Glu Gly Glu Gly Leu 310 Val Thr Ala Lys Asn Lys Ala Gly Arg Met Lys Thr Thr 330 Thr Ala Asn Gly Gin Thr 335 Gly Gin Ala Phe Ala Ser 355 Asp 340 Lys Phe Glu Thr Thr Ser Gly Thr Asn Val Thr 350 Asp Asp Gin Gly Lys Gly Thr Thr 360 Ala Thr Val Ser Gly Asn 370 Ile Thr Val Lys Asp Val Asn Val Gly Asp Ala Leu 380 Asp Ser Lys Ala Asn Val 400 Val 385 Asn Gin Leu Gin Asn 390 Ser Gly Trp Asn Ala Gly Ser Ser Gly 405 Lys Val Ile Ser Gly 410 Asn Val Ser Pro Ser Lys 415 Gly Lys Met Ile Thr Arg 435 Glu Thr Val Asn Asn Ala Gly Asn Asn Ile Glu 430 Met Thr Pro Asn Gly Lys Asn Ile 440 Asp Ile Ala Thr Gin Phe 450 Ser Ser Val Ser Gly Ala Gly Ala Asp 460 Ala Pro Thr Leu Val Asp Asp Glu Ala Leu Asn Val Gly 475 Ser Lys Asp Ala Asn 480 Lys Pro Val Arg Ile 485 Thr Asn Val Ala Gly Val Lys Glu Gly Asp 495 Val Thr Asn His Ile Asp 515 Ala Gin Leu Lys Gly 505 Val Ala Gin Asn Leu Asn Asn 510 Ala Gin Ala Asn Val Asp Gly Ala Arg Ala Gly Ile 525 Ile Ala 530 Thr Ala Gly Leu Val Gin Ala Tyr Leu 535 Pro Gly Lys Ser Met 540 81 Met Ala Ile Gly Gly Gly 545 550 Thr Tyr Arg Gly Giu Ala Gly Tyr 555 Trp Ile Ile Lys Ala Gly Tyr Ser Ser Ser Asp Gly Gly Asn 570 Gly Thr 575 Ala Ser Gly Asn 580 Ser Arg Gly His Gly Ala Ser Ala Ser Val Gly 590 Tyr Gin Trp 595 <210> 41 <211> 600 <212> PRT <213> Neisseria meningitidis (W00066741-0021) <400> 41 Met 1 Asn Lys Ile Arq Ile Ile Trp Asn 10 Ile Ala Leu Asn Ala Trp Val Val Val Thr Val Ala Ser Giu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Vai Gin Thr Ala Val Leu Ala 40 Thr Leu Leu Ser Ala Ala Asn Ala Thr Asp Giu Asp Asn Giu Asp Giu Pro Val Val Arg Thr Ala Pro Val Leu 70 Ser Phe His Ser Asp Lys Glu Gly Thr Giu Lys Giu Glu Gly Ala Ser Ser As n Leu Thr Vai Tyr Phe Asp Lys Asn Arg Asn Leu Lys 115 Val1 100 Leu Lys Ala Gly Ile Thr Leu Lys Ala Gly Asp 110 Glu Asn Thr Ile Lys Gin Asn Thr 120 Asn Glu Asn Thr Asn Ala 130 Her Ser Phe Thr Ser Leu Lys Lys Asp 140 Leu Thr Gly Leu Asn Val Giu Thr Giu Lys Leu Ser Phe Gly 150 155 Ala Asn Giy Lys Lys 160 -82- Val Asn Ile Ile Ser 165 Asp Thr Lys Gly Asn Phe Ala Lys Glu Thr 175 Ala Gly Thr Thr Leu Thr 195 Gly Asp Pro Thr Val 185 His Leu Asn Gly Ile Gly Ser 190 Val Asp Ala Asp Thr Leu Ala Gly 200 Ser Ser Ala Ser His 205 Gly Asn 210 Gin Ser Thr His Tyr 215 Thr Arg Ala Ala Ser 220 Ile Lys Asp Val Leu 225 Gly Asn Ala Gly Trp Gin Ser Glu Asn 245 Asn 230 Ile Lys Gly Val Thr Gly Ser Thr Thr 240 Val Asp Phe Val Arg 250 Thr Tyr Asp Thr Val Glu 255 Phe Leu Ser Asp Asn Gly 275 Ala 260 Asp Thr Lys Thr Thr 265 Thr Val Asn Val Glu Ser Lys 270 Thr Ser Val Lys Arg Thr Glu Lys Ile Gly Ala Lys 285 Ile Lys 290 Glu Lys Asp Gly Lys 295 Leu Val Thr Gly Gly Lys Gly Glu Asn 305 Gly Ser Ser Thr Glu Gly Glu Gly Leu 315 Val Thr Ala Lys Glu 320 Val Ile Asp Ala Val 325 Asn Lys Ala Gly Arg Met Lys Thr Thr Thr 335 Ala Asn Gly Gly Thr Lys 355 Thr Gly Gin Ala Asp 345 Lys Phe Glu Thr Val Thr Ser 350 Ala Thr Val Val Thr Phe Ala Gly Asn Gly Thr Thr 365 Ser Lys 370 Asp Asp Gin Gly Asn 375 Ile Thr Val Lys Asp Val Asn Val