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

专利摘要:

公开号:AU2009200692A1
申请号:U2009200692
申请日:2009-02-19
公开日:2009-03-19
发明作者:Marzia Giuliani；Mariagrazia Pizza；Rino Rappuoli
申请人:Novartis Vaccines and Diagnostics SRL；Novartis Vaccines and Diagnostics Inc；
IPC主号:A61K39-095

专利说明:
AUSTRALIA
Patents Act 1990 FB RICE CO Patent and Trade Mark Attorneys NOVARTIS VACCINES AND DIAGNOSTICS 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:- OUTER MEMBRANE VESICLE (OMV) VACCINE COMPRISING N.MENINGITIDIS SEROGROUP B OUTER MEMBRANE PROTEINS RELATED APPLICATIONS This application is a divisional application under S.79B of the Patents Act 1990 of Australian Patent Application No. 2006200732 filed February 22, 2006, which is a divisional application of Australian Patent No. 784518 filed January 17, 2001, which corresponds to International Application No. PCT/IB01/00166 in the Australian national phase, and claims priority from United Kingdom Patent Application No. 0001067.8 filed January 17, 2000 and United Kingdom Patent Application No. 0005699.4 filed March 9, 2000. The contents of each of the foregoing applications is hereby incorporated in its entirety by way of reference into this divisional application.
TECHNICAL FIELD This invention relates to vaccines against Neisseria meningitidis, serogroup B (NmB).
BACKGROUND ART 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 JMed 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 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.
I Serogroup B remains a problem, however, and it is currently responsible for Sapproximately 50% 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 Sresponse were elicited, it would be anti-self, and therefore undesirable. In order to 0 avoid induction of autoimmunity and to induce a protective immune response, the C capsular polysaccharide has, for instance, been chemically modified substituting the N- Sacetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered C 10 (Romero Outschoorn (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 g. Bjune et al.
(1991) Lancet 338 (8775):1093-96]. Whilst this vaccine is safe and prevents NmB disease, its efficacy is 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.
DISCLOSURE OF THE INVENTION Surprisingly, it has been found that the addition of further defined components to OMV vaccines significantly broadens their efficacy.
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 WO99/57280, or an immunogenic fragment thereof; a protein disclosed in W099/36544, or an immunogenic fragment thereof; a protein disclosed in WO99/24578, 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, 10, 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, 92, 94, 96, 98, 100, 102, 128, 130, 132, 134, 136, 162, 164, 166, 168, 170, 196, 198, 200, 202, 204, 230, 232, 234, 236, 238, 264, 266, 268, 270, 272, 298, 300, 302, 304,306, 332, 334, 336,338, 340, 366, 368,370,372, 374, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 342,344, 346, 348, 350, 352, 354, 356,358, 360, 376,378, 380, 382, 384,386, 388, 390, 392, 394, 124, 126, 158, 160, 192, 194, 226, 228, 260, 262, 294, 296, 328,330, 362,364, 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, 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 1O protein comprising a sequence having sequence identity (preferably greater than eg. 60%, 70%, 80%, 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, 10, 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 as disclosed in W099/36544 (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).
If the composition comprises a protein disclosed in Tettelin et al. e. a protein encoded by one of the genes disclosed therein), said protein preferably comprises an amino acid sequence selected from the group consisting of NMB0001 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 eg. 60%, 70%, 80%, 90%, 95%, 99% or more) to one of these 2160 genes).
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 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 (preferably greater than 50% eg. 60%, 70%, 80%, 90%, 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, 10, 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,
O
92, 94, 96, 98, 100, 102, 104, 128, 130, 132, 134, 136, 138, 162, 164, 166, 168, 170, 172, 196, 198, 200, 202, 204, 206, 230, 232, 234, 236, 238, 240, 264, 266, 268, 270, 272, 274, 298, 300, 302, 304, 306, 308, 332,334, 336, 338,340, 342, 366, 368, 370, 372, 374, 376, 400, 402, 404, 406, 408, 410, 434, 436, 438, 440, 442, 444, 468, 470, 472, 474, 476, 478, 502, 504, 506, 508, 510, 512, 536, 538, 540, 542, 544, 546, 570, 572, 574, 576, 578, 580, 604, 606, 608, 610, 612, 614, 638, 640, 642, 644, 646, 648, 672, 674, 676, 678, 680, 682, 706, 708, 710, 712, 714, 716, 740, 742, 744, 746, 748, 750, 774, 776, 778, 780, 782, 784, 808, 810, 812, 814, 816, 818, 842, 844, 846, 848, 850, 852, 876, 878, 880, 882, 884, 886, 910, 912, 914, 916, 918, 920, 944, 946, 948, 950, 952, 954, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 514, 516, 518, 520, 522, 524, 548, 550, 552, 554, 556,558, 582, 584, 586, 588, 590, 592, 616, 618, 620, 622, 624, 626, 650, 652, 654, 656, 658, 660, 684, 686, 688, 690, 692, 694, 718, 720, 722, 724, 726, 728, 752, 754, 756, 758, 760, 762, 786, 788, 790, 792, 794,796, 820, 822, 824, 826, 828, 830, 854, 856, 858, 860, 862, 864, 888, 890, 892, 894, 896, 898, 922, 924, 926, 928, 930, 932, 956, 958, 960, 962, 964, 966, 526, 528, 530, 532, 534, 560, 562, 564, 566, 568, 594, 596, 598, 600, 602, 628, 630, 632, 634, 636, 662, 664, 666, 668, 670, 696, 698, 700, 702, 704, 730, 732, 734, 736, 738, 764, 766, 768, 770, 772, 798, 800, 802, 804, 806, 832, 834, 836, 838, 840, 866, 868, 870, 872, 874, 900, 902, 904, 906, 908, 934, 936, 938, 940, 942, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1 1038, 1 1066,1 1094,1 1122, 1 1150, 1178,: 1206, 1234, 1262, 1290, 1318, 1346, 1374, 1402, 1430, 1458, 1486, 1514, 1542, 1570, 1598, 1626, 1654, 1682, 1710, 1738, 1766, 1794, 1822, 012, [040, [068, 096, 1124, 1152, 1180, 1208, 1236, 1264, 1292, 1320, 1348, 1376, 1404, 1432, 1460, 1488, 1516, 1544, 1572, 1600, 1628, 1656, 1684, 1712, 1740, 1768, 1796, 1824, 1014, 1042, 1070, 1098, 1126, 11 54, 1182, 1210, 1238, 1266, 1294, 1322, 1350 1378, 1406, 1434, 1462, 1490, 1518, 1546, 1574, 1602, 1630, 1658, 1686, 1714 1742 1770 1798 1826 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034,1 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1100, 1102,1104,1106, 1108,1110,1112,1114,1116, 1118, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1156, 1158,1160,1162, 1164,1166,1168,1170,1172, 1174, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1352, 1354, 1356, 1358, 1360, 1362, 1364,1366,1368,1370, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, .1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 91688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, ,1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 91744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 91772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 11800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 91828, 1830, 1832,91834, 1836, 1838, 1840, 1842, 1844, 1846, 036, 1064, 1092, 1120, 1148, 1176, 1204, 1232, 1260, 1288, 1316, 1344, 1372, 1400, 1428, 1456, 1484, 1512, 1540, 1568, 1596, 1624, 1652, 1680, 1708, 1736, 1764, 1792, 1820, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1906, 1934, 1962, 1990, 2018, 2046, 2074, 2102, 2130, 1880, 1882, 1908, 1910, 1936, 1938, 1964, 1966, 1992, 1994, 2020,2022, 2048,2050, 2076,2078, 2104,2106, 2132,2134, 1884,1886,1888, 1912,1914,1916, 1940, 1942,1944, 1968,1970,1972, 1996,1998,2000, 2024,2026,2028, 2052,2054,2056, 2080,2082,2084, 2108,2110,2112, 2136,2138,2140, 1890, 1892, 1918, 1920, 1946, 1948, 1974, 1976, 2002,2004, 2030,2032, 2058,2060, 2086,2088, 2114,2116, 2142,2144, 1894, 1896, 1922, 1924, 1950, 1952, 1978, 1980, 2006,2008, 2034,2036, 1898,1900,1902, 1926,1928,1930, 1954,1956,1958, 1982,1984,1986, 2010,2012,2014, 2038,2040,2042, 1904, 1932, 1960, 1988, 2016, 2044, 2062,2064,2066, 2090,2092,2094, 2118,2120,2122, 2146,2148,2150, 2068,2070,2072, 2096,2098,2100, 2124,2126,2128, 2152,2154,2156, 2180,2182,2184, 2208,2210,2212, 2236,2238,2240, 2158,2160,2162,2164,2166,2168,2170,2172,2174,2176,2178, 2186, 2214, 2188,2190,2192, 2216,2218,2220, 2194,2196,2198,2200,2202, 2222,2224,2226,2228,2230, 2204,2206, 2232,2234, 2242,2244,2246,2248,2250,2252,2254,2256,2258,2260,2262,2264,2266,2268, 2270,2272,2274,2276,2278,2280,2282,2284,2286,2288,2290,2292,2294,2296, 2298,2300,2302,2304,2306,2308,2310,2312,2314,2316,2318,2320,2322,2324, 2326, 2328,2330,2332,2334,2336,2338,2340,2342,2344,2346,2348,2350,2352, 2354,2356,2358,2360,2362,2364,2366,2368,2370,2372,2374,2376,2378,2380, 2382,2384,2386,2388,2390,2392,2394,2396,2398,2400,2402,2404,2406,2408, 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, 2774, 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%, 70%, 80%, 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.
If 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 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).
If the composition comprises a protein disclosed in W095/03413, said protein preferably comprises an amino acid sequence selected from the group consisting of SEQ IDs 1-23 disclosed in W095/03413 (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).
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%, 70%, 80%, 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%, 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%, 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: protein 919', typified by SEQ IDs 3069-3074 and 3207-3241 of W099/57280 (see also Figure 23 and Example 15 therein).
protein'235', typified by SEQ IDs 869-874 and 3149-3178 of W099/57280 (see also 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 WO99/57280 (see also Figure 19 and Example 11 therein).
protein'ORF40', typified by example 1 (SEQ IDs 1-6) of W099/36544 (see also Figure 1 of WO00/66741 see also W099/31132 and W099/58683).
protein'287', typified by example 9 of W099/57280 (see SEQ IDs 1199-1204,3103 3108 and 3179-3184 therein).
protein'ORFl', typified by example 77 (SEQ IDs 647-654) of WO99/24578 (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 of WO00/66741).
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) 14:1001-1008]; Cartwright et al. [Vaccine (1999) 17:2612-2619]; Peeters et al.
[Vaccine (1996) 14:1009-1015]; Fu et al. [Biotechnology NY (1995) 12:170-74]; Davies et al. Immunol. 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]; Andersen et al. [Vaccine (1997) 15:1225-34]; Bjune et al. [Lancet (1991) 338:1093-96] etc.
OMVs are preferably a deoxycholate extract from NmB obtained from NmB by deoxycholate extraction). The preferred extraction protocol is that described by Fredriksen et al. [Production, characterization and control of MenBvaccine"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 15: P1. 7,16: 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 25, gg protein, 2itg LPS and 1.67mg Al
(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. [ages 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 SEQ ID 7, SEQ ID 9, SEQ ID 11, or SEQ ID 13 depending on the particular SEQ ID, the degree of sequence identity is preferably greater than 50% (eg. 60%, 70%, 95%, 99% or more), which includes mutants and allelic variants, or (ii) a protein comprising an immunogenic fragment of 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, depending on the particular sequence, n is 7 or more (eg. 8,10,12,14,16,18,20 or more).] Combining components and (b) Components and can be combined by simply mixing component with an outermembrane preparation g. 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 outermembrane 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; a protective antigen against Helicobacterpylori; a protective antigen against polio; and/or a protective antigen against hepatitis B virus.
Preferred examples of these optional components are: a polysaccharide antigen against Neisseria meningitidis serogroup A; a polysaccharide antigen against Neisseria meningitidis serogroup C, such as that described in Costantino et al. (1992) Vaccine 10:691-698; a polysaccharide 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.
Pertussis 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 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 mature form of the protein is preferably used. For example, where NspA is included, (W096/29412 see also Martin et al. (1997) J. Exp. Med 185 1173-1183) the mature form of the protein lacking the signal peptide is preferably used.
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.
SEQUENCE LISTING The sequences in the sequence listing are SEQ ID DESCRIPTION 1 N-terminal sequence of N.meningitidis serogroup B protein, 80-85kDa 2 Complete gene from N.meningitidis serogroup B 3 Encoded protein from SEQ ID 2 4 Signal peptide protein from SEQ ID 3 Mature protein from SEQ ID 3 6 Complete gene from N.gonorrhoeae, homologous to SEQ ID 2 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.
17 c 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 SEdition (1989); DNA Cloning, Volumes I and ii N Glover ed. 1985); Oligonucleotide Synthesis J. Gait ed, 1984); Nucleic Acid Hybridization D.
Hames S. J. Higgins eds. 1984); Transcription and Translation D. Hames S. J.
0Higgins eds. 1984); Animal Cell Culture 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 H. 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, N. and Handbook of Experimental Immunology, Volumes I-IV M. Weir and C. C. Blackwell eds 1986).
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.).
They are preferably prepared in substantially pure form (ie. substantially free from other Neisseria or host cell proteins).
Nucleic acid used with the invention can be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single 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 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.
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 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 3' untranslated regions of the gene, such as in regulatory control regions (eg. see US patent 5,753,235).
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 SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), 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 downstream 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 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 terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci.
79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521].
Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].
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 SV40 [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 cells 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 replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and 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 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 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. Baculovirus 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 baculovirus/insect 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").
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 transcription termination sequence, are usually assembled into an intermediate transplacement construct (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 maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
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 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 of Baculoviruses (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 a-interferon, 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, 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 proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
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 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 gm 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 absence (indicative of recombinant virus) of occlusion bodies. "Current Protocols in Microbiology" Vol. 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. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (WO 89/046699 Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol.
Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol.
25:225).
Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression 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 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 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 metabolicallyregulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447- 3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987).
Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation-in plants. The 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, 28 N, for example for the members of the grass family, is found in Wilmink and Dons, 1993, SPlant 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 Sinsertion 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 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 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 signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein 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 scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.
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, sitedirected 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, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, 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 shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The 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 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.
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 sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; US patent 4, 738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. 1. 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 Dalgamo (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 Nterminus will always be a methionine, which 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).
Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the Nterminal portion of an endogenous bacterial 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 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 NI processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign D 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 ("1 may or may not encode a cleavable site. Another example is a ubiquitin fusion protein.
O 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 Sfrom the foreign protein. Through this method, native foreign protein can be isolated 10 [Miller et al. (1989) Bio/Technology 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 (gramnegative 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. Natl. 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 et al. (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 the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples 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. 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 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 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, 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. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], 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) Appl. Environ. Microbiol. 54:655] Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [US patent 4,745,056].
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaC12 or other agents, such as divalent cations and DM SO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc.
Natl. Acad. Sci. 69:2110; 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-phosphatedehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
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 the ADH regulatory sequence linked to the GAP transcription activation region (US Patent Nos.
4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). 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.
Immunol.. 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 Nterminus 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 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.
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 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide 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 can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as 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 8:17-241, pCI/I [Brake et al. (1984) Proc.
Natl. 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 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 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 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 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 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 CUP1 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) J. 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. Natl. 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]; [Das et al. (1984) J.
Bacteriol. 158:1165 De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135 Kluyveromyces] [Cregg et al. (1985) Mol.
Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; US Patent Nos.
4,837,148 and 4,929,555; Pichia]; [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. (1985) Curr. Genet.
10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].
Antibodies 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 binding space with an 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 distinguishing/identifying 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 200 pg/injection 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 0 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 ofnonspecifically 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 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 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 hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 12551), 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, 125I 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, MAbs 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 1251I, 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 a given situation can be determined by routine experimentation and is within the judgement of the clinician.
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.
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 antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical 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, 44 slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, Spolyglycolic acids, polymer 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 N 5 such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like and the salts Sof 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).
O 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 immunogen polypeptide protein (s) or nucleic acid, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymer 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 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.
Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; 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 MF59TM (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 Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y 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 RibiTM 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 (DetoxTM); saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); cytokines, such as interleukins (eg. IL-I, 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 T M are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-Disoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-Disoglutamine (nor-MDP), N-acetylmuramylL-alanyl-D-isoglutaminyl-L-alanine-2- 2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- 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.
Typically, the immunogenic 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. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This 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 transdermally/transcutaneously (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 vivo 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.
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-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous 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 W096/37626). 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 W092/05266), 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) J 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 33:493-503; Baba (1993) JNeurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl 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 employable in this invention include those described in the above referenced documents and in W094/12649, W093/03769, W093/19191, W094/28938, W095/11984, W095/00655, W095/27071, W095/29993, W095/34671, W096/05320, W094/08026, W094/11506, W093/06223, W094/24299, W095/14102, W095/24297, W095/02697, W094/28152, W094/24299, W095/09241, W095/25807, W095/05835, W094/18922 and W095/09654. 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 nucleotides, 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 AAV 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 W092/07945, HSV Us3:: pgC-lacZ 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.
Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR- 370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC 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 WO95/07994 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-1110 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 Natl 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 virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC 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 example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and 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 Hamre (1966) Proc Soc Exp Biol Med 121:190.
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. 08/366,787, 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 Natl 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, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in USSN.
60/023, 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 polymer DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, 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 for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad.
Sci. USA 91 (24):11581-11585. Moreover, the coding sequence and the product of 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 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 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 (3) 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 WO98/20734), 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. WO93/14778. 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, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
Polynucleotide and polvpeptide pharmaceutical compositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.
A. Polypeptides 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. Polvalkvlenes, Polysaccharides, 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 (Feigner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. 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 3-dioleyloxy) propyl]-N, N, N-triethylammonium (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 DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc.
Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP 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. Immunol. 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 polynucleotide/polypeptide 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 Cl, CII, CIII.
A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL 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) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151 :162 Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.
Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, 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) J 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. Polycationic 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, polyomithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-l, Oct-i, 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 above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.
Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene.
LipofectinTM, and lipofectAMINETM are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.
Immunodiagnostic 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 Hvbridisation "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 solution.
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.
"Stringency" refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration 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.
Variables to consider when performing, for example, a Southern blot are the complexity of the DNA being blotted and the homology between the probe and the sequences being detected. The total amount of the fragment to be studied can vary a magnitude of 10, from 0.1 to lpg for a plasmid or phage digest to 10 9 to 10 8 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 1 tg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/Lg. For a single-copy mammalian gene a conservative approach would start with 10 gtg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/tg, 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) 0.4 (G 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.
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 0 C for to 95% homology, and 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 conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.
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.
62 0 NC 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 5 sequencing described above, a signal peptide (SEQ ID 4) and a mature sequence (SEQ ID 5) are inferred.
O Identification of corresponding genes in N.meningitidis serogroup A and N.gonorrhoeae C, 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.
The Mmeningitidis serogroup B sequence and the N-gonorrhoeae sequence show 95.4% identity in 797 aa overlap: orf21 .pep orf21ng.pep orf2l.pep orf2lng.pep orf2l .pep orf2lng.pep Ai orf2l .pep orf2lng.pep orf2l .pep orf2lng.pep orf2l .pep orf2lng .pep 20 30 140 s0 MKLKQIASALMM1GISPLALADFTIQDIRVEGLQRTEPSTVFNYLPVKVGDYNTHGSA
MKLKIASALMGISPLAFADEIDIRVEGLQRTEPSTFNYLPVVGD~YNJHGSA
20 30 40 50 80 90 100 110 120 IIKSLYATGFFDDVRVETAGQLLLTVIERPTIGSLNIT
QAIYESGLBQ
IIKSLYAWFFDDVRVETADGQLLLVIERPTIGSLNIJ Q AIKMESGLQ .80 90 100 110 120 130 140 150 160 170 180 SQYFNQATLNQAVAGLKEEYLGRGKLNIQITPKVTKRNRDIDITIDMJeSAJ(ITDIE 130 140 150 160 170 180 1.90 200 210 220 230 240 190 200 210 220 230 240 250 260 270 280 290 300 DTDIQTEDKTRTIKITVHEGGRFRWGKVSI
EVPEEKRJTMPGKJYEQQ
250 260 270 280 290 300 310 320 330 340 350 360 310 320 330 340 350 360 370 380 390 400 410 420 RDEVVRRELRQMESAPYDTSKliQRSKERVELLWYFDNVQFDAVPLAGTPDKVDLN14SLTE
RDEVVRRELRQMESAPYDTSKIJQRSKERVELLGYFDNVQFDAVPLAGTPDVDLMSLTE
370 380 390 400 410 420 430 440 450 460 470 480 430 440 450 460 470 480 490 500 510 520 530 540 DGVSLGYDIYGKAPDPRKASTSVKQYKTTTAGGGVRMGI
PVTEYDRVNFGLAAEHLTVNT
490 500 510 520 530 540 550 560 570 580 590 600
YNAKYDIKGTGDSKWYKTGGNTSLPRYIIAI
YNAKYDIKGTGDSKLYKTGGNTSSPRYTVAI
550 560 570 580 590 600 610 620 630 640 650 660
LPSLYSTNMPSTTM<GVIGYRKIFEFGGGV
orf2 pep orf2lng .pep orf2l.pep orf2lng .pep orf2l .pep orf2lng.pep orf21 .pep orf2lzig.pep orf2l .pep orf2Thg-pep orf 21.pep orf2lng.pep orf2l .pep orf21ng.pep orf2l .pep orf2lng .pep LPGSKLQYYSATQQWF
FPLSK~L(QVIGGGTE
1 FE~~rGGV 610 620 630 640 650 660 GYESGTLGPKVYDEyGEKISYGNKKANSAIJLLPGAArnSADSV 670 680 6.90 700 710 720 73 4 5 6 770 780 730 740 750 760 770 790 KPEDEIQRFQQGTp 780 790 The NMmeningitids serogroup A and B sequences show 99.9% identity in 797 aa overlap: 25orf2l .pep a~e 30orf2la.pep 30orf2la.pep orf2l .pep ox-f2la .pep orf2l .pep orf2la .pep 1KLKQIASALVLGISPLADFTIQDIVELQ 40 50 10 20 30 40 50 80 90~v~ET 100 110 120 80 90 100 110 120 13 4 S 6 170 180 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 310 3260 330 340 350 3600 250 2603 20282930 370 380 390 400 410 420 orf21.pep Orf2la.pep orf2l-.pep orf2la .pep orf2l.pep orf2la.pep
NO
or 2 l.pep RSTGSLDLSAGWQDTLVMSAGVSQDtLFGTGKSAALRASRSKTTLNGSLSFTPYFTA orf 2la pep RSTGSLDLSAGWVQDTGLVMSAGVSQDNLFGTGKSAALRASRSKTTLNGSLSPITDPYFTA 430 440 450 460 470 480 490 500 510 520 530 540 or 21. pep DGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRH4SVPVTEYDRVNFGLVAEHLrTVT orf 2la .pep DGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRNSVPVTEYDRVNFGLVAEHLTVNT 490 500 510 520. 530 540 550 560 570 580 590 600 orf2 1.pep YNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSAILWPTRGYLTVNAEIA orf 2la .pep YNKAPKHYADFIKKYGKTDGTDGSFKGWLYKG'IVGWGRNKTDSALWPTRGYLTGVNAEIA 550 560 570 580 590 600 610 620 630 640 650 660 orf2l pep LPGSKLQYYSATHNQTWFFPLSKTFTLMLGGEvGIAGGYGRTKEIPFFEN*yGGGLGSVR orf2 la pep LPGSKL#QYYSATHNQWF~PLSKFTLNL#GGEVGIAGGYGRTKEIPFFENFYGGGLGSVR 610 620 630 640 650 660 670 680 690 700 710 720 :)rf21 .pep GYESGTLGPKYDEYGEKISYGGNKKANVSAELLFPPGAKDARlTVRLSLFADAGSVWDG )rf2la pep GYESGTL#GPKVYDEYGEKISYGGNK@JqVSAELLFPHPGAKDARTVRLSLFADAGSVWDG 670 680 690 700 710 720 730 740 750 760 770 780 rf 21. pep KTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGGAVTWIISPLGPMKFSYAYPLKK irf2 la.pep KTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGG;AVTWLSPLGPMKFSYAYPLKK 730 740 750 760 770 780 790 'rf2l .pep KPEDEIQRFFQLG7TF illittiii, 111111II 'rf2la .pep ICPEDEIQRFQFQLGTTFX 790
C
0 0 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.
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% of patients Rosenqvist et al. (1995) Infect. Immun.
63: 4642-4652].
Surprisingly, it has been found that the addition of further defined components to the Norwegian OMV vaccine significantly broadens its efficacy.
The Norwegian vaccine does not elicit protection against NmB strain 2996. Defined proteins from 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: Group Norwegian NmB NmC Bactericidal activity OMV antigen* antigen against NmB strain 2996 1 <4 2 #1 512 3 #2 >2048 4 #3 1024 #3 256 6 #3 2048 7 2048 Three different NmB antigens were used: ORFI e.g. example 77 of W099/24578 (see also W099/55873) #2 protein '287'- e.g. Figure 21 of W099/57280 (also SEQ IDs 3103-3108) #3 a mixture in Al (OH) 3 of#1, #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 vaccines, prepared using Titres were as follows: protection against heterologous 2996 strain proteins, was tested MenB strains. The same against five other strains.
Group 2996 BZ133 BZ232 1000 MC58 NGH38 1 <4 1024 <4 >2048 >2048 32 2 512 512 <4 >2048 >2048 256 3 4096 4096 256 1024 >2048 1024 4 1024 2048 <4 2048 >2048 64 256 >32000 <4 >2048 >2048 128 6 2048 2048 4 <4 64 4 7 2048 >32000 4 128 1024 128 Control* 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 ORFI, 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 The 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. antigen 287 was combined with Norwegian OMP vaccine (15 Rg OMP Al (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 MC58 NGH38 NZ F6124 C11 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 In almost all cases, therefore, the combination of OMVs protein 287 surprisingly gives better results that the OMVs alone.
Recombinant OMVs E.coli were transformed to express ORFI, ORF40 and ORF46. OMVs prepared from the recombinant E. coli were able to induce bactericidal antibodies against N.meningitidis.
ORF1, 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: EMI52.2 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.

