Outer membrane vesicle vaccine (omv) containing proteins of serogroup b n.meningitis outer membrane

FIELD: immunology.

SUBSTANCE: invention relates to composition containing as the first component serogroup B N.meningitis outer membrane vesicle, and as the second one treatment immunogenic component associated with brain fever or other Nisseria infections.

EFFECT: immunotherapy for treatment of Nisseria infections with increased effectiveness.

9 cl, 5 tbl

 

The technical FIELD

This invention relates to vaccines against Neisseria meningitidis, serological group b (NmB).

BACKGROUND of the INVENTION

All documents cited here included by reference in their entirety.

Neisseria meningitidis is a gram-negative stationary diplokokkovyh human pathogen. She colonize the throat, causing meningitis, and occasionally in the absence of meningitis septicaemia. In the United States, the level of infection equal to 0.6 to 1 per 100,000 people per year, and it may be higher during outbreaks of the epidemic (see Lieberman et al. (1996) JAMA 275 (19). 1499-1503; Schuchat et al. (1997) N Engl J Med 337 (14):970-976). In developing countries the rate of endemic diseases are much higher during epidemics levels of morbidity can reach 500 cases per 100,000 people per year. Mortality is very high, 10-20% in the United States and much higher in developing countries. After the introduction of conjugate vaccines against Haemophilus influenzae, N. meningitidis is the leading cause of bacterial meningitis for all ages in the United States (Schuchat et al. (1997) supra).

Based on the capsular polysaccharide of this organism have been identified 12 serological groups of N. meningitidis. Meningococcal vaccine currently used is a tetravalent vaccine, composed of serolog the economic groups And, C, Y and W135. However, after the success of vaccination against N. influenzae were developed conjugate vaccine serological groups a and C.

However, serological group remains a challenge, and she is currently responsible for approximately 50% of cases overall meningitis in the United States, Europe and South Africa. Polysaccharide approach cannot be used because menB capsular polysaccharide is a polymer α (2-8)-linked N-acetylneuraminic acid, which is also present in mammalian tissues. This leads to resistance to the antigen; in fact, if the response was induced, it was a reaction against autoantigen and, therefore, undesirable. In order to avoid induction of autoimmunity and for the induction of protective immune response this capsular polysaccharide was, for example, chemically modified by replacing the N-acetyl group of N-propylaniline groups, keeping unchanged the specific antigenicity (Romero & Outschoorn (1994) Clin Rev Environ 7(4):559-575).

An effective vaccine bubbles (vesicles) of the outer membrane (OMV) against serological group b was obtained from the Norwegian National Institute of Public Health [see, for example, Bjune et al. (1991) Lancet 338(8775):1093-96]. Although this vaccine is safe and prevents disease NmB, its effectiveness is limited to the s strain, used to prepare the vaccine. It was also reported for other vaccines on the basis of preparations of outer membrane. The purpose of this invention is the extension of these vaccines for other strains.

Description of the INVENTION

Unexpectedly, it was found that further addition of certain components to the OMV vaccines greatly enhances their effectiveness.

Thus, this invention provides a composition comprising (a) a drug NmB outer membrane and (b) immunogenic component is selected from one or more of the following components:

protein disclosed in WO 99/57280, or immunogenic fragment;

protein disclosed in WO 99/36544, or immunogenic fragment;

protein disclosed in WO 99/24578, or immunogenic fragment;

protein disclosed in WO 99/66791, or immunogenic fragment;

protein, revealed in the work Tettelin et al. [Science (2000) 287:1809-1815], or immunogenic fragment;

protein, revealed in the work of Parkhill et al. [Nature (2000) 404:502-506], or immunogenic fragment;

protein disclosed in WO 97/28273, or immunogenic fragment;

protein disclosed in WO 96/29412, or immunogenic fragment;

protein disclosed in WO 95/03413, or immunogenic fragment;

protein disclosed in WO 99/31132, or immunogenic fragment;

protein disclosed in WO 99/58683, or immunogenic fragment

protein disclosed in WO 99/55873,or immunogenic fragment; and/or

protein PorA, TbpA, TbpB, PilC, Ora or Neisseria meningitidis Omp85.