Gly 385 Asp Ala Leu Asn Asn Gin Leu Gin Asn 395 Ser Gly Trp Asn Leu 400 Asp Ser Lys Ala Val Ala Gly Ser Ser Gly 405 410 Lys Val Ile Ser Gly Asn 415 83 Val Ser Pro Gly Asn Asn 435 Se r 420 Lys Gly Lys Met Giu Thr Val Asn Ile Asn Ala 430 Asp Ile Ala Ile Giu Ile Thr Arg 440 Asn Gly Lys Asn Thr Ser 450 Met Thr Pro Gin Ser Ser Val Ser Leu 460 Gly Ala Gly Ala Ala Pro Thr Leu Ser 470 Val Asp Asp Glu Ala Leu Asn Val Ser Lys Asp Ala Lys Pro Val Arg Ile 490 Thr Asn Val Ala Pro Gly 495 Val Lys Giu Gin Asn Leu 515 Gly 500 Asp Val Thr Asn Val1 505 Ala Gin Leu Lys Gly Val Ala 510 Ala Arg Ala Asn Asn Arg Ile Asp 520 Asn Vai Asp Gly Asn 525 Gly Ile 530 Ala Gin Ala Ile Ala 535 Thr Ala Gly Leu Gin Ala Tyr Leu Pro 545 Gly Lys Ser Met Met 550 Ala Ile Gly Gly Giy 555 Thr Tyr Arg Gly Ala Gly Tyr Ala Ile 565 Gly Tyr Ser Ser Ser Asp Giy Giy Asn Trp 575 Ile Ile Lys Ser Ala Ser 595 Thr Ala Ser Gly Asn 585 Ser Arg Gly His Phe Gly Ala 590 Vai Gly Tyr Gin <210> <211> <212> 42 594 PRT <213> Neisseria meningitidis (W09931132-0002) <400> 42 Met Aso Lys Ile 1 Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 5 10 Val Val Val Ser Giu Leu Thr Arg Asn His Thr Lys Arg Aia Ser Ala 84 00 Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Thr Val Gin Ala Ser Ala Asn Asn Glu Pro Arg Lys Lys Asp Leu Tyr Leu Asp Val Gin Arg Thr Val1Ala Val Leu Ile Asn Ser Asp Lys Gly Thr Gly Glu Glu Lys Val Glu Giu Asn Ser Asp Trp Ala Val Tyr Phe Asn Ala Gly Asp 115 Giu 100 Lys Gly Val Leu Ala Arg Giu Ile Thr Leu Lys 110 Phe Thr Tyr Asn Leu Lys Ile Lys 120 Gin Asn Gly Thr Ser Leu 130 Lys Lys Asp Leu Asp Leu Thr Ser Val Gly Thr Glu 140 Ile Thr Ser Asp Lys Ser Phe Ser Ala As n 150 Gly Asn Lys Val Asn 155 Thr 160 Lys Giy Leu Asn Phe 165 Ala Lys Glu Thr Ala 170 Gly Thr Asn Gly Asp Thr 175 Thr Val His Asn Thr Giy 195 Asn Gly Ile Giy Ser 185 Thr Leu Thr Asp Thr Leu Leu 190 Thr Asp Asp Ala Thr Thr Asn Thr Asn Asp Asn Val1 205 Giu Lys 210 Lys Arg Ala Aia Ser 215 Vai Lys Asp Val Asn Ala Gly Trp As n 225 Ile Lys Gly Val Pro Gly Thr Thr Ala 235 Ser Asp Asn Val Phe Vai Arg Thr Tyr 245 Asp Thr Val Giu Leu Ser Ala Asp Thr Lys 255 Thr Thr Thr Val1 260 Asn Val Giu Ser Lys 265 sAsp Asn Gly Lys Lys Thr Giu 265 270 oo e, a, 00 Cu U9 Val Lys Ile 275 Gly Val Lys Thr Ser 280 Val lie Lys Glu Lys 285 Asp Gly Lys Leu Val 290 Thr Gly Lys Asp Lys 295 Gly Glu Asn Gly Ser 300 Ser Thr Asp Glu Gly 305 Glu Gly Leu Val Ala Lys Glu Val Ile 315 Asp Ala Val Asn Ala Gly Trp Arg Met Lys Thr Thr Thr 325 Asn Gly Gin Thr Gly Gin 335 Ala Asp Lys Ser Gly Lys 355 Phe 340 Glu Thr Val Thr Ser 345 Gly Thr Asn Val Thr Phe Ala 350 Gin Gly Asn Gly Thr Thr Ala Val Ser Lys Asp Asp 365 Ile Thr 370 Val Met Tyr Asp Val 375 Asn Val Gly Asp Leu Asn Val Asn Gin 385 Ala Leu Gly Leu Gin Gly Ser Ser Gly 405 Ser Gly Trp Asn Leu 395 Asp Ser Lys Ala Lys Val Ile Ser Gly 410 Asn Val Ser Pro Ser Lys 415 Gly Lys Met Ile Thr Arg 435 Asp 420 Glu Thr Val Asn Ile 425 Asn Ala Gly Asn Asn Ile Glu 430 Met Thr Pro Asn Gly Lys Asn Ile 440 