权利要求:
Claims (13)
[1] 2. The composition of claim l, wherein component is a NmB protein.
[2] 3. The composition of claim 2, wherein the NmB protein is protein 919, protein 235, protein 519, protein 225, protein ORF40, protein 287,ORF1, ORF4, or ORF46.
[3] 4. The composition of any preceding claim, wherein component includes a protein from a different NmB strain from that from which component is derived.
[4] 5. The composition of any preceding claim, wherein one or more of its components is adsorbed on Al (OH)3.
[5] 6. The composition of any preceding claim, wherein component comprises OMVs.
[6] 7. The composition of claim 6, wherein the OMVs are a deoxycholate extract from NmB
[7] 8. The composition of any preceding claim, wherein component is adsorbed on Al (OH) 3.
[8] 9. The composition of any preceding claim, wherein 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 Helicobacterpylori; a protective antigen against polio; and/or a protective antigen against hepatitis B virus. The composition of any preceding claim, wherein the composition is a vaccine.
[9] 11. The composition of any preceding claim, for use as a medicament.
[10] 12. The use of composition according to any preceding claim 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.
[11] 13. A method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition according to any one of claims 1 tol0.
[12] 14. A bacterial outer membrane preparation comprising 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 W099/66791, 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 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, DATED this NINETEENTH day of February 2009 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> P023785WO <140> <141> 2001-01-17 <150> GB-0001067.8 <151> 2000-01-17 <150> GB-0005699.4 <151> 2000-03-09 <160> 14 <170> SeqWin99, version 1.02 <210> <211> <212> <213> 1 PRT Neisseria meningitidis <400> Asp Phe 1 1 Thr Ile Gin Asp Ile Arg Val Glu Gly Leu Gin Arg Thr 5 10 <210> 2 <211> 2394 <212> DNA <213> Neisseria meningitidis <400> 2 atgaaactga gccgacttca gtattcaact atcatcaaaa gggcagctcc gcaaaaatgc tcgcaatact Ctcgggcgcg cgcgtcgaca tttgaaggca ggcggcaettt gatatggaaa gataccgaca gaaggcggac aaagccgaac atgaccgccg gaaa tcagcg atcgaaccgg cgcgacgaag aagctgcaac gatgctgtcc cgttccaccg tccgcaggcg tccaggagca aacagattgc ttccgcactg atgatgttgg gcatatcgcc ccatccaaga acctgcccgt gcctgtacgc tgctgaccgt tgcaaaacga ttaatcaggc gcaaactcaa tcgacatcac accaagtcta ggacatggct aagtaaccga tccaaaccaa gtttccgttg tggaaaaact ttttgggtga tacagccgct gccggaaaat tcgtccgccg gttccaaaga cgcttgccgg gttccctgga tttcccaaga aaaccacgct catccgcgtc caaagtcggc caccggtttc tatcgaacgc cgccattaag gacactcaat tatccaaatc gat tgacgag ttccgaccgc gacacgaagc cttctaccaa cgaagacaaa gggcaaagtc gctgaccatg gattcagaac gccgaacgct ctacgtcaac tgaattacgc gcgcgtcgag cacgcccgac tttgagcgcg caacctgttc taacggctcg gaaggcttgc gacacctaca tttgacgacg cccaccatcg aaaaacctcg caggcagtcg acgcccaaag ggcaaatccg aaactgatgc aaccaattca aataacggct accaagcaga tccatcgaag aagcccggca cgcatgggct gaaaccaaaa gaaatacaca caaatggaat Cttttgggct aaagtcgatt ggttgggt tc ggtacgggca ctgtcgttta agcgtaccga acgacacaca tacgcgtcga gctcgctcaa aatcgttcgg ccggcctgaa taaccaaac t ccaaaatcac ggcaaatgtc acgagcagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggc ta ccgtcgattt tcaccggcaa ccgcacctta acttcgacaa tgaaca tgag aagataccgg agtcggccgc ctgacccgta tt tggcac tt gccgagtacc cggcagtgcc aac tgcggac catcaccggc gctggcgcag agaagaatac cgcccgcaac cgacatcgaa cctgaccgaa atttgcccaa ccgtatcctc caccgtccac cgaagtcccc acgccagcag cgcatacagc cgtcctgcac caacaaaacc cgacacctcc tgtccagttt tctgaccgaa gttggtcatg actgcgcgcc cttcacggca 120 IS0 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 gacggggtca accagcatca gttaccgaat tacaacaaag acagacggca accgacagcg ctgcctggca ctgagcaaaa agaaccaaag ggatacgaaa tacggcggca aaagacgcgc aaaacctacg gccggcaata gttacctggc aaaccggaag gcctgggcta aacaatataa acgaccgcgt cgcccaaaca gcttcaaagg cgttatggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgct acaaaaaagc gcaccgtccg acgacaacag cccataaatc tctcgccttt acgaaatcca cgatgtttac aaccaccacg gaatttcggt ctatgccgac ctggctgtac gacgcgcggc atactactcc gatgctcggc ctttgaaaac cggtccgaaa caacgtctcc cctgagcctg cagttccgcg cacctttacc aggcccgatg acgct tccaa ggaaaagcct gcaggcgcag ttggtggcag tttatcaaga aaaggtaccg tacctgacgg gccacccaca ggcgaagtcg ttctacggcg gtctatgacg gccgagctgc tttgccgacg accggcggca aacgaat tgc aaattcagct ttccaactcg tcgacccgcg gcatccgcat aacacctgac aataCggCda tcggctgggg gcgtgaacgc accaaacctg gcattgcggg gcggcctggg aatacggcga tcttcccgat caggcagcgt gggttcaaaa gctattccgc acgcctaccc gcacgacgt t caaagcatcg gagcgtgcct cgtcaacacc aaccgacggc gcgcaacaaa cgaaatcgcc gttcttcccc cggctacggc ttcggtgcgc aaaaatcagc gcccggcgcg gtgggacqgc catttacggc cggcggcgcg gctgaagaaa ctaa 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2394 NO <210> <211> <212> PRT <213> Neisseria meningitidis <400> 3 Met Lys Leu 1 Pro Leu Ala Leu GIn Arg Val Gly Asp Lys Gin 5 Ile Ala Ser Ala Leu Met 10 Met Leu Gly Ile Ser Ala Asp Phe Thr Ile Gin Asp Ile Arg Val Giu Gly Pro Val Lys Thr Giu Pro Ser Thr Val Phe Asn Tyr Thr Tyr Asn Thr His Gly Ser Ile Ile Lys Ser Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Vai Glu Thr Ala Gly Gin Leu Leu Leu Thr Val Ile Glu Pro Thr Ile Gly Ser Leu Asn Ile Thr Leu Giu Ser 115 Ala Lys Met Leu Asn Asp Ala Ile Lys Lys Asri 110 Phe Gly Leu Ala Gin 120 Ser Gin Tyr Phe Asn 125 Gin Ala Thr Leu Asn 130 Lys Leu 145 Gin Ala Val Ala Gly 135 Leu Lys Glu Giu Leu Gly Arg Gly Asn Ile Gin Ile 150 Thr Pro Lys Val Lys Leu Ala Arg Arg Val Asp Ile Asp 165 Ile Thr Ile Asp Gly Lys Ser Ala Lys Ile 175 Thr Asp Ile Giu Phe Glu Gly Asn Val Tyr Ser Asp Arg Lys Leu 190 Met Arg Gin Met Ser Leu Thr Giu Gly Gly Ile Trp Thr Trp Leu Thr Arg Ser Asn Gin Phe Asn Giu Gin Lys 210 Val1 225 Asp Ile Giu Thr Leu 305 Glu Phe H-is Leu Ser 385 Asp Ser Val1 Leu Thr 465 Asp Arg Thr Thr Thr Giy Met 290 Gly Ile Val Ile Arg 370 Lys Ala Leu Gin Phe 450 Thr Gly Lys Asp Asp Vali Asp 275 Lys GiU Ser Leu Thr 355 Gin Giu Val1 Thr Asp 435 Gly Leu Val Ala Phe Ile His 260 Thr Pro Ile Val1 His 340 Gly Met Arg Pro Giu 420 Thr Thr Asn Ser Ser Tyr Gin 245 Giu Asn Gly Gin Gin 325 Ile Asn Giu Val1 Leu 405 Arg Giy Gly Gly Leu 485 Thr Gin 230 Thr Giy Giu Lys Asn 310 Pro Giu Asn Ser Giu 390 Aia Ser Leu Lys Ser 470 Gly Ser 215 Asn Asn Asn Giu Giy Arg Vai Pro 280 Trp Tyr 295 Arg Met Leu Pro Pro Giy Lys Thr 360 Aia Pro 375 Leu Leu Gly Thr Thr Giy Val Met 440 Ser Ala 455 Leu Ser Tyr Asp Ile Lys Giy Asp Phe 265 Lys Glu Gly Asn Arg 345 Arg Giy Pro Ser 425 Ser Ala Phe Val Gin Phe Tyr Lys 250 Mrg Ala Arg Ser Aia 330 Lys Asp Asp Tyr Asp 410 Leu Ala Leu Thr Tyr 490 Tyr Gin 220 Asp Lys Giy Leu Gin 300 Gly Thr Tyr Val Ser 380 Asp Val Leu Val Ala 460 Pro Lys Thr 205 Asp Phe Gin Lys Giu 285 Met Tyr Lys Val Val 365 Lys Asn Asp Ser Ser 445 Ser Tyr Ala Thr Met Arg Thr Val 270 Lys Thr Ala Thr Asn 350 Arg Leu Val Leu Ala 430 Gin Arg Phe Phe Thr 510 500 505 Ala Gly Ilie Arg Met Ser Val Pro Val Thr Giu Tyr Asp Arg Val Asn 515 520 525 Phe Gly Leu Val Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala 530 535 540 Pro Lys His Tyr Ala Asp Phe Ile Lys Lys Tyr Gly Lys Thr Asp Gly 545 550 555 560 Thr Asp Gly Ser Phe Lys Gly Trp Leu Tyr Lys Gly Thr Val Gly Trp 565 570 575 Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu 580 585 590 Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gln Tyr 595 600 605 Tyr Ser Ala Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr 610 615 620 Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly 625 630 635 640 Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu 645 650 655 Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr 660 665 670 Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn 675 680 685 Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg 690 695 700 Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly 705 710 715 720 Lys Thr Tyr Asp Asp Asn Ser Ser Ser Ala Thr Gly Gly Arg Val Gin 725 730 735 Asn Ile Tyr Gly Ala Gly Asn Thr His Lys Ser Thr Phe Thr Asn Glu 740 745 750 Leu Arg Tyr Ser Ala Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly 755 760 765 Pro Met Lys Phe Ser Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp 770 775 780 Glu Ile Gin Arg Phe Gin Phe Gin Leu Gly Thr Thr Phe 785 790 795 <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 Pro Leu <210> <211> <212> <213> Ala Leu Ala 776 PRT Neisseria meningitidis <400> Asp Phe Thr Ile Gin Asp Ile Arg Val Giu Gly Leu Gln Arg Thr Glu 1 Pro Asn Phe Thr Lys Leu Ala Ile Ile 145 Glu Leu Asn Gin Thr 225 Gly Ser Asp Phe Val Met Ala Gly Thr 130 Thr Gly Thr Glu Asn 210 Asn 31 y Thr Thr Asp Ile Leu Gin Leu 115 Pro Ile Asn Glu Gin 195 Asn Glu Arg Val His Asp Glu Gin Ser 100 Lye Lys Asp Gin Gly 180 Lyes Gly Asp Phe 5 Phe Gly Val Arg Asn Gin Glu Val Glu Vai 165 Gly Phe Tyr Lys Arg 245 Asn Ser Arg Pro 70 Asp Tyr Glu Thr Gly 150 Tyr Ile Ala Phe rhr 230 rrp Tyr Ala Vai 55 Thr Ala Phe Tyr Lys 135 Lys Ser Trp Gin Asp 215 Lys Gly 10 Leu Ile 40 Glu Ile Ile Asn Leu 120 Leu Ser Asp Thr Asp 200 Phe Gin Lyes Pro 25 Ile Thr Gly Lys Gin 105 Gly Ala Ala Arg Trp 185 Met Arg Thr Val Val Lys Ala Ser Lys 90 Ala Arg Arg Lys Lys 170 Leu Glu Ile Ile Ser Lys Ser Asp Leu 75 Asn Thr Gly Asn Ile 155 Leu Thr Lys Leu Lys 235 Ile Va1 Leu Gly Asn Leu Leu Lys Arg 140 Thr Met Arg Va1 Asp 220 Ile Glu Gly Tyr Gin Ile Glu Asn Leu 125 Val Asp Arg Ser Thr 205 Thr Thr Gly Asp Ala Leu Thr Ser Gin 110 Asn Asp Ile Gin Asn 190 Asp Asp Val Asp Thr Thr Leu Gly Phe Ala Ile Ile Glu Met 175 Gin Phe Ile His Thr Tyr Gly Leu Ala Gly Val Gin Asp Phe 160 Ser Phe Tyr Gin Glu 240 Asn 250 255 Glu Val Pro Lye Ala Giu Leu Giu Lye Leu Leu Thr Met Lys Pro Gly Lys Asn Pro 305 Glu Asn Ser Glu Ala 385 Ser Leu Lys Ser Gly 465 Ser Ser Giu Asp Lys 545 Asp Giu Trp Arg 290 Leu Pro Lys Ala Leu 370 Giy Tbr Val Ser Leu 450 Tyr Ile Val His Phe 530 Giy Ser Ile Tyr 275 Met Pro Gly Thr Pro 355 Leu Thr Gly Net Ala 435 Ser Asp Lys Pro Leu 515 Ile Trp Ala Ala 260 Giu Arg Gly Ser Asn Ala Arg Lys 325 Arg Asp 340 Tyr Asp Gly Tyr Pro Asp Ser Leu 405 Ser Ala 420 Ala Leu Phe Thr Val Tyr Gin Tyr 485 Val Thr 500 Thr Val Lys Lys Leu Tyr Leu Trp, 565 Leu Pro Gin Aia Glu 310 Ile Glu Thr Phe Lys 390 Asp Giy Arg Asp Gly 470 Lys Giu Asn Tyr Lys 550 Pro Gly Gin Gly 295 Thr Tyr Val Ser Asp 375 Val Leu Val Ala Pro 455 Lys Thr Tyr Thr Gly 535 Gly Thr Ser Met 280 Lys Val Val Lys 360 Asn Asp Ser Ser Ser 440 Tyr Ala Thr Asp Tyr 520 Lys Thr Arg Lys Thr Ala Thr Asn Arg 345 Leu Val Leu Ala Gin 425 Arg Phe Phe Thr Arg 505 Asn Thr Val Gly Leu Ala Tyr Val Glu 330 Arg Gin Gin Asn Gly 410 Asp Ser Thr Asp Ala 490 Val Lys Asp Gly Tyr 570 Gin Val1 Ser Asp 315 Ile Glu Arg Phe Met 395 Trp Asn Lys Ala Pro 475 Gly Asn Ala Gly Trp 555 Leu Tyr Gin Gin Ile 320 Asn Glu Val Leu Arg 400 Gly Gly Gly Leu Thr 480 Met Ala Ala Phe Thr 560 Ala His Asn Gin Thr 595 Trp Phe Phe Pro Ser Lays Thr Phe Thr 605 Leu Met Leu Gly Gly 610 Giu Val Gly Ile Gly Gly Tyr Gly Arg Thr Lays Glu Ile 620 Gly Ser Val Arg Gly Phe Phe Glu Asn Tyr Gly Gly Gly Tyr Glu Ser Lys Ile Ser Leu Phe Pro 675 Gly Thr 645 Leu Gly Pro Lys Tyr Asp Glu TPyr Gly Glu 655 Gly Gly Asn Lys Ala Asn Val Ser Ala Glu Leu 670 Arg Leu Ser Met Pro Gly Ala Asp Ala Arg Thr Leu Phe 690 Ala Asp Ala Gly Val Trp Asp Gly Thr Tyr Asp Asp Ser Ser Ser Ala Gly Gly Arg Val Asn Ile Tyr Gly Gly Asn Thr His Ser Thr Phe Thr Giu Leu Arg Tyr Ser Ala 735 Gly Gly Ala Tyr Ala Tyr 755 Thr Trp Leu Ser Leu Gly Pro Met Lys Phe Ser 750 Gin Arg Phe Pro Leu Lys Lys Lys 760 Pro Giu Asp Glu Ile 765 Gin Phe Gin Leu Giy Thr Thr Phe 770 775 <210> 6 <211> 2379 <212> DNA <213> Neisseria gonorrhoeae <400> 6 atgaaactga gccgacttca gtattcaact atcatcaaaa gggcagc ttc gccaaaatgc tcgcaatact ctcgggcgtg cgcgtcgaca tttgaaggca ggcggcattt gacatggaaa gataccgaca gaaggcggac aaggccgaac atgaccgccg aacagattgc ccatccaaga acc tgcccgt gcctgtacgc tgctgaccgt tgcaaaacga ttaatcaggc gcaaactcaa tcgacatcac accaagtcta ggacatggct aagtaaccga tccaaaccaa gtttccgctg tggaaaaact ttttgggtga ctccgcactg catccgtgtc caaagtcggc caccggtttc tatcgaacgc cgcc atcaag gacactcaac tatccaaatc gattgacgag tatccgaccgc gacacgaagc cttctaccag cgaagacaaa gggcaaagtg gc tgaccatg gattcagaac atgatgttgg gaaggcttgc gacacctaca ttLatgacgacg cccaccatcg aaaaacctcg caggcagtcg acgcccaaag ggcaaatccg aaactgatgc gaccggttcg aacaacggct accaggcaga tcgattgaag aagcccggca cgcatgggc t gcatatcgcc agcgtaccga acgacacaca tacgagtcga gctcgctcaa aatcgttcgg ccggcctgaa taaccaaact ccaaaatcac ggcagatgtc accgccagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggcta tttggcattt gccgagcacc cggcagtgcc aactgcggac catcaccggc gc tggcgcag agaagaatac cgcccgcaac cgacatcgaa gctgaccgaa attcgcccaa ccgtatcctc caccgtccac cgaagtccc acgccagcag cgcatacagc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 gaaatcagcg atcgaaccgg cgcgacgaag aagctgcaac gatgccgtcc cgctccaccg tccgccggcg tcgcgaagca gacggggtca accagcgtca gttaccgaat tacaacaaag gcagacggca accgacagcg ctgcccggca ttaagcaaaa agaaccaaag ggctacgaaa tacggcggca aaagacgcac agaac ctat a aaatccacct cctttgggtc atccaacgct tacagccgct gccggaaaat tcgtgcgccg gctccaaaga cgcttgccgg gctcgctcga tatcgcagga aaaccacgct gcctgggcta aacaatataa acgaccgcgt cacccaaacg gcttcaaagg cgtcatggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgct acaaaaaagc gcaccgtccg ccgccgccga ttaccaacga cgatgaaatt tccaattcca gccgaacgcc ctacgtcaac cgaat tgcgc gcgcgtcgag tacgcccgac cttgagcgcg caacctgttc caacggctcg cgatatttac aaccaccacc caatttcggg ctatgccgac cctgctgtac gacgcgcggc atactactcc gatgc tcggc ctttgaaaac cggcccgaaa caacgtctcc cctgagcctg aaacggtaac attgcgctat cagctacgcc gctcggcacg ggaaccaaaa gaaatccaca caaatggaat cttttgggct aaagtcgatt ggctgggttc ggtacgggca ctgtcgttta ggaaaagcct gccggcggcg ctggcggcgg tttatcagga aaaggcaccg tacctgaccg gccacccaca ggcgaagtcg ttctacggcg gtgtatgacg gccgagctgc tttgccgacg aacaaatcgg tccgccggcg tacccgctga acgttctaa ccgtcgattt tcaccggcaa ccgcgcctta acttcgacaa tgaacatgag aggataccgg agtcggccgc ccgacccgta tcgacccgcg gcgtaaggat aacacc tgac aatacggcaa. tcggctgggg gcgtaaatgc accaaacctg gcattgcggg gcggcctggg aatacggcga tcttcccgat caggcagcgt tttactcgga. gcgcggttac agaaaaaacc cgtcctgcac caacaaaacc cgacacctcc cgtacagttt cc tgaccgaa cttggtcatg cctgcgcgcc cttcacggca caaagcatcg gggtatcccc cgtcaacacc aaccgacggc gcgcaacaag cgaaatcgcc gttcttcccc cggctacggc t tcggtgcgc 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 Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser 1 5 10 Pro Leu Ala Leu Gin Arg Phe Ala Asp Phe Thr Ile Gin Asp Ile Arg Val Glu Gly 25 Thr Giu Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys Val Gly Asp Thr Tyr Asn Thr His Gly Ser Ala Ile Ile Lys Ser Leu T~yr Ala Thr Gly Phe Asp Asp Val Val Giu Thr Ala Asp Gly Gin Leu Leu Leu Thr Val Ile Giu Pro Thr Ile Gly Ser Leu Asn Ile Thr Leu Giu Ser 115 Leu Asn Gin 130 Ala Lys Met Leu Gin 105 Asn Asp Ala Ile Lys Lys Asn 110 Gin Ala Thr Phe Gly Leu Ala Ser Gin Tyr Phe Asn 125 Ala Val Ala Giy 135 Leu Lys Giu Giu Tyr 140 Leu Gly Arg Gly Lys 145 Leu Asn Ile Gin Ile 150 Thr Pro Lys Val Thr Lys Leu Ala Arg 3 P H L IS 3 A S LE Arg Val Asp Ile Asp Ile Thr Ile Asp Giu Gly 165 170 Thr Asp Ile Giu Phe Giu Gly Asn Gin Val Tyr 180 185 Met Arg Gin Met Ser Leu