If this composition contains protein described in WO 99/24578 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 76, 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 and 892, as described in WO 99/24578 (or a protein containing an immunogenic fragment of one or more of these SEQ IDS, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these SEQ ID).

If this composition contains protein described in WO 99/36544 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID NO: 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 and 90, as described in W099/36544 (or a protein containing an immunogenic fragment of one or more of these SEQ IDS, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these SEQ ID).

If this composition contains protein, described in Tettelin et al. (i.e. the protein encoded by one of the genes described in this work), this protein preferably contains an amino acid sequence selected from the group consisting of NMB0001-NMB2160 (or a protein containing an immunogenic fragment of one of the Il is a few of these 2160 genes or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these 2160 genes).

If this composition contains protein, described in Parkhill et al., this protein preferably contains an amino acid sequence selected from the group consisting of 2121 coding sequences described in this work (or a protein containing an immunogenic fragment of one or more of these 2121 sequence, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these 2121 sequences).

If this composition contains protein described in WO 99/57280 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 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, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 10.94, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 136, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, I486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, I860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 19.46, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036,2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 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, 840, 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 and 3020, as described in WO 99/572 .80 (or a protein containing an immunogenic fragment of one or more of these SEQ ID or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these SEQ ID).

If this composition contains protein, described in W099/28273, this protein is preferably a protein presented on figure 4 or figure 13 WO 97/28273.

If this composition contains protein described in WO 96/29412 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID nos:1-8, described in WO 96/29412 (or a protein containing an immunogenic fragment of one or more of these SEQ IDS, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these SEQ ID).

If this composition contains protein described in WO 95/03413 specified protein preferably contains the amino acid th is sequence, selected from the group consisting of SEQ ID NO:1-23, described in WO 95/03413 (or a protein containing an immunogenic fragment of one or more of these SEQ IDS, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with one of these SEQ ID).

If this composition contains protein described in WO 99/31132 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID NO:2 described in WO 99/31132 (or a protein containing an immunogenic fragment of SEQ ID NO:2, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) to SEQ ID NO:2).

If this composition contains protein described in WO 99/58683 specified protein preferably contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 4, described in WO 99/58683 (or a protein containing an immunogenic fragment of SEQ ID NO: 2 or SEQ ID NO: 4, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) SEQ ID NO:2 or SEQ ID NO:4).

If this composition contains protein described in WO 99/55873 specified protein preferably contains the amino acid p is the sequence, selected from the group consisting of SEQ ID NO:2 or SEQ ID NO:4, described in WO 99/55873 (or a protein containing an immunogenic fragment of SEQ ID NO:2 or SEQ ID NO:4, or a protein containing a sequence having sequence identity (preferably higher than 50%, for example, 60%, 70%, 80%, 90%, 95%, 99% or more) to SEQ ID.NO:2 or SEQ ID NO:4).

Details on Ora and PorA can be found in the work Wiertz et al. [Infect. Iinmun. (1996) 61:298-304]. PilC described by Nassif et al. [PNAS USA (1994) 91:3769-73]. Omp85 described in Manning et al. [Microb. Pathog. (1998) 25:11-21]. TbpA and TbpB described in the work of the Ala Aldeen & Borriello [Vaccine (1996) 14:49-53], and Legrain et al. [Protein Expr Purif (1995) 6:570-578].