Asp Ile Ala Thr Gin Phe 450 Ser Ser Val Ser Leu 455 Gly Ala Gly Ala Asp 460 Ala Pro Thr Leu Ser 465 Val Asp Gly Asp Ala 470 Leu Asn Val Gly Lys Lys Asp Asn Pro Val Arg Ile Asn Val Ala Pro Gly 490 Val Lys Glu Gly Asp Val 495 Thr Asn Val Ala 500 Gin Leu Lys Gly Ala Gin Asn Leu Asn Asn Arg 510 Gin Ala Ile Ile Asp Asn 515 Val Asp Gly Asn Ala 520 Arg Ala Gly Ile -86- Ala Thr 530 Ala Gly Leu Val Gin Ala Tyr Leu Pro 535 Lys Ser Met Met Ala 545 Ile Gly Gly Gly Tyr Arg Gly Glu Ala 555 Gly Tyr Ala Ile Tyr Ser Ser Ile Ser 565 Asp Gly Gly Asn Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Arg Gly His Phe Gly 585 Ala Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 43 <211> 594 <212> PRT <213> Neisseria meningitidis (W09958683-0002) <400> 43 Met 1 Asn Lys Ile Tyr 5 Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Ala Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Val Val Leu Phe Arg Ser Asp Lys Giu Gly Thr Giy Lys Giu Val Thr Giu Asp Ser Asn Trp Val Tyr Phe Asp Lys Lys Gly Val Leu Lys Ile Lys 115 Ala Gly Thr Ile Thr 105 Leu Lys Ala Giy Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asn Asn Thr Asn Ala Ser 125 Tyr Ser 130 Leu Lys Lys Asp Leu Thr Asp Leu Thr 135 Ser Val Gly Thr Glu 140 87 Lys 145 Leu Ser Phe Ser Ala 150 Asn Ser Asn Lys Val 155 Asn Ile Thr Ser Thr Lys Gly Leu Phe Ala Lys Lys Thr 170 Ala Glu Thr Asn Gly Asp 175 Thr Thr Val Leu Asn Thr 195 His 180 Leu Asn Gly Ile Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Asn 200 Val Thr Asn Asp Asp Giu 210 Lys Lys Arg Ala Ser Val Lys Asp Val1 220 Leu Asn Ala Gly Asn Ile Lys Gly Val1 230 Lys Pro Gly Thr Ala Ser Asp Asn Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Glu 250 Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Glu Val Lys 275 Thr 260 Val Asn Val Glu Lys Asp Asn Gly Lys Arg Thr 270 Lys Asp Gly Ile Gly Ala Lys Thr 280 Ser Val Ile Lys Lys Leu 290 Val Thr Giy Lys Lys Gly Giu Asn Asp 300 Ser Ser Thr Asp Gly Giu Giy Leu Thr Ala Lys Giu Ile Asp Ala Val Asn 320 Lys Ala Gly Trp Arg 325 Met Lys Thr Thr Ala Asn Gly Gin Thr Giy 335 Gin Ala Asp Ala Ser Gly 355 Phe Giu Thr Val Thr 345 Ser Gly Thr Asn Val Thr Phe 350 Asp Gin Gly Lys Gly Thr Thr Thr Val Ser Lys Asp 365 Asn Ile 370 Thr Val Met Tyr Asp 375 Val Asn Val Gly Ala Leu Asn Val Asn 385 Gin Leu Gin Asn snSer Gly Trp Asn Leu Asp 85390 395 Ser Lys Ala Val 88 Gly Ser Ser Gly Val Ile Ser Gly Asn 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Giu 420 Thr Val Asn Ile Asn 425 Ala Gly Asn Asn Ile Giu Ile 430 Thr Pro Gin Giy Lys Asn Ile Ile Ala Thr Ser Met 445 Phe Ser 450 Ser Val Ser Leu Gly 455 Ala Gly Ala Asp Ala Pro Thr Leu Ser 460 Lys Asp Ala Asn Lys Val1 465 Asp Asp Giu Gly Leu Asn Vai Gly Se r 475 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly 490 Val Lys Glu Gly Asp Val 495 Thr Asn Val Ile Asp Asn 515 Gin Leu Lys Gly Val 505 Ala Gin Asn Leu Asn Asn His 510 Gin Ala Ile Val Asp Gly Asn Arg Ala Gly Ile Al a 525 Ala Thr 530 Ala Giy Leu Vai Gin 535 Aia Tyr Leu Pro Lys Ser Met Met