Thr Giu Gly Gly Ile 195 200 Arg Ser Asp Arg Phe Asp Arg Gin Lys Phe Ala 210 215 Val Thr Asp Phe Tyr Gin Asn Asn Giy Tyr Phe 225 230 235 Asp Thr Asp Ile Gin Thr Asn Giu Asp Lys Thr 245 250 Tie Thr Val His Giu Gly Giy Arg Phe Arg Trp 260 265 Jiu Giy Asp Thr Asn Glu Val Pro Lys Ala Glu 275 280 rhr Met Lys Pro Gly Lys Trp Tyr Giu Arg Gin 290 295 4 eu Gly Giu Ile Gin Asn Arg Met Giy Ser Ala 105 310 315 ;iu Ile Ser Val Gin Pro Leu Pro Asn Ala Gly 325 330 'he Val Leu His Ile Giu Pro Gly Arg Lys Ile 340 345 is Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu 355 360 eu Arg Gin Met Giu Ser Ala Pro Tyr Asp Thr 370 375 er Lys Giu Arg Val Giu Leu Leu Gly Tyr Phe 85 390 395 sp Aia Val Pro Leu Ala Gly Thr Pro Asp Lys 405 410 er Leu Thr Giu Arg Ser Thr Giy Ser Leu Asp I 420 425 al Gin Asp Thr Gly Leu Val Met Ser Ala Gly 435 440 au Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg 1 450 455 4 Lys Ser Trp Gin 220 Asp Arg Gly Leu Gin 300 Gly Thr Tyr Vai Ser 380 ksp lal .eu fal lia E Ser Asp Thr 205 Asp Phe Gin Lys Glu 285 Met Tyr Lys Val Va1 365 Lys Asn ksp 'er 3er jer Ala Arg 190 Trp Met Arg Thr Val 270 Lys Thr Ala Thr Asn I 350 Arg Leu Val Leu I Ala C 430 Gin I Arg 4 Lys 175 Lys Leu Glu Ile Ile 255 Ser Leu Ala Tyr Val 335 lu Arg GIn ,ln ksn 115 :ly ~sp er Ile Leu Thr Lys Leu 240 Lys Ile Leu Val Ser 320 Asp Ile Glu Arg Phe 400 Met Trp Asn Lys Thr 465 Thr Leu Asn Gly Leu Ser Phe Thr Asp 475 Pro Tyr Phe Thr Asp Gly Val Ser Leu Gly Tyr Asp Ile Tyr Gly Lys Ala Phe Asp Pro 485 490 495 Arg Lys Ala Ser Thr Ser Val Lys Gin Tyr Lys Thr Thr Thr Ala Gly 500 505 510 dly Gly Val Arg Met Gly Ile Pro Val Thr Glu Tyr Asp Arg Val Asn 515 520 525 Phe Gly Leu Ala Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala 530 535 540 Pro Lys Arg Tyr Ala Asp Phe Ile Arg Lys Tyr Gly Lys Thr Asp Gly 545 550 555 560 Ala Asp Gly Ser Phe Lys Gly Leu Leu Tyr Lys Gly Thr Val Gly Trp 565 570 575 Gly Arg Asn Lys Thr Asp Ser Ala Ser Trp Pro Thr Arg Gly Tyr Leu 580 585 590 Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gin Tyr 595 600 605 Tyr Ser Ala Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr 610 615 620 Phe Thr Leu Met Leu Gly Gly Giu Val Gly Ile Ala Gly Gly Tyr Gly 625 630 635 640 Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu 645 650 655 Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lye Val Tyr 660 665 670 Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn 675 680 685 Val Ser Ala Giu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg 690 695 700 Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly 705 710 715 720 Arg Thr Tyr Thr Ala Ala Glu Asri Gly Asn Asn Lys Ser Val Tyr Ser 725 730 735 Glu Asn Ala His Lys Ser Thr Phe Thr Asn Glu Leu Arg Tyr Ser Ala 740 745 750 Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Giy Pro Met Lys Phe Ser 755 760 765 Tyr Ala Tyr Pro Leu Lys Lye Lys Pro Glu Asp Glu Ile Gin Arg Phe 770 775 780 Gin Phe Gin Leu Gly Thr Thr Phe 785 790 <210> 8 <211> 21 <212> PRT <213> Neisseria gonorrhoeae <400> 8 Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser 1 5 10 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 Giu Gly 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 Gly Ser Ala Ile Ile Lys Ser Leu Tyr Ala Thr Giy 40 Phe Phe Asp Asp Val Arg Val Glu Thr Ala Asp Gly Gin Leu Leu Leu 55 Thr Val Ile Giu Arg Pro Thr Ile Gly Ser Leu Asn Ile Thr Gly Ala 70 75 Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn Leu Glu 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 Tyr ELeu Gly Arg Gly Lys Leu Asn Ile Gin 115 120 125 Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn Arg Val Asp Ile Asp 130 135 140 Ile Thr Ile Asp Giu Gly Lys Ser Ala Lys Ile Thr Asp Ile Giu Phe 145 150 155 160 Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu Met Arg Gin Met Ser 165 170 175 Leu Thr Giu Gly Gly Ile Trp Thr Trp Leu Thr Arg Ser Asp Arg Phe 180 185 190 Asp Arg Gin Lys Phe Ala Gin Asp Met Giu Lys Val Thr Asp Phe Tyr 195 200 205 Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu Asp Thr Asp Ile Gin 210 215 Thr 225 Gly Glu Lys Asn Pro 305 Glu Asn Ser Glu Ala 385 Ser Leu Lys Ser I 4 Gly 1 465 Ser Gly I Glu H As Gi' Va Tr Ar 29( Let PrC Lys Ala Leu 370 Oly rhr /a 1 ;er ,eu 150 'yr ral le 'is n Giu Asp y Arg Phe 1 Pro Lys 260 P Tyr Glu 275 1 Met Gly Pro Asn Gly Arg Thr Arg 340 Pro Tyr 355 Leu Gly Thr Pro Gly Ser Met Ser 420 Ala Ala 435 Ser Phe Asp Ile Lys Gin 4 Pro Val 1 500 Leu Thr 515 Ly Arc 245 Ale Arg Ser Ala Lys 325 Asp Asp Tyr Asp Leu 405 Ala Leu hr 'yr 'yr 185 'hr ral 5 Th: 23 Tr] Gl Ale Glj 310 Ile Glu Thr Phe Lys 390 Asp Gly Arg Asp Gly 470 Lys Glu Asn r Arg 0 p Gly .i Leu i Gin Gly 295 Thr Tyr I Val Ser Asp 375 Val Leu Val Ala Pro 455 Lys Thr Tyn Thr Gly I 535 Gir Lys Glu Met 280 Tyr Lys Val Val Lys 360 Asn Asp Ser Ser Sen 440 Tyr Ala hr ksp yr 520 I Thr Val Lys 265 Thn Ala Thr Asn Arg 345 Leu Va1 Leu Ala Gin 425 Arg Phe Phe Thr Arg 505 Asn I Ile Ser 250 Leu Ala Tyr Va1 Glu 330 Arg Gin Gin Asn Gly 410 Asp Sen rhr Asp k1a 490 Jal ys Lys 235 Ile Leu Va1 Ser Asp 315 Ile Glu Arg Phe Met 395 Trp Asn Lys Ala Pro 475 Gly Asn Ala Ile Glu Thr Leu Glu 300 Phe His Leu Ser Asp 380 Ser Val Leu Thr Asp 460 Arg Gly Phe Pro Thr Gly Met Gly 285 Ile Val Ile Arg Lys 365 Ala Leu Gin Phe Thr 445 Gly Lys Gly' Gly Lys 525 Val Asp Lys 270 Glu Ser Leu Thr Gin 350 Glu Val Thr Asp Gly 430 Leu Val Ala IVal Leu 510 krg His Thr 255 Pro Ile Val His Gly 335 Met Arg Pro Glu Thr 415 Thr Asn Sen Sen Arg 495 Ala Tyr Glu 240 Asn Gly Gin Gin Ile 320 Asn Glu Val Leu Arg 400 Gly Gly Gly Leu Thr 480 Met Ala Ala Asp Phe 530 Ile Arg Lys Tyr ,ys Thr Asp Gly Ala Asp Gly Sen Phe NO -13- Gly Leu Leu Tyr Lys Gly Thr Val Gly Trp Gly Arg Asn Lys Thr 555 560 Leu Thr Gly Val Asn Ala Asp Ser Ala Glu Ile Ala Asn Gin Thr 595 Ser Trp Pro Thr Arg Gly Tyr 565 570 Leu Pro Giy Ser Lys Leu Gin 580 585 Tyr Tyr Ser Ala Thr His 590 beu Met Leu Trp Phe Phe Pro Leu 600 Ser Lys Thr Phe Gly Giy 610 Giu Val Gly Ile Gly Gly Tyr Gly Arg 620 Thr Lys Glu Ile Phe Phe Glu Asn Tyr Gly Gly Gly Gly Ser Vai Arg Tyr Giu Ser Gly Leu Giy Pro Lys Tyr Asp Giu Tyr Gly Glu 655 Lys Ile Ser Leu Phe Pro 675 Gly Gly Asn Lys Ala Asn Val Ser Ala Giu Leu 670 Arg Leu Ser Met Pro Giy Ala Asp Ala Axg Thr Leu Phe 690 Ala Asp Ala Gly Val Trp, Asp Giy Arg 700 Thr Tyr Thr Ala Ala 705 Giu Asn Giy Asn Lys Ser Val Tyr Ser 715 Giu Asn Ala His Ser Thr Phe Thr Asn 725 Giu Leu Arg Tyr Ser 730 Ala Gly Gly Ala Val Thr 735 Trp Leu Ser Lys Lys Lys 755 Pro 740 Leu Gly Pro Met Lys 745 Phe Ser Tyr Ala TIyr Pro Leu 750 Gin Leu Gly Pro Glu Asp Glu Ile 760 Gin Arg Phe Gin Phe 765 Thr Thr Phe 770 <210> <211> 2394 <212> DNA .<213> Neisseria nieningitidis <400> atgaaactga :gccgacttca gtattcaact atcatcaaaa gggcagctcc gcaaaaatgc tcgcaatact ctcgggcgcg aacagattgc ccatccaaga acctgcccgt gcctgtacgc tgctgaccgt tgcaaaacga ttaatcaggc gcaaactcaa ttccgcactg catccgcgtc caaagtcggc caccggtttc tatcgaacgc cgccattaag gacactcaat tatccaaatc atggtcttgg gaaggcttgc gacacctaca t ttgacgacg cccaccatcg aaaaacctcg caggcagtcg acgcccaaag gcatatcgcc agcgtaccga acgacacaca tacgcgtcga gctcgctcaa aatcgttcgg ccggcc tgaa taaccaaact tttggcactt gccgagtacc cggcagtgcc aactgcggac catcaccggc gctggcgcag agaagaatac cgcccgcaac cgcgtcgaca tttgaaggca ggcggcattt gacatggaaa gataccgaca gaaggcggac aaagccgaac atgaccgccg gaaatcagcg atcgaaccgg cgcgacgaag aagctgcaac gatgccgtcc cgttccaccg tccgcaggcg tcacgaagca gacggggtca accagcatca gttaccgaat tacaacaaag acagacggca accgacagcg c tgcccggca ttaagcaaaa agaaccaaag ggatacgaaa tacggcggca aaagacgcgc aaaacc tacg gccggcaata gttacctggc tcgacatcac accaagtcta ggacatggct aagtaaccga tccaaaccaa gtttccgttg tggaaaaact ttttgggtga tacagccgct gccggaaaat tcgtgcgccg gctccaaaga cgcttgccgg gctcgctcga tttcccaaga aaaccacgct gcc tgggcta aacaatataa acgaccgcgt cgcccaaaca gcttcaaagg cgt ta tggcc gcaaactgca ccttcacgct aaatcccctt gcggcacgct acaaaaaagc gcaccgtccg acgacaacag cc cat aaa tc tctcgccttt gattgacgag ttccgaccgc gacacgaagc cttctaccag cgaagacaaa gggcaaagtc gctgaccatg gattcagaac gccaaacgcc ctacgtcaac cgaattgcgc gcgcgtcgag cacacccgac cttgagcgcg caacctgttc caacggctcg cgatgtttac aaccaccacg gaatttcggt ctatgccgac ctggctgtac gacgcgcggc atactactcc gatgctcggc ctttgaaaac cggtccgaaa caacgtctcc cctgagcctg cagttccgcg cacctttacc aggcccgatg acgcttccaa ggcaaatccg aaactgatgc aaccaattca aacaacggc t accaagcaga tccatcgaag aagcccggca cgcatgggct gaaaccaaaa gaaatccaca caaatggaat cttttgggct aaagtcgatt ggc tgggtac ggtacgggca ctgtcgttta ggaaaagcc t gcaggcgcag ttggtggcag tttatcaaga aaaggtaccg tacctgacgg gccacccaca ggcgaagtcg ttctacggcg gtgtatgacg gccgagctgc t ttgccgacg accggcggca aacgaattgc aaattcagct ttccaactcg ccaaaatcac ggcagatgtc acgagcagaa acttcgattt ccatcaaaat gcgacaccaa aatggtacga cggcaggcta ccgtcgattt tcaccggcaa ccgcgcctta acttcgacaa tgaacatgag aggataccgg agtcggccgc ccgacccgta tcgacccgcg gcatccgcat aacacctgac aatacggcaa tcggc tgggg gcgtgaacgc accaaacctg gcattgcggg gcggcctggg aatacggcga tcttcccgat caggcagcgt gggttcaaaa gctattccgc acgcctaccc gcacgacgtt cgacatcgaa gctgaccgaa atttgcccaa ccgcatcctc caccgtccac cgaagtcccc acgccagcag cgcatacagc cgtcctgcac caacaaaacc cgacacctcc cgtacagttt cc tgaccgaa cctggtcatg cctgcgcgcc cttcacggca caaagcatcg gagcgtgcct cgtcaacacc aaccgacggc gcgcaacaaa cgaaatcgcc gttcttcccc cggctacggc ttcggtgcgc aaaaatcagc gcccggcgcg gtgggacggc catttacggc cggcggcgcg gctgaagaaa ctaa 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 aaaccggaag acgaaatcca <210> 11 <211> 797 <212> PRT <213> Neisseria meningitidis <400> 1i Met Lys Leu 1 Pro Leu Ala Leu Gin Arg Lys Gin 5 Ile Ala Ser Ala Leu 10 Met Val Leu Gly Ile Ser Leu Ala Asp Phe Thr Ile Gin Asp Ile Arg 25 Val Glu Gly Pro Val Lys Thr Glu Pro Ser Thr Val Phe Asn Tyr Val Gly Asp Thr Tyr Asn Thr His Gly Ser Ala Ile Ile Lys Ser Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Giu Thr Ala Gly Gin Leu Leu Thr Val Ile Glu Arg Pro Thr Ile Gly Ser Leu Asn Ile Thr Gly 100 Ala Lys Met Leu Gin 105 Asn Asp Ala Ile Lys Lys Asn Leu Giu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr 115 120 125 Leu Asn 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 Glu 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 Gly Gly Ile Ti-p Thr Trp, Leu Th- 195 200 205 Arg Ser Asn Gin Phe Asn Giu Gin Lys 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 Lys Gin Thr Ile Lys 245 250 255 Ile Thr Val His Giu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile 260 265 270 Giu Gly Asp Thi- Asn Giu Val Pro Lys Ala Giu Leu Giu Lys Leu Leu 275 280 285 Thr Met Lys Pro Giy Lys Ti-p Tyr Giu Arg Gin Gin Met Thr Ala Val 290 295 300 Leu Gly Giu Ile Gin Asri 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 Lys Ile Tyr Val Asn Giu Ile 340 345 350 His Ile Thr Gly Asn Asn Lys Thi- Arg Asp Giu Val 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 Thi- Pro Asp Lys Val Asp Leu Asn Met 405 410 415 Ser Leu Thi- Glu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp 420 425 430 -16- Val Gin Asp 435 Thr Gly Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn Leu Thr 465 Asp Arg Ala Phe Pro 545 Thr Gly Thr Tyr Phe 625 Arg Gly Asp Val Thr 705 Lys Phe 450 Thr Gly Lys Gly Gly 530 Lys Asp Arg Gly Ser 610 Thr Thr Ser Glu Ser 690 lal ['hr Gly *Leu *Val Ala Ile 515 Leu His Gly Asn Val 595 Ala Leu Lys Val Tyr 675 Ala Arg I Tyr Thr Asr Ser Ser 500 Arg Val1 Tyr Ser Lys 580 Asn Thr M1et Glu krg 660 31y 31u ~eu ~sp Gly Gly *Leu 485 *Thi- Met Ala Ala Phe 565 Thr Ala His Leu Ile 645 Gly Glu Leu Ser Asp 725 Lys Ser 470 Gly Ser Ser Giu Asp 550 Lys Asp Giu Asn G ly 630 Pro Tyr Lys Leu Leu 710 Asn Ser 455 *Leu Tyr Sle Val His 535 Phe Gly Ser Ile Gin 615 Gly Phe Giu Ile Phe 695 Phe Ser AiE Sex AsE Lys Pro 520 Leu Ile Ti-p Ala Ala 600 Th- Giu Phe Ser Ser 680 Pro Ala Ser Ala Phe Val Gin 505 Val Thi- Lys Leu Leu 585 Leu Trp Val Giu Gly 665 Tyr Met Asp Ser Leu Thi- Tyr 490 Tyr Thr Val1 Lys Tyr 570 Ti-p Pro Phe Gly Asn 650 Thi- Gly Pro Ala Ala 730 Arg Asp 475 Gly Lys Giu Asn Tyr 555 Lys Pro Gly Phe Ile 635 Phe Leu Gly Gly Giy 715 Thi- Ala 460 Pro Lys Th- Tyr Thi- 540 Gly Gly Thr Ser Pro 620 Ala Tyr Gly Asn Ala 700 Ser Gly 445 Ser Tyr Ala Thr Asp 525 Tyr Lys Thr Arg Lys 605 Leu Gly Gly Pro Lys 685 Lys Val Gly Arg Phe Phe Thr 510 Arg Asn Thr Vai Gly 590 Leu Ser Gly Gly Lys 670 Lys Asp rrp Arg Ser Thr Asp 495 Ala Val Lys Asp Gly 575 Tyr Gin Lys Tyr Gly 655 Val1 Al a Ala Asp Val Lys Ala 480 Pro Gly Asn Ala Gly 560 Ti-p Leu TPyr Thr Gly 640 Leu Tyr Asn Arg Gly 720 Gin 735 Asn Ile Tyr Ala Gly Asn Thi- His Lys Ser Thi- Phe 745 Thr Asn Giu 750 Lau Arg Pro Met 770 Glu Ile 785 <210> <211> <212> <213> <400> Met Lys 1 Pro Leu <210> <211> <212> <213> <400> GI -17- Ser Ala Gly Gly Ala Vai Thr Trp Leu. Ser Pro Leu Gly 760 765 Phe Ser Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp 775 780 Arg Phe Gin Phe Gin Leu Gly Thr Thr Phe 790 795 12 21 PRT Neisseria ineningitidis 12 Leu Lys Gin Ile Ala Ser Ala Leu Met Val Leu Gly Ile Ser 5 10 Ala Leu Ala 13 776 PRT Neisseria. meningitidis 13 Asp Phe Thr Ile Gin Asp Ile Arg Val Giu. Gly Leu Gin Arg Thr Giu. 1 Pro Asn Phe Thr Lys Leu Ala Ile Ile 145 Ser Asp Phe Val1 Met Ala Gly Thr 130 Thr Thr Thr Asp Ile Leu Gin Leu Pro Ile Val1 His Asp Glu Gin Ser 100 Lys Lys Asp 5 Phe Gly Val Arg Asn Gin Giu Val Glu. Asn Ser Arg Pro 70 Asp Tyr Glu Thr Gly Tyr Ala Val 55 Thr Ala Phe Tyr Lys 135 Lys Leu Ile 40 Giu Ile Ile Asn Leu 120 Lau Ser Pro Val 25 Ile Lys Thr Ala Gly Ser Lys Lys Gin Ala 105 Gly Arg Ala Arg Ala Lys Lys Ser Asp Leu 75 Asn Thr Gly Asn Ile 155 Val Leu Gly Asn Leu Leu Lys Arg 140 Thr Giy Gin Ile Giu Asn Lau 125 Val Asp Asp Ala Lau Thr Ser Gin 110 Asn Asp Ile Tyr Gly Leu Ala Gly Val Gin Asp Phe 150 Glu Gly Asn Gin Vai Tyr Ser Asp Arg Lys Leu Met Arg Gin Met Ser Leu Thr Giu Gly Gly Ile Trp, Thr 180 Asn Gin Thr 225 Gay Glu Lys Asn Pro 305 Glu Asn Ser Glu Ala 385 Ser Leu Lys Ser Gly 465 Glu Asn 210 Asn Gly Vali Trp Arg 290 Leu Pro Lys Ala Leu 370 Gly Thr Val1 Ser Leu 450 T'yr IGin 195 IAsn Giu Arg Pro Tyr 275 Met Pro Gly Thr Pro 355 Leu Thr Giy Met Ala 435 Ser Asp Lys Gly Asp Phe Lys 260 Giu Gly Asn Arg Arg 340 Tyr Gly Pro Ser Ser 420 Phe Ja 1 Phe Tyr Lys Arg 245 Ala Arg Ser Ala Lys 325 Asp Asp Tyr Asp Leu 405 Ala Leu Thr Tyr Ala Phe Thr 230 Trp Giu Gin Ala Giu