Preferred proteins for the component (b) are:

protein '919', submitted SEQ ID NO:3069-3074 and 3207-3241 WO 99/57280 (see also figure 23 and example 15 in it).

protein '235', submitted SEQ ID NO:869-874 and 3149-3178 WO 99/57280 (see also figure 20 and example 12 in it).

protein '519', submitted SEQ ID NO:3045-3056 and 3185-3206 WO 99/57280 (see also figure 22 example 14).

protein '225', submitted SEQ ID NO:793-804 and 3115-3148 WO 99/57280 (see also figure 19 and example 11).

protein 'ORF40', represented by example 1 (SEQ ID NO:1-6) WO 99/36544 (see also figure 1 WO 00/66741; see also WO 99/31132 and WO 99/58683).

protein '287', represented by the example 9 W099/57280 (see SEQ ID NO:1199-1204, 3103-3108 and 3179-3184 in it).

protein 'ORF1'submitted by example 77 (SEQ ID NO:647-654) WO 99/24578 (see also WO 99/55873 and the access number AJ 242535).

- the trees 'ORF4', submitted by example 26 (SEQ ID NO:215-2 .26) WO 00/24578 (see also figure 2 WO 00/66741).

protein 'ORF46'submitted by example 55 (SEQ ID. NO:457-466) WO 99/24578 (see also figure 12 WO 00/66741).

Component (b) of this composition is preferably a protein NmB. Preferably component (b) includes protein from strain NmB different from the strain that the OMV component (a), i.e. the OMV component (a) preferably supplemented immunogenic component (b) from a different strain NmB.

One or more components (or all components) can be adsorbed on Al(OH)3.

Component of the outer membrane preparation

The compositions of this invention include the preparation NmB outer membrane as a component (a). Preferably it is in the form of bubbles (vesicles) of the outer membrane (OMV).

Getting OMV from NmB is well known in this field. Methods of obtaining suitable preparations are described, for example, in the works: 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. [J.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. (1998) 66:959-965]; Rosenqvist et al. [Dev. Biol. Stand. (1998) 92:323-333]; Haneberg et al. [Infect. Iinmun. (1994) 62:4419-4424]; Naess et al. [Infect. Immun. (1998).66:1334-41]; Andersen et al. [Vaccine (1997) 15:1225-34]; Bjune et al. [Lancet (1991) 338:1093-96] ets.

OMV represent is preferably deoxycholate extract from NmB (i.e. obtained from NmB extraction dezoksiholatom). The preferred extraction Protocol is a Protocol that describes Fredriksen et al. [Production, characterization and control of MenB-vaccine "Folkehelsa": an outer membrane vesicle vaccine against group In meningococcal disease (1991) NIPH Ann. 14(2):67-80].

The preferred strain, which should be extracted OMV is the strain 44/76 (B:15:R,16:P5.5:L3,7,9) N. meningitidis.

Additional details concerning OMV component can be found, for example, in the works 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 In meningococcal disease. Pages 818-824 of Pathobiology and Immunology of Neisseriaceae (eds. Conde-Glez et al.) ISBN 968-6502-13-0].

Component OMV can be adsorbed on the Freund aluminum hydroxide. The preferred ratio of protein : adjuvant is 1:67 (mass/mass).

The typical dose human vaccine contains 25 micrograms of protein, 2 μg LPS and 1.67 mg Al(OH)3and can be injected in volumes of 0.5 ml in the deltoid muscle.

Component OMV (e.g., obtained by extraction dezoksiholatom) can be processed to remove certain components. For example, can be removed pyrogens or toxic components (e.g., LPS).

Preferably component OMV should retain antigenic component 80 kDa described Fredriksen et al. [pages 818-824 of Pathobiology and Immunobiology of Neisseriaceae].

More preferably component OMV should save the take protein, containing one or more of the following amino acid sequences: SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 [or (i) a protein having sequence identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13, depending on the specific SEQ ID degree of sequence identity, preferably is greater than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more), which includes mutants and allelic variants, or (ii) a protein containing immunogenic fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13 fragment must contain at least n consecutive amino acids from this sequence, and, depending on the particular sequence, n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more)].

Combining components (a) and (b)

Components (a) and (b) can be combined by simple mixing of the component (a) preparation of outer membranes (for example, by mixing ORF4 with the Norwegian OMV).

Alternatively, they can be combined" manipulation of bacteria in such a way that it produces (preferably overproducer) component (a) in its outer membrane - preparation of outer membranes of such recombinant bacteria will contain as component (a)and component (b).