Ala 545 Ile Gly Gly Gly Tyr Arg Gly Giu Ala 555 Gly Tyr Ala Ile Gly 560 Tyr Ser Ser Ile Ser 565 Asp Giy Gly Asn T rp 570 Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Arg Gly His Phe Gly 585 Ala Ser Ala Ser Vai Giy Tyr 590 Gin Trp <210> 44 <211> 591 <212> PRT <213> Neisseria meningitidis (W09958683-0004) <400> 44 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 -89- Val Ala Val Thr Val Lys Ser Glu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Ser Ala Asn Asn Glu Gin Giu Giu Asp Leu Tyr Leu Asp Pro Val1Gin Arg Thr Val Ala Val Leu Ile Val Ser Asp Lys Giu Thr Gly Giu Lys Lys Val Glu Giu Asn Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Asp Asn 115 Lys 100 Gly Val Leu Thr Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Leu Lys Ile Lys Gin 120 Asn Gly Thr Asn Phe 125 Leu Lys 130 Lys Asp Leu Thr Leu Thr Ser Val Giy 140 Thr Giu Lys Leu Phe Ser Ala Asn Giy 150 Asn Lys Val Asn Thr Ser Asp Thr Gly Leu Asn Phe Lys Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Thr Gly Ala 195 As n 180 Gly Ile Gly Ser Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Giu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Aia Ala Ser Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Asp Asn Val Asp Val Arg Thr Tyr Thr Val Giu Phe Leu 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Giu Val 90 00 Lys Ile Gly 275 Ala Lys Thr Ser Ile Lys Giu Lys Asp 285 Gly Lys Leu Val Thr 290 Gly Lys Asp Lys Gly 295 Glu Asn Gly Ser Thr Asp Giu Gly Giu 305 Gly Leu Val Thr Lys Giu Val Ile Asp 315 Ala Val Asn Lys Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Thr Ser Gi y 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 Thr Vai 370 Met Tyr Asp Vai As n 375 Val Gly Asp Ala Asn Val Asn Gin Leu 385 Gin Aso Ser Gly Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser 400 Ser Gly Lys Val Ile 405 Ser Gly Asn Vai Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asn Giy Lys 435 As Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Ser Val 450 Ser Leu Giy Ala Gly 455 Ala Asp Ala Pro Leu Ser Val Asp Gly 465 Asp Ala Leu Asn Gly Ser Lys Lys Asp 475 Aso Lys Pro Val Arg 480 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Gly Asp Val Thr Asn Val 495 Ala Gin Leu Lys 500 Gly Val Ala Gin Asn 505 Leu Aso Asn Arg Ile Asp Asn -91- Val Asp Gly 515 Asn Ala Arg Ala Ile Ala Gin Ala Ile 525 Ala Thr Ala Gly Leu 530 Val Gin Aia Tyr Leu 535 Pro Gly Lys Ser Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Giu Ala Gly Tyr Ala 555 Ile Giy Tyr Ser Ser 560 Gly Asn 575 Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile Gly Thr Ala Ser Ser Arq Gly Phe Gly Ala Ser Ala 585 Ser Vai Gly Tyr Gin Trp 590
AU2006200732A 2000-01-17 2006-02-22 Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins Ceased AU2006200732B2 (en)

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AU28754/01A AU784518B2 (en) 2000-01-17 2001-01-17 Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins
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