[13] 310. Ile Giu Thr Phe Lys 390 Asp Gly Arg Asp Gly 470 Gin Asp 215 Lys Gly Leu Gin Giy 295 Thr Tyr Val1 Ser Asp 375 Val Leu Val Ala Pro 455 Lys Asp 200 Phe Gin Lys Giu Met 280 Tyr Lys Val Val Lys 360 Asn Asp Ser Ser Ser 440 Tyr Ala Trp Leu 185 Met Giu Arg Ile Thr Ile Val Ser 250 Lys Leu 265 Thr Ala Ala Tyr Thr Val Asn Giu 330 Arg Arg 345 Leu Gin Val Gin Leu Asn Ala Gly 410 Gin Asp 425 Arg Ser Phe Thr Phe Asp Thr Lys Leu Lys 235 Ile Leu Val Ser Asp 315 Ile Giu Arg Phe Met 395 Trp Asn Lys Ala Pro 475 Arg Val1 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 Giy 285 Ile Val Ile Arg Lys 365 Ala Leu Gin Phe Thr 445 Giy Lys Asn 190 Asp Asp Val1 Asp Lys 270 Giu Ser Leu Thr Gin 350 Giu Val Thr Asp Giy 430 Leu Val1 Ala Gin Phe Ile His Thr 255 Pro Ile Val1 His Gly 335 Met Arg Pro Giu Thr 415 Thr Asn Ser Ser Phe Tyr Gin Giu 240 Asn Gly Gin Gin Ile 320 ASn Glu Val Leu Arg 400 Gly Gly Gly Leu Thr Ser Ile Lys Gin Tyr Lys Thr Thr Thr Ala Giy Ala Gly Ile Arg Met -19- Ser Val Pro Val Thr Giu Tyr Asp 500 Giu Asp Lys 545 Asp Glu Asn Gly Pro 625 Tyr Lys Leu Leu Asn 705 Gly Gly Tyr Gin His Phe 530 Gly Ser Ile Gin Gly 610 Phe Giu Ile Phe Phe 690 Ser Asn klia he Leu 515 Ile Trp Ala Ala Thr 595 Giu Phe Ser Ser Pro 675 Ala Ser Thr Ala Tyr 755 Gin Thr Lys Leu Leu Leu 580 Trp Val1 Glu Gly Tyr 660 Met Asp Ser His Val 740 Pro Leu Val1 Lys Tyr Trp 565 Pro Phe Gly Asn Thr 645 Gly Pro Ala Ala Lys 725 Thr Leu Gly Asn Tyr Lys 550 Pro Gly Phe Ile Phe 630 Leu Gly Gly Gly Thr 710 Ser Trp Lys Thr Thr Gly 535 Gly Thr Ser Pro Ala 615 Tyr Gly Asn Ala Ser 695 Gly Thr Leu Lys Thr 775 Tyr 520 Lys Thr Arg Lys Leu 600 Gly Gly Pro Lys Lys 680 Val1 Gly Phe Ser Lys 760 Phe Arg Val 505 Asn Lys Thr Asp Val Gly Gly Tyr 570 Leu Gin 585 Ser Lys Gly Tyr Gly Gly Lys Val 650 Lys Ala 665 Asp Ala Trp Asp Arg Val Thr Asn 730 Pro Leu 745 Asn Phe Ala Pro Gly Thr 540 Trp Giy 555 Leu Thr Tyr Tyr Thr Phe Giy Arg 620 Leu Gly 635 Tyr Asp Asn Val Arg Thr Giy Lys 700 Gin Asn 715 Giu Leu Giy Pro Gly Leu 510 Lys His 525 Asp Gly Arg Asn Gly Val Ser Ala 590 Thr Leu 605 Thr Lys Ser Val Giu Tyr Ser Ala 670 Val Arg 685 Thr Tyr Ile Tyr Arg Tyr Met Lys 750 Val Ala Tyr Ala Ser Phe Lys Thr 560 Asn Ala 575 Thr His Met Leu Giu Ile Arg Gly 640 Gly Glu 655 Giu Leu Leu Ser Asp Asp Gly Ala 720 Ser Ala 735 Phe Ser Pro Giu Asp Glu Ile Gin Arg Phe <210> 14 <211> 441 <212> PRT <213> Neisseria meningitidis <400> 14 Met Lys Lys Tyr Leu Phe Arg Ala Ala Leu Tyr Gly Ile Ala Ala Ala 1 5 10 Ile Leu Ala Ala Cys Gin Ser Lys Ser Ile Gin Thr Phe Pro Gin Pro 25 Asp Thr Ser Val Ile Asn Gly Pro Asp Arg Pro Val Gly Ile Pro Asp 40 rN Pro Ala Gly Thr Thr Val Gly Gly Gly Giy Ala Val Tyr Thr Val Val 55 Pro His Leu Ser Leu Pro His Trp Ala Ala Gin Asp Phe Ala Lys Ser 70 75 Leu Gin Ser Phe Arg Leu Gly Cys Ala Asn Leu Lys Asn Arg Gin Gly 90 Trp Gin Asp Val Cys Ala Gin Ala Phe Gin Thr Pro Val His Ser Phe 100 105 110 Gin Ala Ljys Gin Phe Phe Giu Arg Tyr Phe Thr Pro Trp Gin Val Ala 115 120 125 Giy Asn Gay Ser Leu Ala Gly Thr Val Thr Gly Tyr Tyr Giu Pro Val 130 135 140 Leu Lys Gly Asp Asp Arg Arg Thr Ala Gin Ala Arg Phe Pro Ile Tyr 145 150 155 160 Gly Ile Pro Asp Asp Phe Ile Ser Val Pro Leu Pro Ala Gly Leu Arg 165 170 175 Ser Gly Lys Ala Leu Val Arg Ile Arg Gin Thr Gly Lys Asn Ser Gly 180 185 190 *Thr Ile Asp Asn Thr Gly Gly Thr his Thr Ala Asp Leu Ser Arg Phe 195 200 205 Pro Ile Thr Ala Arg Thr Thr Ala Ile Lys Gly Arg Phe Giu Gly Ser 210 215 220 Arg Phe Leu Pro Tyr His Thr Arg Asn Gin Ile Asn Gly Gly Ala Leu 225 230 235 240 Asp Gly Lys Ala Pro Ile Leu Gly Tyr Ala Giu Asp Pro Val Giu Leu 245 250 255 Phe Phe Met His Ile Gin Giy Ser Gly Arg Leu Lys Thr Pro Ser Gly 260 265 270 Lys Tyr Ile Arg Ile Gly Tyr Ala Asp Lys Asn Glu His Pro Tyr Val 275 280 285 Ser Ile Gly Arg Tyr Met Ala Asp Lys Gly Tyr Leu Lys Leu Gly Gin 290 295 300 Thr Ser Met Gin Gly Ile Lys Ala Tyr Met Arg Gin Asn Pro Gin Arg 305 310 315 320 -21 Leu Ala Glu Val Leu Ala Gly Ser 340 Leu Met Gly Glu 355 Gly Ala Pro Leu Le u 325 Ser Gly Gin Asn Pro Asn Asp Gly Se r 330 Val1 Asp Tyr IIle Phe Phe Arg Glu 335 Gly Ala Leu Gly Thr Pro 350 Arg His Tyr Ile Thr Leu Tyr Ala Gly 365 Thr Phe Val 370 Leu Asn Ala 375 Ala Ala His Pro Val1 380 Ser Arg Lys Ala Arg Leu Ile Gin Asp Thr Ala Ile Lys Gly 400 Val Arg Val Asp 405 Gin Phe Trp Gly Tyr 410 Tyr Asp Glu Ala Gly Glu 415 Leu Pro Le'u Ala Gly Asn Gly Met 435 Lys 420 Lys Lys Thr Thr Val Trp Gin Pro Glu Tyr Arg 440 <210> <2 11> 736 <212> DNA <213> Neisseria meningitidis (W09936544-0001) <400> acactgttgt ttgcaacggt atatttagac cccgtacaac cacgggagaa aaagaaaaag aggagtacta acagccagag aaacggcaca aacttcacct aactgaaaaa ttatcgttta aggcttgaat tttgcgaaag cggtattggt tcgactttga caacgacaac gttaccgatg cgdtggctgg aacattaaag cgtccgcact tacgacacag tgtggaaagc aaagacaacg tattaaagaa aaagac tcaggcaagt gcactgttgc tagaagaaaa aaatcaccyt actcgctgaa g ogoa aa cgg aaacggctgg ccgatacgct acgagaaaaa gcgttaaacc tcgagttctt gcaagaaaac gctaaccaat cgtgttgata ttcagattgg caaagccggc aaaagacctc caataaagtc sacgaacggc gctgaatacc acgtgcggca cggtacaaca gagcgcagat cgaagttaaa gaaqagcaag gtcaattccg gcagtatatt gacaacctga acagatctga aacatcacaa gacaccacgg ggagcgacca agcgttaaag gcttccgata acgaaaacaa atcggtgcga aagaagattt 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> 16 245 PRT 22 <213> Neisseria meningitidis (W09936544-0002) <220> <221> misc feature <222> (70)7..(70) <223> Xaa can be any naturally occurring amino acid <400> 16 Thr Leu Leu Phe Ala Thr Val Gin Ala Ser Ala Asn Gin Glu Giu Gin NO) Glu Glu Asp Ile Val Asn Glu Asn Ser Leu Tyr Leu Asp Pro Gin Arg Thr Val Ala Val Leu Lys Val Glu Ser Asp Lys Glu Asp Trp Ala Val Gly 40 Thr Giy Giu Lys Glu Tyr Phe Asn Giu Gly Val Leu Thr Ala Arg Giu Ile Thr Lys Ala Gly Asp Asn Leu Lys Ile Lys Asn Gly Thr Asn Phe Thr Tyr Ser Leu Lys Asp Leu Thr Asp Leu Thr Ser Val Val Asn Ile 115 Gly 100 Thr Giu Lys Leu Phe Ser Ala Asn Gly Asn Lys 110 Lys Giu Thr Thr Ser Asp Thr Lys 120 Gly Leu Asn Phe Ala 125 Aia Gly 130 Thr Asn Gly Asp Thr Val His Leu Gly Ile Gly Ser Thr 145 Leu Thr Asp Thr Leu 150 Leu Asn Thr Gly Ala 155 Thr Thr Asn Val Thr 160 Asn Asp Asn Val Asp-Val Leu Asn 180 Thr Ala Ser Asp 195 Thr 165 Asp Asp Giu Lys Lys 170 Arg Ala Ala Ser Val Lys 175 Ala Gly Trp Asn Lys Gly Val Lys Pro Gly Thr 190 Thr Val Glu. Asn Val Asp Phe 200 Val Arg Thr Tyr Asp 205 Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys 23 z 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 tataccgcat catttggaat agtgccctca atgcctgggt cgtcgtatc No gagctcacai acactgttgl tatttagac( acgggagaai ggagtactaz aacggcacaz actgaaaaat ggcttgaatt ggtattggtt aacgacaacg gctggctgga gtccqcactt gtggaaagca attaaagaaa acagacgaag ggttggagaa accgttacat agtaaagatg aacgtcaatc tcgggcaaag aacattaatg cgcaaccacac t ttgcaacggt ccgtacaac i aagaaaaagt icagccagaga iacttcaccta tatcgtttag ttgcgaaaga cgactttgac ttaccgatga acattaaagg acgacacagt aagacaacgg aagacggtaa gcgaaggctt tgaaaacaac caggcacaaa atcaaggcaa agctgcaaaa tcatcagcgg ccggcaacaa caaacgcgcc tcaggcaagt fcactgttgcc agaagaaaat *aatcaccctc ctcgctgaaa cgcaaacggc aacggctggg cgatacgctg cgagaaaaaa cgttaaaccc cgagttcttg caagaaaacc gttggttact agtgactgca aaccgctaat tgtaaccttt catcactgtt cagcggttgg caatgtttcg catcgagatt tccgcaaccg gctaacaatg gtgttgatag tcagattggg aaagccggcg aaagacctca aataaagtca acgaacggcg ctgaataccg cgtgcggcaa. ggtacaacag agcgcagata gaagttaaaa ggtaaagaca aaagaagtga ggtcaaacag gctagtggta atgtatgatg aatttggatt ccgagcaagg acccgcaacg tgaagaccgc aagagcaaga tcaattccga. cagtatattt acaacctgaa. cagatctgac acatcacaag acaccacggt gagcgaccac gcgttaaaga. cttccgataa. cgaaaacaac tcggtgcgaa aaggcgagaa ttgatgcagt gtcaagctga. aaggtacaac taaatgtcgg ccaaagcggt gaaagatgga gtaaaaatat cgtattggcg agaagattta taaagaaggc caacgagaaa. aatcaaacaa cagtgttgga cgacaccaaa tcatctgaac aaacgtaacc cgtattaaac cgttgatttc gactgttaat gacttctgtt tggttcttct aaacaaggct caagtttgaa tgcgactgta cgatgcccta tgcaggttct tgaaaccgtc cgacatcgcc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 acttcgatga ccccgcagtt ttccagcgtt tcgctcggcg cgggggcgga tgcgcccact 24 ttgagcgtgg a ttaccaatg ggcgtggcgc atcgcccaag atggcgatcg atttccgacg ttcggtgctt a tggggacgc tcgccccggg aaaacttgaa cgattgcaac gcggcggcac gcggaaattg ccgcatctgt attgaatgtc cgttaaagag caaccgcatc cgcaggtctg ttatcgcggc gattatcaaa cggttatcag ggcagcaaga ggggatgtta qacaatgtgg gttcaggcgt gaagccggtt ggcacggctt tggtaa aggacaacaa caaacgtcgc acggcaacgc atttgcccgg acgccatcgg ccggcaattc acccgtccgc acaacttaaa gcgtgcgggc caagagtatg ctactccagt gcgcggccat 1440 1500 1560 1620 1680 1740 1776 <2.10> 18 <211> 591 <212> PRT <213> Neisseria meningitidis (W09936544-0004) <400> 18 Met Asn Lys Ile Tyr Arg Ile Ile 1 Val Val Val Ser Glu Leu Thr Arg Trp Asn Ser Ala Leu Asn Ala Trp Asn His 25 Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Val Lys Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Ser Ala Asn Asn Giu Giu 55 Gin Giu Giu Asp Leu Tyr Leu Asp Pro Ser Asp Lys Giu Gly Val1Gin Arg Thr Vai Ala 70 Val Leu Ile Val Asn Thr Gly Giu Lys Lys Val Giu Giu Ser Asp Trp Ala Val Tyr Phe Asn Giu Lys 100 Gly Val Leu Thr Ala 105 Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Gly Asp Asn Leu 115 Lys Ile Lys Gin 120 Asn Gly Thr Asn Phe 125 Leu Lys 130 Lys Asp Leu Thr Asp Leu 135 Thr Ser Val Gly 140 Thr Giu Lys Leu Ser 'Phe Ser Ala Asn Gly 145 150 Asn Lys Val Asn Ile Thr Ser Asp Thr 155 Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Gly Thr Asn Giy Asp Thr Thr 170 175 Val His Leu Thr Gly Ala 195 Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 185 190 Thr Thr Asn Val Asn Asp Asn Val Thr 205 Asp Asp Glu Lys Lys 210 Arg Ala Ala Ser Lys Asp Val Leu Ala Gly Trp Asn Ile 225 Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Ser 235 Asp Asn Val Asp Val Arg Thr Tyr Asp 245 Thr Val Giu 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 Ile Lys Glu Lys Val Thr 290 Gly Lys Asp Lys Gly 295 Glu Asn Gly Ser Ser Thr Asp Glu 300 Ala Val Asn Lys Gly Ala 320 Gly Leu Val Thr Ala 310 Lys Glu Val Ile Asp 315 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 Met 370 Glu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val1 360 Ser Lys Asp Asp Gin 365 Tyr Asp Val Asn 375 Val Gly Asp Ala Leu 380 Asn Val Asn Gin Leu Gin Asn Ser Gly Trp Asn 385 390 Leu Asp Ser Lys 395 Ala Val Ala Gly Ser Gly Lys Val Ile Ser Gly Asn Vai 405 Ser 410 Pro Ser Lys Gly Lys Met 415 26 Asp Glu Thr Val Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Thr Arg NO Asn Gly Lys 435 Asn Ile Asp Ile Thr Ser Met Thr Pro 445 Gin Phe Ser Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Aia Pro Leu Ser Vai Asp Gly 465 Asp Ala Leu Asn Gly Ser Lys Lys Asp 475 Asn Lys Pro Val Arg 480 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Giy Asp Val Thr Asn Vai 495 Ala Gin Leu Val Asp Gly 515 Giy 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 Aia Tyr Leu 535 Pro Gly Lys Ser Met Met Ala Ile Giy 540 Giy 545 Gly Thr Tyr Arg Gly 550 Giu Ala Gly Tyr Ile Giy Tyr Ser IlieSer Asp Gly SerArg Gly His 580 Gly Asn 565 Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Phe Gly Ala Ser Ala 585 Ser Val Gly Tyr Gin Trp 590 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <22 3> <220> <22 1> 19 1779 DNA Neisseria meningitidis (W09936544-0005) misc feature n is a, C. g, or t misc feature (420) (420) n is a, C, g, or t misc feature 27 <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <2'21> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> (608) (608) n is a, c, g, or t misc feature (682) .(684) n is a, C, g, or t misc feature (7 63) (763) n is a, c, g, or t misc feature (774) (774) n is a, C, g, 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 tataccgcat gagctcacac gcaaccacac acactgttgt ttgcaacggt gaatccgtac aacgctctgt ttggaaacga tatcattatc gtagttaccc tcaaagccgg aatgccagta gcttcaccta actgaaaaat tatcgtttgg ggcttgaatt tcgcgaaaga ggtatcggtt cgactttgac ggtaaccnaa gtacacatta tggaatatta agggtgttaa gtccgcactt acgacacagt gtggaaagca aagacaacgg attaaagaaa aagacggtaa catttggaat caaacgcgcc tcaggcgaat cgtagggagc aatgactaac cgacaacctg ctcgctgaaa cgcaaacggc aacggctggg cgatacgctt cactcgtgca annnggctca cgagttcttg caagagaacc gttggttact agtgccctca tccgcaaccg gctaccgatg attcaagcca gacagcaagg aaaatcaaac aaagacctca aagaaagtca acgaacggcg gcgggttctt gcaagtatta acaactggtc agcgcagata gaagttaaaa ggtaaaggca atgcctgngt tgaagaccgc aaqatgaaga gtatggaagg aatttgtaga aaaacaccaa caggcctgat acatcataag acaccacggt ctgcttctca aggatgtgtt aatcagaaaa cgnaaacaac tcggtgcgaa aaggcgagaa cgccgtatcc cgtattggcg agaagagtta cagcggcgaa cccatacata tgaaaacacc caatgttgan cgacaccaaa tcatctgaac cgttgatgcg gaatgcgggt tgtcgatttc gacngttaat gacttctgtt tggttcttct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 28 acagacgaag qcgaaggctt agtgactgca aaagaagtga ttgatgcagt aaacaaggct ggt tqgaqaa accgttacat agtaaagatg aacgtcaatc tcgggcaaag aacattaatg acttcgatgg ttaagcgtg cgcattacca aaaggcgtgg ggcatcgccc atgatggcga agtatttccg catttcggtg *tgaaaacaac caggcacaaa atcaaggcaa agctgcaaaa tcatcagcgg ccggcaacaa cgccgcagtt atgacgaggg atgtcgcccc cgcaaaactt aagcgattgc tcggcggcgg acggcggaaa cttccgcatc aaccgctaat tgtaaccttt catcactgtt cagcggttgg caatgtttcg catcgagatt ttccagcgtt cgcgttgaat gggcgttaaa gaacaaccgc aaccgcaggt cacttatcgc ttggattatc tgtcggttat ggtcaaacag gctagtggta atgtatgatg aatttggatt ccgagcaagg agccgcaacg tcgctcggcg gtcggcagca ganggggatg atcgacaatg ctggttcagg ggcgaagccg aaaggcacgg cagtggtaa gtcaagctga aaggtacaac taaatgtcgg ccaaagcggt gaaagatgga gtaaaaatat cgggggcaga aggatgccaa ttacaaacgt tggacggcaa cgtatctgcc gttacgccat cttccggcaa caagtttgaa tgcgactgta cgatgcccta tgcaggttct tgaaaccgtc cgacatcgcc tgcgcccact caaacccgtc cncacaactt cgcgcgtgcn cggcaagagt cggctactcc ttcgcgcggc 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> <211> <212> <213> 592 PRT Neisseria meningitidis (W09936544-0006) <220> <221> <222> K <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 naturally occurring amino acid naturally occurring amino acid naturally occurring amino acid misc feature (228)..(228) Xaa can be any naturally occurring amino acid misc feature (255)..(255) Xaa can be any naturally occurring amino acid -29- <220> <221> <222> <223> misc_ feature (4 91) (4 91) Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (4 98) (4 98) <223> Xaa can be any naturally occurring amino acid <400> Met Asn Lys Ile Tyr Arg Ile Ile NO) Trp Asn Ser Ala Leu. Asn Ala Xaa 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 Thr Leu Leu Phe Ala Asn Ala Thr Asp Giu Asp Glu Giu Giu Giu 55 Leu Giu. Ser Val Gin Arg Ser Val Val Gly Leu Giu Thr Ile Ser Ile Gin Ala Ser Met 75 Glu. Gly Ser Gly Glu Leu Ser Met Thr Asn 90 Asp Ser Lys Giu Phe Val Asp Pro Tyr Lys Gin Asn 115 Val Val Thr Leu. Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Glu Asn Th r 120 Asn Ala Ser Ser Leu Lys 130 Lys Asp Leu Thr Leu Ile Asn Val Thr Glu Lys Leu Ser 145 Phe Gly Ala Asn Gi y 150 Lye Lye Vai Asn Ile 155 Ile Ser Asp Thr Gly Leu Asn Phe Val His Leu Asn 180 Ser Ser Ala Ser 195 Ala 165 Lye Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Gly Ile Gly Ser Th r 185 Leu Thr Asp Thr Leu. Ala Gly 190 His Tyr Thr His Val Asp Ala Gly Asn Xaa Ser 200 Thr 205 30 Arg Ala Ala Ser Ilie Lys Val Leu Asn Ala Trp Asn Ile Lys Gly 225 Val Lys Xaa Gly Ser 230 Thr Thr Gly Gin Ser 235 Glu Asn Val Asp Phe 240 Val Arg Thr Tyr Asp 245 Thr Val Glu Phe Ser Ala Asp Thr Xaa Thr 255 Thr Thr Val Lys Ile Gly 275 Val Glu Ser Lys Asp 265 Asn Gly Lys Arg Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Gly Lys Gly 295 Giu Asn Gly Ser Thr Asp Glu Gly Glu 305 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 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giu Thr Val Thr Ser Gly 340 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Gly Lys Gly 355 Thr Thr Ala Thr Ser Lys Asp Asp Gin 365 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 Gly Ser 400 Ser Gly Lys Val Ile 405 Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Val 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Ser Arg 430 Asn Gly Lys Asn Ile Asp Ile Ala 435 440 Ser Val Ser Leu Gly Ala Gly Ala 450 455 Thr Ser Met Ala Pro Gin Phe Ser 445 Asp Ala Pro Thr 460 Leu Ser Val Asp -31!