Suitable bacteria for manipulation, so about the time, include Neisseria meningitidis (any serological group or strains), Neisseria lactamica, Neisseria cinerea, or any other which does not allow typing of Neisseria. Can also be used gram-negative bacteria, such as E. coli, Salmonella, Shigella, Bordetella, Yersinia, Helicobacter, etc. Methods of transformation are well known in this field.

Multivalent vaccines

Optionally, the composition of this invention may also contain one or more of the following components:

- protective antigen against serological group a Neisseria meningitidis;

- protective antigen against serological group C Neisseria meningitidis;

- protective antigen against serological group Neisseria meningitidis;

- protective antigen against serological group W Neisseria meningitidis;

- protective antigen against Haemophilus influenzae;

- protective antigen against Pneumococcus;

- protective antigen against diphtheria;

- protective antigen against tetanus;

- protective antigen against pertussis;

- protective antigen against Helicobacter pylori;

- protective antigen against polio; and/or

- protective antigen against hepatitis C.

Preferred examples of these optional components are:

- polysaccharide antigen against serological group a Neisseria meningitidis;

- polysaccharide antigen against serological group C Neisseria meningitidis, such as antigen described Constantino et al. (1992) Vaccine 10:691-698;

- polysaccharide antigen against serological group Neisseria meningitidis;

- polysaccharide antigen against serological group W Neisseria meningitidis;

- polysaccharide antigen against Haemophilus influenzae;

- polysaccharide antigen against Pneumococcus;

- protective antigen against diphtheria, consisting of diphtheria toxoid, for example mutant CRM197 [see, for example, Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70].

- protective antigen against tetanus, consisting of tetanus toxoid [see, for example, Wassilak & Orenstein, Chapter 4 of Vaccine (eds. Plotkin &Mortimer), 1988].

- protective antigen against pertussis containing holotoxin pertussis (PT) and filamentous hemagglutinin (FHA); optionally containing additional pertactin and/or agglutinogens 2 and 3 [see, e.g., Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238].

- protective antigen against N. pylori containing one or more of CagA (for example, WO 93/18150), VacA (for example, WO 93/18150), NAP (for example, WO 99/53310), Nor (for example, WO 98/04702), HopY (for example, WO 98/04702), urease.

- protective antigen against hepatitis b virus, consisting of a surface antigen of HBV and/or crustal antigen of HBV.

If the composition comprises an antigen against diphtheria, it preferably also contains antigens against tetanus and polio. If the composition comprises an antigen against tetanus, it preferably contains takeanother against diphtheria and polio. If the composition comprises an antigen against polio, it preferably also contains antigens against diphtheria and tetanus.

The pertussis toxin is toxic protein and, if it is present in the composition, it is preferably detoxificating. Detoxification can be chemical and/or may be genetic methods. Preferred detoxificating mutant is a double mutant 9K/129G [see, for example, Rappuoli (1997) Nature Medicine 3:374-376].

If the composition includes a protein that exists in a variety of nascent and Mature forms, use preferably the Mature form of this protein. For example, if you have enabled NspA protein (WO 96/29412; see also Martin et al. (1997) J. Exp. Med. 185 1173-1183), preferably using Mature form of this protein lacking the signal peptide.

If the composition includes a polysaccharide antigen, the polysaccharide preferably anywhereman with protein carrier.

Therapy, prevention, diagnosis

The composition of this invention preferably is a vaccine. Vaccines in accordance with this invention may either be prophylactic (i.e. to prevent infection)or therapeutic (i.e. to treat disease after infection).

This invention also provides compositions of the present invention for use in ka is este drugs (preferably vaccines or diagnostic reagents. It also provides the use of compositions in accordance with this invention in the preparation of: (i) a medicinal product for treating or preventing infection caused by the bacteria Neisseria; (ii) a diagnostic reagent for detecting the presence of bacteria Neisseria or antibodies induced against bacteria Neisseria; and/or (iii) reagent, which can induce antibodies against the bacterium Neisseria. These bacteria Neisseria can be any species or strain (such as Neisseria gonorrhoeae), but preferably they are N. meningitidis, in particular, serological group b (NmB).