- Asp Giu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val 465 470 d7rap Arg Ile Thr Asn Val Ala Pro Gly Val 485 Xaa Gly Asp Val Thr Asn 495 Val Xaa Gin Lys Gly Val Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr NO Asn Val Asp 515 Giy Asn Ala Arg Gly Ile Ala Gin Ala Giy 530 Leu Val Gin Ala Tyr 535 Leu Pro Giy Lys Ser 540 Met Met Ala Ile Gly 545 Ser Gly Gly Thr Tyr Arg 550 Gly Giu Ala Giy Ala Ile Gly Tyr Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Giy 575 Asn Ser Arg Gly 580 His Phe Gly Ala Ser 585 Ala Ser Val Gly Tyr Gin Trp 590 <210> <211> <212> 21 591 PRT <213> Neisseria meningitidis (W00066741-0001) <400> 21 Met Asn Lys Ile Tyr 1 5 Arg Ile Ile Trp Asn Ser Ala Leu Asn 10 Ala Trp Val Val Val Thr Val Lys Ser Giu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Ala 40 Thr Leu Leu Phe Ala Ala Ser Val Gin Ala Asn Asn Giu Arg Thr Val Ala 70 Glu 55 Gin Giu Glu Asp Leu Tyr Leu Asp Pro Ser Asp Lys Giu Gly Val Leu Ile Val Asn Thr Gly Giu Lys Glu Lys Val Glu Giu Asn Ser Asp Trp Ala Val Tyr 32 Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile Lys Gin 120 Asn Gly Thr Asn Thr Tyr Ser Leu Lys 130 Lys Asp Leu Thr Asp 135 Leu Thr Ser Val Gly 140 Thr Glu Lys Leu Cl) Ser 145 Phe Ser Ala Asn Asn Lys Val Asn Thr Ser Asp Thr Lys 160 Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Gly Ile Gly Ser Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Glu Thr Gly Ala 195 Thr Thr Asn Val Thr 200 Asn Asp Asn Val Th r 205 Lys Lys 210 Arg Ala Ala Ser Val1 215 Lys Asp Val Leu Ala Gly Trp Asn Lys Gly Vai Lys Giy Thr Thr Ala Ser 235 Asp Asn Val Asp Phe 240 Vai Arg Thr Tyr Thr Vai Giu 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 Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Val1 280 Ile Lys Glu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Ser 300 Thr Asp Giu Gly Glu Gly Leu Val Thr 305i 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 Thr Val Thr As Ls heGu hrVa TrSer Giy Thr Asn Val Thr Phe Ala Ser 33 340 Giy Lys Gly Thr Thr Ala Thr Ser Lys Asp Asp Gly Asn Ile Thr Val 370 Leu Gin 385 Met Tyr Asp Val As n 375 Val Gly Asp Ala Leu 380 Asn Val Asn Gin Asn Ser Gly Trp 390 Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser 400 Ser Gly Lys Val 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 Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Ser Val 450 Gly Asp 465 Ser Leu Giy Ala Gly 455 Ala Asp Ala Pro Thr 460 Leu Ser Val Asp Ala Leu Asn Gly Ser Lys Lys Asn Lys Pro Val Arg 480 Ile Thr Asn Val Ala 485 Pro Gly Val Lys Giu 490 Gly Asp Val Thr Asn Vai 495 K. Ala Gin Leu Val Asp Gly 515 Lys 500 Gly Val Ala Gin Asn 505 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 Leu 535 Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Gly 550 Giu Ala Giy Tyr Ala 555 Ile Gly Tyr Ser Ser 560 Ile Ser Asp Gly Gly Asn 565 Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Gly His 580 Phe Giy Ala Ser Ala Ser Val Giy Tyr 585 Gin Trp 590 34 <210> 22 <211> 591 <212> PRT <213> Neisseria meningitidis (WO006674i-0002) <400> 22 Met Asn Lys Ile Tyr Ar Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Lys Ala Ser Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Thr Val Gin Asn Asn Glu Gin Giu Glu Asp Leu Tyr Leu Asp Pro Val Gin Arg Thr Val Thr Gly Giu Lys Glu Val Leu Ile Val Ser Asp Lys Glu Lys Val Giu Glu Asn 90 Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Asp Asn 115 Lys 100 Gly Val Leu Thr Ala 105 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 Asp Leu Thr Ser Val 135 Thr Glu Lys Leu Ser 145 Phe Ser Ala Asn Gly 150 Asn Lys Val Asn Ile 155 Thr Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Asn Gly Ile Gly Ser 180 Thr 185 Leu Thr Asp Thr Thr Gly Ala Thr 195 Thr Asn Val Thr Asn 200 Asp Asn Val Thr 205 Leu Leu Asn 190 Asp Asp Glu Gly Trp Asn Lys'Lys Arg Ala Ala Ser Val 210 215 Lys Asp Val Leu Asn Ala 220 35 Ile Lys 225 Gly Val Lys Pro Gly 230 Thr Thr Ala Ser Asp Asn Val Asp 235 Val Arg Thr Tyr Thr Val Glu Phe Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 As n 260 Val Giu Ser Lys Asp 265 Asn Gly Lys Lys Thr Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Giy Ser Thr Asp Giu Gly Glu 305 Gly Leu Val Thr Ala 310 Lys Glu Vai Ile Asp 315 Ala Val Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giy Lys Giy 355 Giu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Giy Asn Ile Thr Thr Ala Thr Ser Lys Asp Asp Thr Val 370 Met Tyr Asp Vai Asn 375 Val Giy Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Giy T rp 390 Asn Leu Asp Ser Ala Val Ala Gly Ser Gly Lys Val Ser Gly Asn Val Pro Ser Lys Giy Lys Met 415 Asp Glu Thr Asn Giy Lys 435 Vali 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Pro 445 Ser Val 450 Ser Leu Giy Ala Gly 455 Ala Asp Ala Pro Thr Leu Ser Vai Asp 460 Gly Asp Ala Leu Asn Val 465 470 Gly Ser Lys Lys Asp 475 Asn Lys Pro Vai Arg 480 Ile Thr Asn Val Ala Pro Gly Val Lys Giu 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 Leu Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Glu Ala Gly Tyr Ala Ile Gly Tyr Ser 555 Ile Ser Asp Gly Gly 565 Asn Trp Ile Ile Lys 570 Gly Thr Ala Ser Gly Asn 575 Ser Arg Gly <210> 23 <211> 591 <212> PRT Hius Phe 580 Gly Ala Ser Ala Ser Val Gly Tyr Gin Trp 585 590 <213> Neisseria meningitidis (W00066741-0003) <400> 23 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val 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 Thr Leu Leu Phe Ala Ser Val Gin Ala Asn Asn Giu Gin Glu Giu Asp Leu Tyr Leu Asp Pro Arg Thr Val Ala 70 Val Leu Ile Val As n Ser Asp Lys Giu Thr Gly Giu Lys Giu Lys Val Giu Val Leu Thr Glu Asn 90 Ala Arg 105 Ser Asp Trp Ala Giu Ile Thr Leu 110 Val Tyr Lys Ala Phe Asn Giu Lys Gly 100 -37- Gly Asp Asn Leu Lys 115 Ile Lys Gin 120 Asn Gly Thr Asn Phe Thr Tyr Ser 125 Thr Glu Lys Leu Leu Lys 130 Lys Asp Leu Thr Asp Leu 135 Thr Ser Val Gly 140 Phe Ser Ala Asn Gly 150 Asn Lys Val Asn Thr Ser Asp Thr Gly Leu Asn Phe Lys Glu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Gly Ile Gly Ser Leu Thr Asp Thr Leu Leu Asn 190 Thr Gly Ala 195 Thr Thr Asn Val Asn Asp Asn Val Asp Asp Glu Lys Lys 210 Arg Ala Ala Ser Val1 215 Lys Asp Val Leu Ala Gly Trp Asn Ile 225 Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Ser 235 Asp Asn Val Asp Val Arg Thr Tyr Asp 245 Thr Val Glu Phe 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 Val1 280 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 Ala 310 Lys Glu Val Ile Ala Val Asn Lys Gly Trp Arg Met Lys 325 Thr Thr Thr Ala As n 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 Asn Ile Gly Lys Gly Thr Thr Ala Thr Val 355 360 Ser Lys Asp Asp Gin 365 38 Thr Val 370 Leu Gin 385 Met Tyr Asp Val Asn Val 375 Gly Asp Ala Asn Val Asn Gin Asn Ser Gly Trp Asn Leu Asp Ser Lys 390 395 Ala Vai Ala Gly Ser Gly Lys Val Ile 405 Ser Giy Asn Vai Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Val1 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Thr Ser Met Thr Pro 445 Ser Vai 450 Ser Leu Giy Ala Giy 455 Ala Asp Ala Pro Thr Leu Ser Val 460 Asn Lys Pro Val Asp Giy 465 Asp Ala Leu Asn Val1 470 Giy Ser Lys Lys Asp 475 Arg 480 Ile Thr Asn Val Ala 485 Pro Giy Vai Lys Giu 490 Gly Asp Vai Thr Asn Vai 495 Ala Gin Leu Val Asp Gly 515 Lys 500 Gly Val Ala Gin As n 505 Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Asn Aia Arg Ala Ile Ala Gin Ala Giy Leu 530 Val Gin Ala Tyr Leu 535 Pro Gly Lys Ser Met 540 Met Ala Ile Gly Gly 545 Gly Thr Tyr Arg Gly 550 Glu Ala Giy Tyr Ala 555 Ile Gly Tyr Ser Ser 560 Ile Ser Asp Gly Gly Asn 565 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> 24 <21 1> 591 <212> PRT <213> Neisseria meningitidis (WO0066741-0004) 39 <400> 24 Met 1 Asn Lys Ile Tyr Arg Ile Ile Trp 5 Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Glu Ser Giu Leu Thr Arg Asn His Thr Lys Arg 25 Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Ser Ala Asn Asn Giu Glu Gin Glu Giu Asp Tyr Leu Asp Pro Val Gin Arg Thr Vai Vai Leu Ile Val Asn Ser Asp Lys Glu Thr Gly Glu Lys Lys Val Glu Glu Ser Asp Trp Ala Val Tyr Phe Asn Glu Gly Asp Asn 115 Gly Val Leu Thr Arg Glu 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 Asp 135 Leu Thr Ser Val Gly Thr Glu Lys Leu 140 Thr Ser Asp Thr Lys Ser Phe Ser Ala Asn 145 Gly Leu Asn Phe Ala 165 Asn Lys Val Asn 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 Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Glu Thr Thr Asn Val Thr 200 Asn Asp Asn Val Thr 205 Lys Lys 210 Ile Lys 225 Arg Ala Ala Ser Val1 215 Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Gly Val Lys Pro 230 Gly Thr Thr Ala Ser 235 Asp Asn Val Asp Phe 240 Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 40 c) 245 Thr Thr Val Asn Val Glu Ser Lys 260 Asp 265 Asn Gly Lys Lys Thr Glu Val 270 Gly Lys Leu Lys Ile Gly 275 Ala Lys Thr Ser Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Ser 300 Thr Asp Glu Gly Giu 305 Gly Leu Val Thr Ala 310 Lys Glu Val Ile Ala Val Asn Lys Gly Trp Arg Met Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giy 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 Thr Val 370 Met Tyr Asp Val As n 375 Val Gly Asp Ala Leu 380 Asn Val Asn Gin Leu 385 Gin Asn Ser Gly Trp 390 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 Giti Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Thr Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Pro 445 Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Thr 460 Leu Ser Val Asp Gly Asp Ala Leu Asn Val 465 470 Gly Ser Lys Lys Asp 475 Asn Lys Pro Val Arg 480 Ile Thr Asn Val Ala Pro Gly Val Lys Giu Gly Asp Val 485 490 Thr Asn Val 495 -41- Ala Gin Leu Lys Gly Val Ala Gin Asn 500 505 Val1 Asp Gly Asn Ala Arg Ala Gly Ile 515 520 Leu Asn Asn Arg Ile Asp Asn 510 Ala Thr Ala Ala Gln Ala Giy Leu 530 Val Gin Ala Tyr Pro Gly Lys Ser Met Ala Ile Gly Giy 545 Gly Thr Tyr Arg Gly 550 Giu Ala Gly Tyr Ile Giy Tyr Ser Ile Ser Asp Gly Giy Asn Trp Ile Ile 565 Lys 570 Gly Thr Ala Ser Giy Asn 575 Ser Arq Gly His 580 Phe Giy Ala Ser Ala 585 Ser Val Giy Tyr Gin Trp 590 <210> <211> 591 <212> PRT <213> Neisseria meningitidis (W00066741-0005) <400> Val Val Val Ile Tyr Arg Ile Ile Trp Asn 10 Ser Ala Leu Asn Ala Trp Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Vai Lys Thr Ala Vai Leu Ala 40 Thr Leu Leu Phe Ala Thr Val Gin Ala Ser Ala Asn Asn Giu Gln Giu Glu Asp Tyr Leu Asp Pro Val1Gin Arg Thr Val Ala 70 Val Leu Ile Val Asn 75 Ser Asp Lys Giu Gly Thzr Gly Giu Lys Glu Lys Val Glu Giu Ser Asp Trp Ala Val Tyr Phe Asn Giu Lys 100 Gly Val Leu Thr Ala 105 Arg Giu Ile Thr Leu Lys Ala 110 Thr Tyr Ser Gly Asp Asn Leu Lys Ile Lys Gln 120 Asn Giy Thr Asn Phe 125 42 Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser 130 135 Val Gly Thr Giu Lys Leu 140 145 Gly Leu Asn Phe Asn Gly Asn 150 Lys Val Asn Thr 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 Thr 185 Leu Thr Asp Thr Leu Leu Asn 190 Asp Asp Glu Thr Thr Asn Val Asn Asp Asn Val Thr 205 Lys Lys 210 Arg Ala Ala Ser Lys Asp Vai Leu Ala Giy Trp Asn Ile 225 Lys Gly Val Lys Pro 230 Gly Thr Thr Ala Ser 235 Asp Asn Val 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 Gly 275 Asn 260 Val Glu Ser Lys Asp 265 Asn Giy Lys Lys Thr Giu Vai 270 Giy Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser Ser 300 Thr Asp Glu Gly Gly Leu Val Thr Lys Glu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Gly Trp Arg Met Thr Thr Thr Ala As n 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Giy Asn Ile Giy Lys Giy Thr Thr Ala Thr Val 355 360 Ser Lys Asp Asp Gin 365 Thr Val Met 370 Tyr Asp Val Asn Val Gly Asp Ala Leu 375 380 Asn Val Asn Gin -43- Leu Gin Asn Ser Gly Trp Asn Leu Asp Ser Lys 395 Ser Pro 410 Ala Val Ala Gly Ser Gly Lys Asp Glu Thr Val Ile 405 Ser Gly Asn Val Ser Lys Gly Lys Met 415 Asn Ile Asn Ala Gly Asn Asn Ile Giu 425 Ile Thr Arg 430 Gin Phe Ser Asn Gly Lys 435 Asn Ile Asp Ile Thr Ser Met Thr Pro 445 Ser Val 450 Ser Leu Gly Ala Gly 455 Ala Asp Ala Pro Leu Ser Vai Asp Gly 465 Asp Ala Leu Asn Gly Ser Lys Lys Asp 475 Asn Lys Pro Vai Ile Thr Asn Val Pro Gly Val Lys Giu 490 Giy Asp Val Thr Asn Val 495 Ala Gin Leu Val Asp Gly 515 Giy 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 1530 Val Gin Ala Tyr Pro Gly LYS Ser Met Met Ala Ile Gly 540 Ile Gly Tyr Ser Ser Gly 545 Gly Thr Tyr Arg Gly 550 Glu Ala Gly Tyr Al a 555 Ile Ser Asp Gly Gi y 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> 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 Ser Glu Leu Thr Val Ala Thr Ala Val Ala Ser Thr Thr Asp Asp Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Leu Ala Asp Asp 55 Thr Leu Leu Phe Leu Tyr Leu Glu Pro Val Gin Arg Thr Ala Pro Val Leu Phe His Ala Asp Glu Gly Thr Gly Glu Lys Glu Val Thr Glu Asp Ser Asn Trp Gly 90 Val Tyr Phe Asp Lys Lys Gly Val Leu Lys Ile Lys 115 Ala Gly Thr Ile Leu Lys Ala Gly Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asp Glu 120 Asn Thr Asn Ala Tyr Ser Leu Lys Lys Asp Leu .130 135 Thr Asp Leu Thr Ser 140 Val Glu Thr Giu Lys 145 Leu Ser Phe Gly Ala 150 Asn Gly Lys Lys Val1 155 Asn Ile Thr Ser Asp 160 Thr Lys Gly Leu Phe Ala Lys Glu 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 Asn 200 Val Thr Asn Asp Asn 205 Asp Glu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val 220 Leu Asn Ala Gly Trp 225 Asn Ile Lys Gly Val 230 Lys Pro Gly Thr Thr 235 Ala Ser Asp Asn Val 240 Asp Phe Val Arg Thr Tyr Asp Thr Val Glu 245 250 Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr 260 Giu Vai Lys Ile Gly Ala Lys Thr 275 280 Ser Val Ile Lys Giu 285 Lys Asp Gly Lys Leu Val 290 Thr Gly Lys Asp 295 Lys Giy Glu Asn Ser Ser Thr Asp Glu 305 Gly Giu Gly Leu Val 310 Thr Ala Lys Giu Ile Asp Ala Val Asn 320 Lys Ala Gly Trp Met Lys Thr Thr Thr 330 Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Ala Ser Gly 355 Phe Giu Thr Val Ser Gly Thr Lys Val Thr Phe 350 Asp Gin Gly Asn Gly Thr Thr Ala 360 Thr Val Ser Lys Asn Ile 370 Thr Val Lys Tyr Asp 375 Val Asn Val Gly Asp 380 Ala Leu Asn Val 385 Gly Gin Leu Gin Asn Ser Ser Gly Lys 405 Gly Trp Asn Leu Ser Lys Ala Val Val Ile Ser Gly 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 460 Lys Asp Ala Asn Ser Val Asp Asp Giu Gly Ala 465 470 Leu Asn Val Gly Ser 475 Lys 480 Asp Val 495 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly Val 490 Lys Glu Gly Thr Asn Val Ala 500 Gin Leu Lys Gly Val 505 Ala Gin Asn Leu Asn Asn His 510 46 Ile Asp Asn Val Asp Gly Asn 515 Ala Thr Ala Gly Leu Val Gin .530 535 Arg Ala Gly lie Ala Gin Ala Tie Ala Tyr Leu Pro Gly Lys 540 Ser Met Met Ala 545 Ile Gly Gly Gly Tyr Arg Giy Giu Ala Giy Tyr Ala Ile 555 Tyr Ser Ser Ile Ser 565 Asp Gly Gly Asn Ile Ile Lys Gly Thr Ala 575 Ser Giy Asn Ser 580 Arg Giy His Phe Gly 585 Aia Ser Ala Ser Val Giy Tyr 590 Gin Trp <210> 27 <21i> 594 <212> PRT <213> Neisseria meningitidis (w00066741-0007) <400> 27 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Ala 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 40 Thr Leu Leu Phe Ala Ala Ser Thr Ala Thr Thr Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Pro Val Leu Phe His Ala Asp Ser Giu Gly Thr Gly Lys Giu Val Thr Giu Asp Ser Asn Trp Gly Val Leu Thr Ala Gly Thr Ile Thr 100 105 Gly 90 Val Tyr Phe Asp Lys Lys Leu Lys Ala Gly Asp Asn Leu 110 Lys Ile Lys Gin Asn Thir Asp Giu Asn Thr 115 120 Asn Ala Ser Ser Phe Thr -47- Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Glu Thr Glu IND Leu Ser Phe Gly Ala 150 Asn Gly Lys Lys Va1 155 Asn Ile Thr Ser Thr Lys Gly Leu Phe Ala Lys Glu 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 Asn 200 Val Thr Asn Asp Asp Glu 210 Lys Lys Arg Ala Ser Val Lys Asp Val 220 Leu Asn Ala Gly Trp 225 Asn Ile Lys Gly Lys Pro Gly Thr 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 Thr 260 Val Asn Val Glu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Gly Ala Lys Thr 280 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 Giu Gly Leu Thr Ala Lys Glu Ile Asp Ala Val Lys Ala Gly Trp Met Lys Thr Thr Thr 330 Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Lys 340 Phe Glu Thr Val Thr 345 Ser Gly Thr Lys Ala Ser Gly Asn 355 Gly Thr Thr Ala 360 Thr Val Ser Lys Asp 365 Asp Ala 380 Val Thr Phe 350 Asp Gin Gly Leu Asn Val Asn Ile Thr Val Lys Tyr Asp 370 375 Val Asn Val Gly -48- Asn Gin Leu Gin Asn Ser Gly Trp Asn Len Asp Ser 395 Lys Ala Val Ala 400 Gly Ser Ser Gly Lys 405 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 Giu Ile 430 Thr Pro Gin Giy Lys Asn Ile Asp 440 Ile Ala Thr Ser Met 445 Phe Ser 450 Ser Val Ser Leu Gly 455 Aia Gly Aia Asp Pro Thr Leu Ser Val1 465 Asp Asp Glu Gly Ala 470 Leu Asn Val Gly Ser 475 Lys Asp Ala Asn Lys 480 Pro Val Arg Ile Thr 485 Asn Vai Ala Pro Gly 490 Val Lys Giu Giy Asp Val 495 Thr Asn Val Ile Asp Asn 515 Ala 500 Gin Leu Lys Giy Vai 505 Ala Gin Asn Leu Asn Asn His 510 Gin Ala Ile Val Asp Gly Asn Arg Ala Gly Ile Ala Thr 530 Ala Gly Leu Val Ala Tyr Leu Pro Gly 540 Lys Ser Met Met Ala 545 Ile Gly Gly Giy Tyr Arg Gly Glu Ala 555 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 Gly 585 Ala Ser Ala Ser Vai Gly Tyr 590 Gin Trp <210> 28 <211> 594 <212> PRT <213> Neisseria meningitidis (W00066741-0008) <400> 28 -49 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Ser Giu Leu Thr Arg Asn His Thr Lys Arg 25 Ala Ser Ala Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gin Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Pro Val Leu Phe His Ala Asp Giu Gly Thr Gly Giu Lys Giu Val Thr Asp Ser Asn Trp Gly 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 Tyr Ser 130 Leu Lys Lys Asp Thr Asp Leu Thr Ser 140 Val Glu Thr Giu Leu Ser Phe Gly Ala 150 Asn Gly Lys Lys Val1 155 Asn Ile Thr Ser Asp 160 Thr Lys Gly Leu Asn 165 Phe Ala Lys Giu Thr 170 Ala Gly Thr Asn Gly Asp 175 Thr Thr Val Leu Asn Thr 195 His 180 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Asn 200 Val Thr Asn Asp Asn 205 Asp Giu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val 220 Leu Asn Ala Gly Trp 225 Asn Ile Lys Gly Val 230 Lys Pro Gly Thr Tb r 235 Ala Ser Asp Asn Val 240 Asp Phe Val Arg Thr Tyr Asp Thr Val Giu Phe Leu Ser Ala Asp Thr 245 250 255 50 Lys Thr Thr Thr Val Asn Val Glu 260 Giu Val Lys Ile Gly Ala Lys Thr 275 280 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ser Val Ile Lys Lys Leu 290 Val Thr Gly Lys Lys Gly Glu Asn Gly 300 Ser Ser Thr Asp Giu 305 Gly Glu Gly Leu Val1 310 Thr Ala Lys Giu Ile Asp Ala Val Lys Ala Gly Trp Met Lys Thr Thr Thr 330 Ala Asn Gly Gin Thr Gly 335 Gin Ala Asp Ala Ser Gly 355 Phe Giu 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 Se r 390 Gly Trp Asn Leu Asp 395 Ser Lys Ala Val Giy Ser Ser Gly Lys 405 Val Ile Ser Gly As n 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Thr Val Asn Ile Ala Gly Asn Asn Ile Giu Ile 430 Thr Pro Gin Gly Lys Asn Ile Asp 440 Ile Ala Thr Ser Met 445 Phe Ser .450 Val Asp 465 Ser Val Ser Leu Gly 455 Ala Gly Ala Asp Ala Pro Thr Leu Ser 460 Lys Asp Ala Asn Lys Asp Glu Gly Ala 470 Leu Asn Val Gly Ser 475 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Giy Val 490 Lys Giu Gly Asp Val 495 Thr Asn Val Ala Gin Leu Lys Gly Val Ala Gin Asn Leu Asn Asn His -51 Ile Asp Asn 515 Ala Thr Ala 530 Val Asp Gly Asn Arg Ala Gly Ile Ala 525 Gln Ala Ile Gly Leu Val Gln Ala Tyr Leu Pro 535 Lys Ser Met Met Ala Ile Gly Gly Giy Thr 545 550 Tyr Arg Gly Glu Ala 555 Gly Tyr Ala IleGly 560 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 Gin Trp <210> 29 <211> 594 <212> PRT <213> Neisseria meningitidis (W00066741-0009) <400> 29 Met Asn Lys Val Val Val Thr Val Ala Ile Tyr Arg Ile Ile Trp Ser Ala Leu Asn Ala Trp 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 Asn Ala Thr Asp Asp Asp Leu Tyr Leu Giu Pro Val Gin Arg Thr Ala Val Val Leu Ser 70 Phe Arg Ser Asp Lys Glu Gly Thr Gly Lys Giu Gly Thr Giu Asp Ser Asn Trp Ala 90 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 Giu Asn Thr Asn Asp Ser Ser Phe Thr 52 115 Tyr Ser Leu Lys Lys Asp Leu 130 135 Lys Leu Ser Phe Gly Ala Asn 145 150 Thr Asp Leu Thr Ser Val 140 Glu Thr Glu Gly Asn Lys Val 155 Asn Ile Thr Ser Thr Lys Gly Leu Phe Ala Lys Glu Ala Gly Thr NO Pro Thr Val Leu Asn Thr 195 His 180 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asn Gly Asp 175 Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Asn 200 Val Thr Asn Asp Asn 205 Asp Glu 210 Lys Lys Arg Ala Ser Val Lys Asp Val1 220 Leu Asn Ala Gly Trp 225 Asn Ile Lys Gly Val1 230 Lys Pro Gly Thr Th r 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 Thr 260 Val Asn Val Glu Ser 265 Lys Asp Asn Gly Lys Lys Thr 270 Lys Asp Gly Ile Gly Ala Lys Thr 280 Ser Val Ile Lys Glu 285 Lys Leu 290 Val Thr Gly Lys Lys Asp Glu Asn Gly 300 Ser Ser Thr Asp Glu 305 Gly Glu Gly Leu Thr Ala Lys Glu Ile Asp Ala Val 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 Thr Ser Gly Thr Asn Val Thr Phe 350 Asp Asp Gin Gly 365 Lys Gly Thr Thr Ala 360 Thr Val Ser Lys 53 Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala 375 380 Leu Asn Val Asn Gin Leu Gin Asn Ser Gly Trp Asn 385 390 Leu Asp 395 Ser Lys Ala Vai Gly Ser Ser Gly Lys 405 Val Ile Ser Gly Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Glu 420 Thr Val Asn Ile Asn Ala Gly Asn Asn 425 Ile Glu Ile 430 Ala Pro Gin Gly Lys Asn Ile Ile Ala Thr Ser Met 445 Phe Ser 450 Ser Val Ser Leu Ala Gly Ala Asp Pro Thr Leu Ser Val 465 Asp Asp Giu Gly Leu Asn Val Gly Ser 475 Lys Asp Thr Asn Lys 480 Pro Vai Arg Ile Thr 485 Asn Vai Ala Pro Giy 490 Val Lys Giu Giy Asp Val 495 Thr Asn Vai Ile Asp Asn 515 Gin Leu Lys Gly Ala Gin Asn Leu Asn Asn Arg 510 Gin Ala Ile Val Asp Gly Asn Al a 520 Arg Ala Gly Ile Ala Thr 530 Ala Gly Leu Val Gin 535 Ala Tyr Leu Pro Gly 540 Lys Ser Met Met Ile Gly Gly Asp Thr 550 Tyr Arg Gly Giu Ala 555 Trp Ile 570 Gly Tyr Ala Ile Tyr Ser Ser Ile Ser Asp 565 Gly Gly Asn Ile Lys Gly Thr Ala 575 Ser Gly Asn Ser Arg Gly His Phe 580 Gly Ala Ser Ala Ser Val Gly Tyr 585 590 Gin Trp <210> <211> <212> 594 PRT 54 <21 3> Neisseria meninqitidis (W00066741-0Ol0) <400> Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Thr Val Ala Ser Glu 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 Asp Asp Asp Leu Tyr Leu Pro Val Gin Arg Thr Ala Val Val Leu Se r Phe Arg Ser Asp Giu Gly Thr Giy Lys Giu Giy Thr Giu Asp Ser Asn Trp Val Tyr Phe Asp Glu Lys Arg Val Leu Lys Ile Lys 115 Ala Gly Ala Ile Th r 105 Leu Lys Ala Gly Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asn Giu 120 Asn Thr Asn Asp Ser 125 Tyr Ser 130 Leu Lys Lys Asp Leu 135 Thr Asp Leu Thr Val Glu Thr Giu Lys 145 Leu Ser Phe Giy Ala 150 Asn Gly Asn Lys Asn Ile Thr Ser Asp 160 Thk Lys Giy Leu Asn 165 Phe Ala Lys Glu Thr 170 Ala Gly Thr Asn Gly Asp 175 Pro Thr Val Leu Asn Thr 195 His 180 Leu Asn Gly Ile Gly 185 Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr Asn 200 Val Thr Asn Asp Asn 205 Asp Giu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val Leu 220 Asn Ala Gly Trp Asn Ile Lys Giy Val 225 230 Lys Pro Gly Thr Thr Ala Ser Asp Asn Val 235 240 Asp Phe Val Arq Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 255 Asp Asn Gly Lys Lys Thr 270 Lys Thr Thr Thr Val Asn Val Giu 260 Glu Vai Lys Ile Gly Ala Lys Thr 275 280 Ser Lys 265 Ser Val Ile Lys Lys Asp Giy Lys Leu 290 Val Thr Gly Lys Gly Lys Asp Giu Asn Gly 295 300 Ser Ser Thr Asp Giu 305 Gly Giu Giy Leu Thr Ala Lys Giu 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 Aia Ser Giy 355 Phe Glu 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 Lys Tyr Val Asn Val Gly Asp Ala Leu Asn Val 380 Ser Lys Ala Val Ala Asn 385 Gin Leu Gin Asn Ser Ser Giy Lys 405 Giy Trp Asn Leu Asp 395 Gly Val Ile Ser Gly Asn 410 Val Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Giu 420 Thr Vai Asn Ile Ala Gly Asn Asn Ile Giu Ile 430 Ala Pro Gin Gly Lys Asn Ile Asp 440 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 Thr Asn Lys Val Asp Asp Giu Gly Ala 465 470 Leu Asn Val Gly Ser 475 Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val 490 Lys Giu Gly Asp Val 495 -56- Thr Asn Val Ala Gin Leu Lys Gly Val 500 505 Ala Gin Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gin Ala Ile 525 Lys Ser Met Met Ala Thr Ala 530 Gly Leu Val Ala Tyr Leu Pro Gly 540 Ala Ile Gly Gly Asp Thr 545 550 Tyr Arg Gly Giu Ala Gly Tyr Ala 555 Ile Ile Lys Gly Ile Tyr Ser Ser Ile Ser Asp 565 Gly Gly Asn Trp 570 Thr Ala 575 Ser Gly Asn Se r 580 Arg Gly His Phe Gly Ala 585 Ser Ala Ser Val Gly Tyr 590 Gin Trp <210> 31 <211> 598 <212> PRT <213> Neisseria meningitidis (wo0066741-0011) <400> 31 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn 1 5 10 Ser Ala Leu Asn Ala Trp Val Val Val 2 Ser 20 Glu Leu Thr Arg Asn His Thr Lys Arg 25 Ala Ser Ala Thr Val Gin Thr Val Ala Thr Ala Val Leu Thr Leu Leu Phe Ala Ala Asn Thr Ala Ala Thr Asp Asp Asp 55 Asp Leu Tyr Leu Giu Pro Val Gin Arg Val Val Leu Ser 70 Phe Arg Ser Asp Lys Glu Gly Thr Gly Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp S90 Glu Lys Arg Val Leu Lys Ala Gly Ala Ile Thr 100 105 Leu Lys Ala Gly Asp Asn Leu 110 57 Lys Ile Lys Gin Asn Thr Asn Asn Thr Asn Glu Asn 125 Thr Asn Asp Leu Thr Ser Ser Ser 130 Phe Thr Tyr Ser Leu 135 Lys Lys Asp Leu Thr Asp 140 Val Glu Thr Glu Lys 145 Leu Ser 150 Phe Gly Ala 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 Asp 180 Pro Thr Val His Asn Gly Ile Gly Ser Thr Leu 190 Thr Asn Asp Leu Leu Asn Thr Gly 200 Ala Thr Thr Asn Asn V~al 210 Thr Asp Asp Giu Lys Arg Ala Ala Se r 220 Val Lys Asp Val Asn Ala Gly Trp Asn 230 Ile Lys Gly Val Pro Gly Thr Thr Ala 240 Ser Asp Asn Val Asp 245 Phe Val Arg Thr Asp Thr Val Glu Phe Leu 255 Ser Ala Asp Gly Lys Lys 275 Lys Thr Thr Thr Asn Val Giu Ser Lys Asp Asn 270 Val Ile Lys Thr Giu Val Lys Gly Ala Lys Thr Ser 285 Glu Lys 290 Asp Gly Lys Leu Val1 295 Thr Gly Lys Gly Asp Glu Asn Gly Ser 305 Ser Thr Asp Giu Gly 310 Giu Gly Leu Val Th r 315 Ala Lys Glu Val Ile 320 Asp Ala Vai Asn Gl Gin Thr Gly 340 Lys Ala Gly Trp Arg 325 Gin Ala Asp Lys Phe 345 Met 330 Lys Thr Thr Thr Ala Asn 335 Glu Thr Val Thr Ser Gly Thr 350 Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 58 355 Asp Asp Gin 370 365 Lys Tyr Asp Val Asn Val Gly Asp 380 Gly Asn Ile Thr Val 375 Ala 385 Leu Asn Val Asn Leu Gin Asn Ser Gly 395 Trp Asn Leu Asp Lys Ala Val Ala Gly Ser 405 Ser Gly Lys ValI 410 Ile Ser Gly Asn Vai Ser 415 NO) Pro Ser Lys Asn Ile Giu 435 Gly 420 Lys Met Asp Glu Val Asn Ile Asn Ala Gly Asn 430 Ala Thr Ser Ile Thr Arg Asn Giy 440 Lys Asn Ile Asp Ile 445 Met Thr 450 Pro Gin Phe Ser Ser 455 Val Ser Leu Gly Ala Giy Ala Asp Ala 460 Asn Val Giy Ser Lys Thr Leu Ser Val Asp 470 Asp Glu Giy Ala Leu 475 Asp Ala Asn Lys Pro 485 Val Arg Ile Thr Asn 490 Val Ala Pro Gly Val Lys 495 Glu Gly Asp Leu Asn Asn 515 Val1 500 Thr Asn Val Ala Leu Lys Gly Val Ala Gin Asn 510 Ala Gly Ile Arg Ile Asp Asn Asp Gly Asn Ala Arg 525 V. I Ala Gin 530 Ala Ile Ala Thr Gly Leu Ala Gin Tyr Leu Pro Gly Lys 545 Ser Met Met Ala Ile 550 Gly Gly Gly Thr Tyr 555 Arg Giy Glu Ala Gly 560 Tyr Ala Ile Gly Tyr 565 Ser Ser Ile Ser Asp 570 Thr Gly Asn Trp Val Ile 575 Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala 580 585 590 Ser Val Gly Tyr Gin Trp 595 -59- <210> 32 <211> 599 <212> PRT <213> Neisseria meninqitidis (WO006674i-0012) <400> 32 met Asn Lys 1 Val Ala Vai Thr Vai Lys Ile Tyr Arg Ile Ile Trp 5 Asn 10 Ser Ala Leu Asn Ala Trp 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 Giu Asp 55 Giu Giu Glu Giu Leu Giu Pro Val Val Arg Ser Ala Leu Val Leu 70 Gin Phe Met Ile Lys Giu Gly Asn Gly Giu Asn Giu Ser Thr Gly Asn Ile Gly Trp 90 Ser Ile Tyr Tyr Asp Asn His Asn Thr Leu Lys Ile 115 Leu 100 His Gly Ala Thr Val1 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 *I Leu Thr Asp Leu Thr 140 Asn Gly Asn Lys Val Val Giu Thr Giu Leu Ser Phe Gly Ala 155 Asn Ile Thr Ser Asp 165 Thr Lys Gly Leu Asn 170 Phe Ala Lys Giu Thr Ala 175 Gly Thr Asn Gi y 180 Asp Thr Thr Val His 185 Leu Asn Gly Ile Gly Ser Thr 190 Leu Thr Asp Thr 195 Asp Asn Val Thr 210 Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn 200 205 Asp Asp Lys 215 Lys Lys Arg Ala Ala 220 Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys 225 230 d) C4 Gly Val 235 Thr Tyr 250 Lys Pro Gly Thr Ala Ser Asp Asn Val 245 Asp Phe Val His Asp Thr Val Glu Phe 255 Leu Ser Ala Thr Lys Thr Thr Val Asn Val Glu Ser Lys Asp 270 Ser Val Ile Asn Gly Lys 275 Arg Thr Glu Val Ile Gly Ala Lys Thr 285 Lys Glu 290 Lys Asp Gly Lys Leu 295 Val Thr Gly Lys Gly 300 Lys Gly Glu Asn Gly 305 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 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 Ile 375 Thr Val Lys Tyr Asp 380 Val Asn Val Gly Asp 385 Ala Leu Asn Val Asn 390 Gin Leu Gin Asn Ser 395 Gly Trp Asn Leu Asp 400 Ser Lys Ala Val Ala 405 Gly Ser Ser Gly 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 Asp 445 Ser Met 450 Thr Pro Gin Phe Ser Ser Val Ser Leu 455 Gly 460 Ala Gly Ala Asp Ala 465 Pro Thr Leu Ser Val Asp Asp Lys Gly Ala 470 475 Leu Asn Val Gly Ser 480 -61 Lys Asp Ala Asn Lys Pro Val Arg Ile Thr 485 490 Asn Val Ala Pro Gly Val 495 Lys Glu Gly Asp 500 Val Thr Asn Val Ala 505 Gin Leu Lys Gly Val Ala Gin 510 Arg Ala Gly Asn Leu Asn 515 Asn Arg Ile Asp Asn 520 Val Asp Gly Asn Ile Ala 530 Gin Ala Ile Ala Ala Gly Leu Val Gin Ala Tyr Leu Pro 540 Gly 545 Lys Ser Met Met Ile Gly Giy Giy Tyr Arg Gly Giu Ala 560 Gly Tyr Ala Ile Gly 565 Tyr Ser Ser Ile Ser 570 Asp Gly Gly Asn Trp Ile 575 Ile Lys Gly Thr Ala Ser Gly Asn 580 Ser Arg 585 Giy His Phe Gly Ala Ser 590 Ala Ser Val 595 Gly Tyr Gin Trp <210> 33 <211> 592 <212> PRT <213> Neisseria meningitidis (WO006674i-0013) <400> 33 Met Asn Lys Ilie Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp -1 Val Ala Val Ser Glu Leu Thr Arg Asn 25 Thr Val Lys Thr Ala Val Leu Ala Thr 40 His Thr Lys Arg Ala Ser Ala Thr Val Gin Leu Leu Phe Ala Ala Asn Ala Thr Asp Giu Asp Giu Giu Glu Giu Leu Giu Ser Val Gin Arg Ser Val Val Gly Ser Ile 70 Gin Ala Ser Met Glu Gly Ser Gly Leu Giu Thr Ile Ser Leu Ser Met Thr Asn 90 Asp Ser Lys Glu Phe Val 62 Asp Pro Tyr Ile Val Val Thr Leu Lys 100 105 Ala Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Lys Gin Asn Thr 115 Asn Glu Asn Thr 120 Asn Ala Ser Ser Phe 125 Leu Lys Lys 130 Asp Leu Thr Leu Ile Asn Val Thr Giu Lys Leu Phe Gly Ala Asn Gly 150 Lys Lys Val Asn Ile Ile Ser Asp 155 Thr Asn Gly Asp Thr Gly Leu Asn Phe Ala 165 Lys Glu Thr Ala Gly 170 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 Val Asp Ala 200 Gly Asn Gin Ser Thr 205 Arg Ala 210 Ala Ser Ile Lys Val Leu Asn Ala Gly Trp Asn Ile Lys 220 Giu Asn Val Asp Phe Gly 225 Val Lys Thr Gly Ser 230 Thr Thr Gly Gin Val Arg Thr Tyr Asp 245 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 Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Val 280 Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Ser Thr Asp Glu Gly 300 Ala Val Asn Lys Ala Giu 305 Gly Leu Val Thr Ala 310 Lys Giu Val Ile Gly Trp Arg Met Lys Thr Thr Thr Ala Asn 325 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giu Thr Val Thr Ser Gly Thr 340 345 Asn Val Thr Phe Ala Ser 350 -63 Gly Lys Gly Thr Thr Ala Thr Ser Lys Asp Asp Gly Asn Ile Thr Val 370 Met Tyr Asp Val As n 375 Val Giy Asp Ala Asn Val Asn Gin Gin Asn Ser Giy Trp 390 Asn Leu Asp Ser Lys 395 Ala Val Ala Gly Ser 400 (Ni) Ser Gly Lys Vai Ser Gly Asn Val Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly Asn Asn Ile Glu 425 Thr Ser Met Ala Pro 445 Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Ser Vai 450 Ser Leu Giy Ala Gly 455 Aia Asp Ala Pro Thr 460 Leu Ser Vai Asp Asp 465 Glu Giy Ala Leu Asn 470 Vai Giy Ser Lys Aia Asn Lys Pro Arg Ile Thr Asn Val Ala Pro Gly Vai Lys 1485 490 Giu Giy Asp Vai Thr Asn 495 Val Ala Gin Asn Val Asp 515 Lys Giy Vai Ala Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Giy Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala 525 Ala Gly 530 Leu Val Gin Ala Tyr 535 Leu Pro Gly Lys 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 Gly 565 Gly Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg Gly His 580 Phe Gly Ala Ser Ala Ser Val Gly 585 Tyr Gin Trp 590 <210> 34 64 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0014) <400> 34 Met Asn Lys 1. Ile Tyr Arg Ile Ile Trp 5 Asn Ser 10 Ala Leu Asn Ala Trp Val Ala Val Ser Giu Leu Thr Arg Asn 25 His Thr Lys Arg Thr Val Gin Cl) Thr Val Lys 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 Ser Val Val Gly Ser 70 Ile Gin Ala Ser Met 75 Giu Gly Ser Gly Giu Leu Giu Thr Ile Leu Ser Met Thr Asn Asp Ser Lys Giu Phe Val Asp Pro Tyr Lys Gin Asn 115 Vai Vai Thr Leu Ala Giy Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Giu Asn Asn Ala Ser Ser Leu Lys 130 Lys Asp Leu Thr Gly 135 Leu Ile Asn Val Giu 140 Thr Giu Lys Leu Phe Gly Ala Asn Giy 150 Lys Lys Val Asn Ile 155 Ile Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Giu Thr Ala Thr Asn Gly Asp Thr Thr 175 Val His Leu Asn 180 Gly Ilie Gly Ser Th r 185 Leu Thr Asp Thr Leu Ala Gly 190 Ser Ser Ala 195 Ser His Val Asp Ala 200 Gly Asn Gin Ser Thr His Tyr Thr 205 Arg Ala Ala Ser Ile Lys Asp Val 210 215 Leu Asn Ala Gly 220 Trp Asn Ile Lys Gly Val Lys Thr Gly Ser Thr Thr Gly Gin Ser Giu Asn Val Asp Phe 225 Val Arg Thr Tyr Asp Thr 245 Val Giu Phe 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 Giu Val 270 Gly Lys Leu Ala Lys Thr Ser Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Gly Lys Gly 295 Glu Asn Gly Ser Thr Asp Glu Gly Gly Leu Val Thr Lys Giu Val Ile Asp 315 Ala Val Asn Lys Giy Trp Arg Met Thr Thr Thr Ala Gly Gin Thr Giy Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Giu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val1 360 Ser Lys Asp Asp Gin 365 Thr Val 370 Met Tyr Asp Val As n 375 Vai Gly 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 Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Asp Gly Lys 435 Val1 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Ala 440 Thr Ser Met Ala Pro 445 Ser Val 450 Asp Glu 465 Ser Leu Gly Gly Ala Leu Ala Gly 455 Ala Asp Ala Pro Thr Leu Ser Val Asp 460 Asn Val Gly Ser Lys Asp 470 475 Ala Asn Lys Pro Val 480 66 Arc Ile Thr Asn Val Ala Pro Gly Val 485 Glu Gly Asp Val Ala Gin Leu 500 Lys Gly Val Ala Asn Leu Asn Asn Val Thr Asn 495 Arg Ile Asp 510 Ile Ala Thr Asn Val Asp Gly Asn Ala Arg Giy Ile Ala Gin Ala 525 Ala Gly 530 Leu Val Gin Ala Leu Pro Gly Lys Met Met Ala Ile Gly 545 Gly Gly Thr Tyr Gly Giu Ala Gly Tyr 555 Ala Ile Gly Tyr Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Phe Gly Ala Ser 585 Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg Gly His 580 Ala Ser Val Gly Tyr Gin Trp 590 <210> <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0015) <400> Met Asn Lys Ile 1 Tyr Arg Ile Ile Trp Asn 5 10 Ser Ala Leu Asn Ala Trp Val Ala Val Thr Val Lys Ser Giu Leu Thr Arg Asn 25 Hius Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Ala Val Leu Thr Leu Leu Phe Ala Asn Ala Thr Asp Glu Asp 55 Giu Giu Glu Glu Leu Giu Ser Val Gin Arg Ser Val Val Gly Ser Ile Gin Ala Ser Giu Gly Ser Gly Leu Glu Thr Ile Ser Leu Ser Met Thr Asn 90 Lys Ala 105 Asp Ser Lys Giu Phe Val Asp Pro Tyr Ile Val Val Thr Leu 100 Gly Asp Asn Leu Lys Ile 110 67 Lys Gin Asn Thr Asn Glu C*N c) Asn Thr 120 Gly Leu 135 Asn Ala Ser Ser Phe Thr Tyr Ser 125 Thr Giu Lys Leu Leu Lys Lys 130 Asp Leu Thr Ile Asn Val Giu 140 Phe Gly Ala Asn Gly 150 Lys Lys Val Asn Ile Ser Asp Thr Gly Leu Asn Phe Lys Glu Thr Ala Gly 170 Thr Asn Gly Asp Thr Thr 175 Val His Leu Ser Ser Ala 195 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 Thr 205 Arg Ala 210 Ala Ser Ile Lys Asp 215 Val Leu Asn Ala Gly 220 Trp Asn Ile Lys Gly 225 Val Lys Thr Gly Ser 230 Thr Thr Gly Gin Ser 235 Giu Asn Val Asp Val Arg Thr Tyr Asp 245 Thr Val Glu Phe Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Lys Ile Gly 275 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 Asp 285 Val Thr 290 Giy Lys Gly Lys Gly 295 Giu Asn Gly Ser Thr Asp Giu Giy Giu 305 Giy Leu Val Thr Ala 310 Lys Giu Val Ile Asp 315 Ala Val Asn Lys Ala 320 Giy Trp Arg Met Lys 325 Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Giu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Giy Lys Giy Thr 355 Thr Ala Thr Ser Lys Asp Asp Gin Gly Asn Ile 365 -68- Thr Val 370 Met Tyr Asp Val Asn Val 375 Gly Asp Ala Asn Val Asn Gin Le u 385 Gin Asn Ser Gly Trp 390 Asn Leu Asp Ser Ala Val Ala Gly Ser 400 Ser Gly Lys Val Ser Gly Asn Val Pro Ser Lys Giy Lys Met 415 Asp Glu Thr Val1 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Ser Arg 430 Asn Gly Lys 435 Asn Ile Asp Ile Ala Thr Ser Met Ala 440 Pro Gin Phe Ser 445 Leu Ser Val Asp Ser Val 450 Ser Leu Gly Ala Giy 455 Ala Asp Ala Pro Thr 460 Giu Giy Ala Leu Asn 470 Val Gly Ser Lys Asp 475 Ala Asn Lys Pro Arg Ile Thr Asn Ala Pro Giy Val Giu Giy Asp Val Thr Asn 495 Val Ala Gin Asn Val Asp 515 Lys Gly Vai 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 Tyr 535 Leu Pro Gly Lys Ser 540 Met Met Ala Ile Gly 545 Ser Gly Gly Thr Tyr Ile Ser Asp Giy 565 Arg 550 Gly Glu Ala Gly Ala Ile Gly Tyr Gly Asn Trp Ile Ile 570 Lys Giy Thr Ala Ser Gly 575 Asn Ser Arg Gly 580 His Phe Giy Ala Ser 585 Ala Ser Vai Gly Tyr Gin Trp 590 <210> 36 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0016) <400> 36 69 Met Asn Lys 1. Val Ala Val Ile Tyr Ar Ile Ilie Trp 5 Ser Glu Leu Thr Arg Asn 25 Ser Ala Leu Asn Ala Trp His Thr Lys Arg Ala Ser Ala Thr Val Lys Thr Ala Val Leu Thr Leu Leu Phe Ala Thr Val Gin Ala Asn Ala Thr Asp Giu Asp Glu Giu Glu Giu Giu Ser Val Gin Arg Ser Val Val Gly Ile Gin Ala Ser Met 75 Glu Gly Ser Giy Giu Leu Giu Thr Ile Leu Ser Met Thr Asp Ser Lys Giu Phe Val Asp Pro Tyr Lys Gin Asn 115 le 100 Val Val Thr Leu Ala Giy 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 Gly 135 Leu Ile Asn Val Giu 140 Thr Giu Lys Leu Ser 145 Phe Gly Ala Asn Lys Lys Val Asn Ile Ser Asp Thr Lys 160 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 Ilie Gly Ser Thr 185 Leu Thr Asp Thr 190 His Tyr Thr Ser His Val Asp Ala 200 Gly Asn Gin Ser Arg Ala 210 Gly Val 225 Ala Ser Ile Lys Thr Gly Lys Asp 215 Ser Thr 230 Val Leu Asn Ala Gly 220 Trp Asn Ile Lys Thr Gly Gin Ser 235 Giu Asn Vai Asp Val Arg Thr Tyr Asp Thr Val Glu Phe Leu 245 250 Ser Ala Asp Thr Lys Thr 255 70 Thr Thr Val Asn Val 260 Giu Ser Lys Asp 265 Asn Gly Lys Arg Thr Giu Val 270 Lys lie Gly 275 Ala Lys Thr Ser Ile Lys Giu Lys Asp Giy Lys Leu 285 Val Thr 290 Gly Lys Giy Lys Giy 295 Giu Asn Giy Ser Thr Asp Giu 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 Thr Thr Thr Ala As n 330 Giy Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Thr Ser Gl y 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val1 360 Ser Lys Asp Asp Gin 365 Thr Val 370 Met Tyr Asp Val Val Gly Asp Ala Asn Val Asn Gin Leu 385 Gin Asn Ser Gly Trp 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 Glu Thr Asn Gly Lys 435 Asn Ile Asn Ala Gly 425 Asn Asn Ile Glu Ile Ser Arg 430 Gin Phe Ser Asn Ile Asp Ile Al a 440 Thr Ser Met Ala Ser Val 450 Ser Leu Gly Ala Gly Ala Asp Ala Pro 455 Thr 460 Leu Ser Vai Asp Asp 465 Glu Gly Ala Leu Asn 470 Val Gly Ser Lys Asp 475 Ala Asn Lys Pro Val1 480 Arg Ile Thr Asn Val Ala 485 Pro Gly Val L~s Glu 490 Gly Asp Vai Thr Asn 495 Vai Ala Gin Leu Lys Gly Val Ala Gin Asn Leu Asn Asn Arg Ile Asp -71 500 Asn Val Asp Gly Asn Ala Arg Ala 515 520 Ala Gly Leu Val Gin Ala Tyr Leu 530 535 Gly Ile Ala Gin Ile Ala Thr Pro Gly Lys Met Met Ala Ile Gly 545 Gly Gly Thr Tyr Arg 550 Gly Glu Ala Gly Ala Ile Gly Tyr NO Ser Ile Ser Asp Giy Asn Trp Ile Ile 570 Lys Gly Thr Ala Ser Gly 575 Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Vai Gly 580 585 <210> 37 <211> 592 <212> PRT <213> Neisseria meningitidis (W00066741-0017) <400> 37 Tyr Gin Trp 590 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Ala Val Ser Giu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe 40 Ala Ala Asn Ala Thr Asp Giu Asp Giu Glu Giu Glu Giu Ser Val Gin Arg Ser Val Val Gly Ile Gin Ala Ser Giu Gly Ser Giy Leu Giu Thr Ile Ser Leu Ser Met Thr Asn 90 Asp Ser Lys Giu Phe Val Asp Pro Tyr Lys Gin Asn 115 Ile 100 Val Val Thr Leu Lys Ala 105 Gly Asp Asn Leu Lys Ile 110 Thr Tyr Ser Thr Asn Giu Asn Th r 120 Asn Ala Ser Ser Phe 125 Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Giu Thr Giu Lys Leu 72 Ser Phe Gly Ala Asn Gly Lys Lys Val Asn Ile Ile Ser Asp Thr Lys 160 Thr Thr 175 Gly Leu Asn Phe Al a 165 Lys Glu Thr Ala Thr Asn Giy Asp Val His Leu Gly Ile Gly Ser (N Thr Leu Thr Asp Met 185 Asn Asp Asn Val Thr Leu Leu Asn 190 Asp Asp Glu Thr Gly Ala 195 Thr Thr Asn Val Thr 200 Lys Lys 210 Arg Ala Ala Ser Val 215 Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Lys Gly Val Lys Gly Thr Thr Ala Asp Asn Val Asp Val Arg Thr Tyr Asp 245 Thr Val Giu Phe 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 Val1 280 Ile Lys Giu Lys Asp 285 Val Thr 290 Gly Lys Gly Lys Gly 295 Giu Asn Gly Ser Ser 300 Thr Asp Glu Gly Glu 305 Gly Leu Val Thr Ala 310 Lys Giu Val Ile Ala Val Asn Lys Gly Trp Arg Met Lys 325 Thr Thr Thr Ala Asn 330 Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Thr Val Met 370 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 Gin 365 Tyr Asp Val Asn Val Gly Asp Ala Leu Asn 380 Val Asn Gin 73 Leu Gin Asn Ser Gly Trp Asn Leu Asp Ser 385 390 Ala Val Ala Gly Ser 400 Ser Gly Lys Val Ile 405 Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Glu Thr Val1 420 Asn Ile Asn Ala Asn Asn Ile Glu Ile Thr Arg 430 Gin Phe Ser (N Asn Gly Lys 435 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 Asp 465 Lys Gly Ala Leu Val Gly Ser Lys Asp 475 Ala Asn Lys Pro Arg Ile Thr Asn Ala Pro Gly Val Glu Gly Asp Val Thr Asn 495 Val Ala Gin Asn Vai Asp 515 Leu 500 Lys Gly Val Ala Gin 505 Asn Leu Asn Asn Arg Ile Asp 510 Ile Ala Thr Gly Asn Ala Arg Ala 520 Gly Ile Ala Gin Ala Giy 530 Leu Val Gin Ala Leu Pro Gly Lys Met Met Ala Ile Gly 545 Gly Gly Thr Tyr Gly Glu Ala Gly Tyr 555 Ala Ile Gly Tyr Ser Ile Ser Asp Gly 565 Gly Asn Trp Ile Ile 570 Lys Giy Thr Ala Ser Gly 575 Asn Ser Arg Gly 580 His Phe Gly Ala Ser 585 Ala Ser Val Gly Tyr Gin Trp 590 <210> 38 <211> 589 <212> PRT <213> Neisseria meningitidis (WO006674i-0018) <400> 38 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 74 Val Val Val. Ser Glu Leu Thr Arg Asn His Thr Lys Arq Ala Ser Ala Thr Val Ala Thr Ala Val Leu Thr Leu Leu Ser Ala Thr Val Gin Ala Asn Ala Thr Asp Thr Asp Glu Asp Giu Glu Glu Ser Val Val Ser Ala Leu Val NO' Leu Gin Phe Met Ile Lys Giu Gly Asn Giy Giu Ile Giu Ser Giy Asp Ile Gly Ser Ile Tyr Tyr Asp Asp His Asn Thr His Gly Ala Thr Vali 105 Thr Leu Lys Ala Giy Asp Asn 110 Leu Lys Lys Leu Lys Ile 115 Lys Gin Ser Giy Asp Phe Thr Tyr Ser 125 Giu Leu 130 Lys Asp Leu Thr Ser 135 Val Giu Thr Giu Leu Ser Phe Gly Ala 145 Asn Gly Asn Lys Val1 150 Asn Ile Thr Ser Asp 155 Thr Lys Giy Leu Phe Ala Lys Giu Thr 165 Ala Gly Thr Asn Gly 170 Asp Pro Thr Val His Leu 175 Asn Gly Ile Ser His Val 195 Giy 180 Ser Thr Leu Thr Asp 185 Thr Leu Ala Gly Ser Ser Ala 190 Arg Ala Ala Asp Ala Gly Asn Ser Thr His Tyr Thr 205 Ser Ile 210 Thr Gly 225 Lys Asp Val Leu Ser Thr Thr Gly 230 Asn 215 Ala Gly Trp Asn Ile 220 Lys Gly Val Lys Gin Ser Giu Asn Val 235 Asp Phe Val Arg Tyr Asp Thr Val Giu 245 Phe Leu Ser Ala Asp Thr 250 Lys Thr Thr Thr Val 255 Asn Val Giu Ser Lys Asp Asn Gly Lys Arg Thr Giu Val Lys Ile Gly 265 270 75 Ala Lys Thr Ser Val Ile Lys Glu 275 280 Lys Asp Gly Lys Val Thr Gly Lys Gly Lys Gly Glu 290 Asn Gly Ser Ser Thr 295 Asp Glu 300 Asn Lys 315 Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val 305 310 Ala Gly Trp Arg 320 Met Lys Thr Giu Thr Vai Thr Thr Ala 355 Thr Thr Ala 325 Asn Gly Gin Thr 330 Gly Gin Ala Asp Lys Phe 335 Thr Ser 340 Gly Thr Lys Thr Phe Ala Ser Gly Asn Gly 350 Thr Vai 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 Asp 390 Ser Lys Ala Val Ala 395 Gly Ser Ser Gly Val Ile Ser Gly Val Ser Pro Ser Lys 410 Gly Lys Met Asp Giu Thr 415 Val Asn Ile K2 Asn Ile Asp 435 Asn 420 Ala Giy Asn Asn Giu Ile Thr Arg Asn Gly Lys 430 Ser Val Ser Ile Ala Thr Ser Thr Pro Gin Phe Leu Gly 450 Aia Gly Ala Asp Ala 455 Pro Thr Leu Ser Asp Asp Giu Gly Ala 465 Leu Asn Vai Giy Ser 470 Lys Asp Ala Asn Pro Val Arg Ile Asn Vai Ala Pro Giy Val Lys Giu Giy Asp 485 490 Val Thr Asn Val Ala Gin 495 Leu Lys Gly Val Ala 500 Gin Asn Leu Asn 505 Asn Arq Ile Asp Asn Val Asp 510 Ala Gly Leu Gly Asn Ala Arg Ala Gly Ile 515 Ala Gin Ala Ile Ala Thr 520 525 76 Ala Gin Ala 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 Asp Thr Gly Asn Trp Val Ile Lys Gly 565 Ala Ser Gly Asn Ser Arg 575 Gly His Phe Gly 580 Thr Ser Ala Ser Val Gly 585 Tyr Gin Trp <210> 39 <211> 589 <212> PRT <213> Neisseria meningitidis (W00066741-0019) <400> 39 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Ser Thr Val Ala Thr Glu Leu Thr Arg His Thr Lys Arg Ala Ser Ala Thr Val Gin Ala Val Leu Ala Thr Leu Leu Ser Ala Asn Ala Thr Asp Thr Asp 55 Glu Asp Glu Glu Leu Glu Ser Val Val Arg Ser Ala Leu Val Leu 70 Gin Phe Met Ile Asp Lys Giu Gly Asn Glu Ile Giu Ser Thr Gly Asp Ile Gly Ser Ile Tyr Tyr Asp Asp His Asn Thr Leu 100 His Gly Ala Thr Thr Leu Lys Ala Gly Asp Asn 110 Leu Lys Lys Leu Lys Ile Lys Gin Ser Gly Lys 120 Asp Phe Thr Tyr Ser 125 Glu Leu Lys Asp Leu Thr Ser Val 130 135 Giu Thr Glu Lys 140 Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn Ile Thr Ser 145 150 Asp Thr Lys Gly Leu Asn 155 160 77 Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Pro Thr Val His Leu 175 Asn Gly Ile Ser His Val 195 Ser Thr Leu Thr Asp 185 Thr Leu Ala Gly Ser Ser Ala 190 Arg Ala Ala Asp Ala Gly Asn Ser Thr His Tyr Thr 205 Ser Ile .210 Lys Asp Val Leu Ala Gly Trp Asn Lys Gly Val Lys Thr 225 Gly Ser Thr Thr Gly 230 Gin Ser Glu Asn Asp Phe Val Arg Thr 240 Tyr Asp Thr Val Glu 245 Phe Leu Ser Ala Asp 250 Thr Lys Thr Thr Thr Val 255 Ile Gly Asn Val Glu Se r 260 Lys Asp Asn Gly Lys 265 Arg Thr Glu Val Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly 1 275 280 285 Lys Gly 290 Lys Gly Giu Asn Ser Ser Thr Asp Gly Giu Gly Leu Val1 305 Thr Ala Lys Giu Ile Asp Ala Val Lys Ala Gly Trp Arg 320 Met Lys Thr Thr Thr 325 Ala Asn Gly Gin Thr 330 Gly Gin Ala Asp Lys Phe 335 Glu Thr Val Thr Thr Ala 355 Thr 340 Ser Gly Thr Lys Val1 345 Thr Phe Ala Ser Gly Asn Gly 350 Thr Val Lys Thr Val Ser Lys Asp 360 Asp Gin Gly Asn Tyr Asp 370 Val Asn Val Gly Ala Leu Asn Val Asn 380 Gin Leu Gin Asn Ser Gly Trp Asn Leu Asp 385 390 Ser Lys Ala Val Ala 395 Giy Ser Ser Gly Lys 400 Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr 78 405 Val Asn Ile Asn Ala Giy Asn 420 Asn Ile Asp Ile Ala Thr Ser 435 Asn Ile Giu Ile Thr Arg 415 Asn Gly Lys 430 Ser Val Ser Met 440 Thr Pro Gin Phe Ser 445 Leu Gly Ala 450 Gly Ala Asp Pro Thr Leu Ser Asp Asp Giu Gly NO) Ala 465 Leu Asn Val Giy Ser 470 Lys Asp Ala Asn Pro Val Arg Ile Asn Vai Aia Pro Gly 485 Val Lys Giu Gly Asp 490 Val Thr Asn Val Ala Gin 495 Leu Lys Gly Gly Asn Ala 515 Val1 500 Ala Gin Asn Leu Asn Asn Arg Ile Asp 505 Asn Val Asp 510 Ala Gly Leu Arg Ala Gly Ile Ala 520 Gln Ala Ile Ala Ala Gin 530 Ala Tyr Leu Pro Lys Ser Met Met Ala 540 Ile Gly Gly Gly Thr 545 Tyr Arg Gly Giu Ala 550 Gly Tyr Ala Ile Gly 555 Tyr Ser Ser Ile Ser 560 Asp Thr Gly Asn Gly His Phe Gly 580 T rp 565 Val Ile Lys Gly Thr 570 Ala Ser Gly Asn Ser Arg 575 Thr Ser Ala Ser Val 585 Gly Tyr Gin Trp <210> <211> 595 <212> PRT <213> Neisseria meningitidis (WO006674i-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 Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 25 Thr Val Glu Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gin 79 Giu Leu Giu Pro Val Val Ala Asn Ala Thr Asp Thr Asp Glu Asp Asp 55 Arg Ser Ala Leu Val Leu Gin 70 Phe Met Ile Asp Lys 75 Giu Gly Asn Gly Glu Ile Giu Ser Gly Asp Ile Giy Trp Ser Ile Tyr Tyr Asp Asp NO) His Asn Thr Leu Lys Ile 115 His Giy Aia Thr Val1 105 Thr Leu Lys Ala Gly Asp Asn 110 Ser Ser Phe Lys Gin Asn Thr Giu Asn Thr Asn Ala 125 Thr Tyr 130 Giu Giu 145 Ser Leu Lys Lys Asp 135 Leu Thr Asp Leu Ser Val Gly Thr Leu Ser Phe Gly 150 Ala Asn Gly Asn Vai Asn Ile Thr Asp Thr Lys Gly Leu 165 Asn Phe Ala Lys Lys 170 Thr Ala Giy Thr Asn Gly 175 Asp Thr Thr Leu Ala Gly 195 Val1 180 His Leu Asn Gly Gly Ser Thr Leu Thr Asp Thr 190 Gin Ser Thr Ser Ser Ala Ser Val Asp Ala Gly K 2 His Tyr 210 Thr Arg Ala Ala Ser 215 Ile Lys Asp Val Leu 220 Asn Ala Gly Trp Asn 225 Ile Lys Gly Val Lys 230 Thr Gly Ser Thr Th r 235 Gly Gin Ser Giu Val Asp Phe Val Arg 245 Thr Tyr Asp Thr Val1 250 Glu Phe Leu Ser Ala Asp 255 Thr Lys Thr Thr 260 Thr Val Asn Val Giu 265 Ser Lys Asp Asn Gly Lys Arg 270 Giu Lys ASP Tht Giu Val Lys Ile Gly Ala Lys 275 280 Thr Ser Val Ile Lys 285 Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn 290 295 300 Gly Ser Ser Thr Asp Giu Gly Giu Giy Leu Val Thr Ala Lys Giu Val Ile Asp Ala 305 310 315 Asn Lys Ala Gly Trp 325 Arg Met Lys Thr Thr Ala Asn NO' Gly Gin Ala Asp Lys Phe Giu 340 Phe Ala Ser Gly Lys Gly Thr 355 Thr Val 345 Thr Ser Gly Thr Gly Gin Thr 335 Asn Vai Thr 350 Asp Asp Gin Ala Thr Vai Ser Giy Asn 370 Ile Thr Val Lys Tyr 375 Asp Val Asn Vai Gly 380 Asp Ala Leu Asn Asn Gln Leu Gin As n 390 Ser Gly Trp Asn Asp Ser Lys Ala Val1 400 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 Asp 420 Glu Thr Val Asn Asn Ala Gly Asn Asn Ile Glu 430 Asn Gly Lys Asn Asp Ile Ala Thr Ser Met Thr Pro 445 K Gin Phe 450 Ser Val 465 Ser Ser Val Ser Gly Ala Gly Ala Asp Ala Pro Thr Leu 460 Ser Lys Asp Ala Asn Asp Asp Glu Gly 470 Ala Leu Asn Val Lys Pro Val Arg Ile 485 Thr Asn Val Ala Pro 490 Gly Val Lys Glu Gly Asp 495 Val. Thr Asn His Ile Asp 515 Val1 500 Ala Gin Leu Lys Gly 505 Val Ala Gin Asn Leu Asn Asn 510 Ala Gin Ala Asn Val Asp Gly Asn 520 Ala Arg Ala Gly Ile 525 Ile Ala Thr Ala Gly Leu Val Gin 530 535 Ala Tyr Leu Pro 540 Gly Lys Ser Met Met Ala Ile Gly Gly Giv Thr Tyr Arg Gly 545 550 Gly Tyr Ser Ser Ile Ser Asp Gly Gly Asn 565 570 Ala Ser Gly Asn Ser Arg Gly His Phe Gly 580 585 Glu Ala Gly Tyr Ala Trp Ile Ile Lys Gly Thr 575 Ala Ser Ala Ser Val Gly 590 Tyr Gin Trp 595 <2.10> 41 <211> 600 <212> PRT <213> Neisseria meningitidis (wo0066741-0021) <400> 41 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ile 1 5 10 Ala Leu Asn Ala Trp Val Val Val Ser Glu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Val Ala Thr Ala Vai Leu Thr Leu Leu Ser Ala Ala Asn Ala Thr Asp Giu Asp Asn Glu Asp Giu Pro Val Val Arg. Thr Ala Pro Val Leu 70 Ser Phe His Ser Asp 75 Lys Glu Gly Thr Giy Giu Lys Giu Giu Gly Ala Ser Ser Leu Thr Val Tyr Phe Asp Lys Asn Arg Asn Leu Lys 115 Val1 100 Leu Lys Ala Gly Thr 105 Ile Thr Leu Lys Ala Gly Asp 110 Glu Asn Thr Ile Lys Gin Asn Thr 120 Asn Glu Asn Thr Asn 125 Asn 'Ala 130 Ser Ser Phe Thr Tyr 135 Ser Leu Lys Lys Asp 140 Leu Thr Gly Leu Ile Asn Val Glu Thr Glu Lys Leu 145 150 Ser Phe Gly 155 Ala Asn Gly Lys Lys 160 82 Val Asn Ilie TIe Ser Asp Thr Lys 165 Ala Gly Thr Asn Gly Asp Pro Thr 180 Gly Leu 170 Val His 185 Asn Phe Ala Lys Glu Thr 175 Leu Asn Gly Ile Gly Ser 190 Thr Leu Thr 195 Asp Thr Leu Ala Gly 200 Ser Ser Ala Ser Val Asp Ala Cl' Gly Asn 210 Gin Ser Thr His Tyr 215 Thr Arg Ala Ala Ser 220 Ile Lys Asp Val Leu 225 Asn Ala Gly Trp Ile Lys Gly Val Thr Gly Ser Thr Thr 240 Gly Gin Ser Giu Asn Val Asp Phe Val 245 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 Giu Val1 280 Lys Ile Gly Ala Lys 285 Ile Lys 290 Glu Lys Asp Gly Leu Val Thr Gly Gly Lys Gly Glu Asn 305 Gly Ser Ser Thr Giu Gly Glu Gly Leu 315 Val Thr Ala Lys Giu 320 Val Ile Asp Ala Asn Lys Ala Gly Arg Met Lys Thr Thr Thr 335 Ala Asn Gly Gly Thr Lys 355 Gin 340 Thr Gly Gin Ala Asp 345 Lys Phe Giu Thr Val Thr Ser 350 Ala Thr Val Val Thr Phe Ala Ser 360 Gly Asn Gly Thr Thr 365 Ser Lys 370 Asp Asp Gin Gly Asn 375 Ile Thr Val Lys Tyr 380 Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn 385 390 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 83 Val Ser Pro Ser Lys Gly Lys 420 Met Asp 425 Giu Thr Val Asn Ile Asn Ala 430 Gly Asn Asn 435 Ile Giu Ile Thr Arg Asn Gly Lys Asn Ile 440 445 Asp Ile Ala Thr Ser Met Thr Pro Gin Phe Ser Ser Val Ser 450 455 Leu Giy 460 Ala Gly Ala Asp 465 Ala Pro Thr Leu Ser 470 Val Asp Asp Giu Ala Leu Asn Val Ser Lys Asp Ala Lys Pro Val Arg Thr Asn Val Ala Pro Gly 495 Val Lys Giu Gin Asn Leu 515 Gi y 500 Asp Val Thr Asn Ala Gin Leu Lys Gly Val Ala 510 Ala Arg Ala Asn Asn Arg Ile Asp 520 Asn Val Asp Gly Asn 525 Gly Ile 530 Ala Gin Ala Ile Ala 535 Thr Ala Gly Leu Val 540 Gin Ala Tyr Leu Gly Lys Ser Met Met 550 Ala Ile Gly Gly Gly 555 Thr Tyr Arg Gly Glu 560 Ala Gly Tyr Aia Ile 565 Gly Tyr Ser Ser Ile 570 Ser Asp Gly Gly Asn Trp 575 1, Ile Ile Lys Giy 580 Thr Ala Ser Gly As n 585 Ser Arg Gly His Phe Gly Ala 590 Ser Ala Ser Val Gly Tyr Gin 595 Trp 600 <210> 42 <211> 594 <212> PRT <213> Neisseria meningitidis (W09931132-0002) <400> 42 Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 84 Thr Val Lys Thr Ala Val Ala Ser Ala Asn Asn Giu Leu Ala 40 Arg Pro Thr Leu Leu Phe Ala Thr Val Gin Arq Lys Lys Leu Tyr Leu Asp Val Gin Arg Thr Val1Ala Val Leu Ile Val Asn Ser Asp Lys 75 Glu Gly Thr Gly Glu Glu Lys Val Glu Giu Asn Ser Asp Trp Ala Val Tyr Phe Asn Aia Giy Asp 115 Glu 100 Lys Gly Val Leu Ala Arg Giu Ile Thr Leu Lys Phe Thr Tyr Asn Leu Lys Ile Gin Asn Gly Thr Ser Leu 130 Lys Lys Asp Leu Thr 135 Asp Leu Thr Ser Val1 140 Gly Thr Glu Lys Leu 145 Ser. Phe Ser Ala Gly Asn Lys Val Asn 155 Ile Thr Ser Asp Thr 160 Lys Gly Leu Asn Ala Lys Glu Thr Ala 170 Gly Thr Asn Gly Asp Thr 175 Thr Val Hi-s Asn Thr Gly 195 Leu 180 Asn Gly Ile Gly Thr Leu Thr Asp Thr Leu Leu 190 Thr Asp Asp Ala Thr Thr Asn Thr Asn Asp Asn ValI 205 Glu Lys 210 Lys Arg Ala Ala Ser 215 Val Lys Asp Val Leu Asn Ala Gly 220 Ser Asp Asn Val Trp Asp 240 As n 225 Ile Lys Gly Val Lys 230 Pro Gly Thr Thr Ala 235 Phe Val Arg Thr Tyr Asp Thr Val 245 Thr ,Thr Thr Val Asn Val Glu Ser 260 Phe 250 Leu Ser Ala Asp Thr Lys 255 Lys Asp Asn Gly Lys Lys Thr Glu 265 270 Val Lys Ile 275 Gly Val Lys Thr Ser Val Ile Lys Glu Lys 285 Gly Ser Ser 300 Asp Gly Lys Thr Asp Glu Leu Val 290 Thr Gly Lys Asp Gly Glu Asn Gly 305 Glu Gly Leu Val Thr 310 Ala Lys Glu Val Asp Ala Val Asn Ala Gly Trp Arg Lys Thr Thr Thr (N a> O ¢o O O Ala 330 Asn Gly Gln Thr Gly Gln 335 Ala Asp Lys Ser Gly Lys 355 Glu Thr Val Thr Ser 345 Gly Thr Asn Val Thr Phe Ala 350 Gln Gly Asn Gly Thr Thr Ala Val Ser Lys Asp Asp 365 Ile Thr 370 Val Met Tyr Asp Asn Val Gly Asp Gly Trp Asn Leu 395 Leu Asn Val Asn Leu Gly Leu Gin Asn Ser 390 Asp Ser Lys Ala Lys Val Ile Ser Ala Gly Ser Ser Gly 405 Gly 410 Asn Val Ser Pro Ser Lys 415 Gly Lys Met Ile Thr Arg 435 Asp 420 Glu Thr Val Asn Asn Ala Gly Asn Asn Ile Glu 430 Met Thr Pro Asn Gly Lys Asn Asp Ile Ala Thr Ser 445 Gin Phe 450 Ser Ser Val Ser Leu 455 Gly Ala Gly Ala Ala Pro Thr Leu Val Asp Gly Asp Ala 470 Leu Asn Val Gly Ser 475 Lys Lys Asp Asn Lys 480 Pro Val Arg Ile Thr 485 Asn Val Ala Pro Gly Val Lys Glu Gly 490 Asp Val 495 Thr Asn Val Ala 500 Gln Leu Lys Gly Val 505 Ala Gin Asn Leu Asn Asn Arg 510 Gin Ala Ile Ile Asp Asn Val Asp Gly Asn 515 Ala Arg 520 Ala Gly Ile Ala 525 -86- Ala Thr 530 Ala Gly Leu Val Ala Tyr Leu Pro Gly 540 Lys Ser Met Met Ala 545 Ile Gly Gly Giy Thr 550 Ser Asp 565 Tyr Arg Gly Giu Giy Tyr Ala Ile Tyr Ser Ser Ile Gly Gly Asn Trp 570 Ile Ile Lys Gly Thr Ala 575 NO' Ser Gly Asn Se r 580 Arg Gly His Phe Giy 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 Asn Lys Ilie Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Ala Val Ser Glu Leu Thr Arg Asn 25 His Thr Lys Arg Ala Ser Ala Thr Val Gin Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe 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 Gly Lys Giu Val Thr Asp Ser Asn Trp Val Tyr Phe Asp Lys Lys Gly Vai Leu Lys Ile Lys Tyr Ser Leu 130 Thr 100 Ala Gly Thr Ile Thr 105 Leu Lys Ala Giy Asp Asn Leu 110 Ser Phe Thr Gin Asn Thr Asn Giu 120 Leu Thr 135 Asn Thr Asn Ala Ser 125 Lys Lys Asp Asp Leu Thr Ser Val Gly Thr Giu 140 87 Ly's Leu Ser Phe Ser Ala Asn Ser Asn Lys Val Asn Ile Thr Ser Asp 160 Thr Lys Gly Leu Phe Ala Lys Lys Thr 170 Ala Glu Thr Asn Gly Asp 175 Thr Thr Val Leu Asn Thr 195 Leu Asn Gly Ile Ser Thr Leu Thr Asp Thr Leu 190 Val Thr Asp Gly Ala Thr Thr As n 200 Val Thr Asn Asp Asn 205 Asp Glu 210 Lys Lys Arg Ala Ala 215 Ser Val Lys Asp Val1 220 Leu Asn Ala Gly T rp 225 Asn Ile Lys Gly Val1 230 Lys Pro Gly Thr Thr 235 Ala Ser Asp Asn Asp Phe Val Arg Thr 245 Tyr Asp Thr Val Giu 250 Phe Leu Ser Ala Asp Thr 255 Lys Thr Thr Glu Val Lys 275 Val Asn Val Giu Lys Asp Asn Gly Lys Arg Thr 270 Lys Asp Gly Ile Gly Ala Lys Th r 280 Ser Val Ile Lys Giu 285 Lys Leu 290 Val Thr Gly Lys Asp 295 Lys Gly Giu Asn Asp 300 Ser Ser Thr Asp ,,Lys 305 Gly Giu 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 Thr 330 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 Asn Val Thr Phe 350 Asp Gin Gly Lys Gly Thr Thr Ala 360 Thr Val Ser Lys Asp 365 Asn Ile 370 Thr Val Met Tyr Asp 375 Val Asn Val Gly Asp 380 Asp Ser 395 Ala Leu Asn Val Lys Ala Val Ala 400 Asn Gin Leu Gin Asn Ser 385 390 Gly Trp Asn Leu 88 Gly Ser Ser Gly Val Ile Ser Gly Asn Val 410 Ser Pro Ser Lys Gly 415 Lys Met Asp Thr Arg Asn 435 Giu 420 Thr Val Asn Ile Ala Gly Asn Asn Ile Giu Ile 430 Thr Pro Gin Gly Lys Asn Ile Asp 440 Ile Ala Thr Ser NO) Phe Ser 450 Ser Val Ser Leu Ala Gly Ala Asp Ala Pro Thr Leu 460 Lys Asp Ala Asn Ser Asp Asp Giu Gly Leu Asn Val Gly Ser 475 Lys 480 Pro Val Arg Ile Th r 485 Asn Val Ala Pro Gly 490 Val Lys Giu Giy Asp Val 495 Thr Asn Val Ile Asp Asn 515 Al a 500 Gin Leu Lys Gly Ala Gin Asn Leu Asn Asn His 510 Gin Ala Ile Val Asp Gly Asn Ala 520 Arg Ala Giy Ile Ala 525 Ala Thr 530 Ala Gly Leu Val Ala Tyr Leu Pro Lys Ser Met Met Ala 545 Ile Gly Gly Gly Th r 550 Tyr Arg Giy Giu Giy Tyr Ala Ile Tyr Ser Ser Ile Ser 565 Asp Gly Gly Asn T rp 570 Ile Ile Lys Gly Thr Ala 575 Ser Gly Asn Ser 580 Arg Gly His Phe Gly 585 Ala Ser Ala Ser Val Gly 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 Ser Thr Val Lys Thr Glu Leu Thr Arg Asn His Thr Lys Thr Leu Leu Phe Arg Ala Ser Ala Ala Val Leu Thr Val Gin Ala Ser Ala Asn Asn Giu Gin Giu Glu Asp Tyr Leu Asp Pro Gin Arg Thr Val Val Leu Ile Val Ser Asp Lys Giu Gly Thr Gly Giu Lys Giu Lys Val Giu Glu Ser Asp Trp Ala Val Tyr Phe Asn Giu Gly Asp Asn 115 Gly Val Leu Thr Ala 105 Arg Glu 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 Asp 135 Leu Thr Ser Val Gly 140 Thr Giu Lys Leu Phe Ser Ala Asn Gly 150 Asn Lys Val Asn Thr Ser Asp Thr Gly Leu Asn Phe Ala 165 Lys Giu Thr Ala Thr Asn Gly Asp Thr Thr 175 K. 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 Val Thr 200 Asn Asp Asn Val Thr 205 Lys Lys 210 Ile Lys 225 Arg Ala Ala Ser Val1 215 Lys Asp Val Leu Asn 220 Ala Gly Trp Asn Gly Val Lys Pro Gly 230 Thr Thr Ala Ser Asp 235 Asn Val Asp Phe 240 Val Arg Thr Tyr Asp Thr Val Giu Phe Leu 245 250 Ser Ala Asp Thr Lys Thr 255 Thr Thr Val Asn Val Giu Ser Lys Asp Asn Gly Lys Lys Thr Giu Val 90 270 Glu Lys Asp Gly Lys Leu 285 Lys Ile Gly Ala Lys Thr Ser Val Ile Lys 275 280 Va1 Thir Gly Lys Asp Lys Gly 295 Giu Asn Gly Ser 300 Ala Val Asn Lys Ala Giu 305 Gly Leu Val Thr Lys Giu Val Ile Gly Trp Arg Met Thr Thr Thr Ala Gly Gin Thr Gly Gin Ala 335 Asp Lys Phe Gly Lys Gly 355 Giu 340 Thr Val Thr Ser Gly 345 Thr Asn Val Thr Phe Ala Ser 350 Gly Asn Ile Thr Thr Ala Thr Val1 360 Ser Lys Asp Asp Thr Val 370 Met Tyr Asp Val Asn 375 Val Gly Asp Ala Leu 380 Asn Val Asn Gin Le u 385 Ser Gin Asn Ser Gly Trp 390 Asn Leu Asp Ser Ala Val Ala Gly Gly Lys Val Ser Gly Asn Val Ser 410 Pro Ser Lys Gly Lys Met 415 Asp Giu Thr Val 420 Asn Ile Asn Ala Gly 425 Asn Asn Ile Giu Ile Thr Arg 430 AsnGiy yLys 435 Asn Ile Asp Ile Ala 440 Thr Ser Met Thr Pro Gin Phe Ser 445 Ser Val 450 Gly Asp 465 Ser Leu Giy Ala Gly 455 Ala Asp Ala Pro Leu Ser Val Asp Ala Leu Asn Val Gly 470 Ser Lys Lys Asp 475 Asn Lys Pro Val Ile Thr Asn Val Ala 485 Pro Gly Val Lys Giu 490 Giy Asp Vai Thr Asn Val 495 Ala Gin Leu Lys Gly Val Ala Gin 500 Asn Leu Asn Asn Arg Ile Asp Asn 510 -91 Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gin Ala 515 520 Giy Leu Val Gin Ala Tyr Leu Pro Giy Lys Ser Met 530 535 540 Ile Ala 'rhr Ala 525 Met Aia Ile Gly Giy 545 Giy Thr Tyr Arg Giy Giu Aia Giy Tyr Aia 550 555 Ile Giy Tyr Ser Ile Ser Asp Giy Gly Asn Trp Ile Ilie Lys Giy 565 570 Thr Ala Ser Giy Asn 575 Ser Arg Giy His Phe Giy Aia Ser Aia Ser Vai Giy Tyr Gin Trp 580 585 590

类似技术:

---

公开号 | 公开日 | 专利标题

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AU2009200692B2|2011-04-14|Outer membrane vesicle | vaccine comprising N. meningitidis serogroup B outer membrane proteins

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EP1228217B1|2012-11-21|Conserved neisserial antigens

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AU2006200732B2|2008-11-20|Outer membrane vesicle | vaccine comprising N. meningitidis serogroup B outer membrane proteins

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ES2348657T3|2010-12-10|EXTERNAL MEMBRANE VESICULA | VACCINE THAT INCLUDES EXTERNAL MEMBRANE PROTEINS OF SEROGROUP B OF N. MENINGITIDIS.

---

AU2004240199B2|2007-05-17|Conserved Neisserial antigens

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法律状态:
2011-08-11| FGA| Letters patent sealed or granted (standard patent)|

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2017-09-28| PC| Assignment registered|Owner name: GLAXOSMITHKLINE BIOLOGICALS S.A. Free format text: FORMER OWNER(S): NOVARTIS VACCINES AND DIAGNOSTICS S.R.L. |

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2019-08-15| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

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