This invention also provides a method of treatment of a patient, introducing a patient a therapeutically effective amount of a composition of the present invention. This method is preferably immunization.

Ways

According to further aspects, the invention provides various methods.

Provided is a method of obtaining the composition of the present invention, providing phase extraction (for example, extraction dezoksiholatom) OMV from N. meningitidis.

The LIST of SEQUENCES

In the list of sequences represented by the following sequence:

SEQ ID NO:DESCRIPTION
1N-terminal placentas is the activity of the protein 80-85 kDa N. meningitidis sero-group
2Full gene from N. meningitidis sero-group
3The encoded protein of SEQ ID NO:2
4The signal sequence of the protein of SEQ ID NO:3
5The Mature protein of SEQ ID NO:3
6Full gene from N. gonorrhoeae, which is homologous to SEQ ID NO:2
7The encoded protein of SEQ ID NO:6
8The signal peptide of the protein of SEQ ID NO:7
9The Mature protein of SEQ ID NO:7
10Full gene from N. meningitidis sero-groups A, homologous to SEQ ID NO:2
11The encoded protein of SEQ ID NO:10
12The signal peptide of the protein of SEQ ID NO:11
13The Mature protein of SEQ ID NO:11
14Protein '919' from strain 2996 NmB

WAYS of CARRYING out the INVENTION

What follows is a summary of standard methods and procedures that can be used to perform the present invention (for example, the application of the described sequences for vaccination or for diagnostic purposes). This summary is not a limitation on this invention, but rather gives examples which may be used, but it is not required.

A common part

The practice of this invention will be used, unless otherwise indicated, conventional molecular biology, Microbiology, recombinant DNA and immunology, which are known to experts in this field. Such techniques are explained fully in the literature, for example, Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait, ed, 1984); Nucleic Acid Hybridization (B.D. Hames &S.J. Higgins, eds. 1984); and reduced Translation (B.D. Hames &S.J. Higgins, eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. 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.Y.), and Handbook of Experimental Immunology, Volumes IV (D.M. Weir and C.C. Blackwell eds 1986).

This application uses standard abbreviations for nucleotides and amino acids.

Proteins used with this invention, can be obtained in various ways (e.g., by recombinant expression, purification from cell culture, chemical synthesis etc) and in various forms (e.g. native proteins, fused proteins, and so on). Preferably, they get essentially pure (i.e. essentially not containing other Belko is Neisseria or proteins of the host cell).

Nucleic acids used with this invention, can be obtained in many ways (for example, by chemical synthesis, from the libraries of genomic DNA or cDNA, from the organism itself, etc. and can be in different forms (for example, in the form of single-stranded, double-stranded DNA, vectors, probes, etc). The term "nucleic acid" includes DNA and RNA, as well as their analogues, such as analogues containing modified skeletons, and peptidoglicanova acid (NCP), etc.

Definition

A composition containing X, is "essentially not containing Y, when at least 85% by weight of the total amount of X+Y in the composition is X. Preferably, X is at least about 90% by weight of the total amount of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term "comprising" means "including"and "comprising", for example, a composition "comprising" X may consist exclusively of X or may include something additional to X, for example, X+y

The term "heterologous" refers to two biological components that are not found together in nature. These components can be cell-hosts, genes or regulatory regions such as promoters. Although heterologous components are not found together in nature, they can function is to make plans in connection together as, for example, when a promoter for heterologous gene functionally linked to the gene. Another example is the case when the sequence of Neisseria is heterologous to the mouse host cell. Additional examples could be two epitope from the same or different proteins, which were assembled into a single protein in a location that is not found in nature.

"The replication origin (origin) is a polynucleotide sequence which initiates and regulates the replication of polynucleotides, such as expressing vector. The replication origin behaves as an Autonomous unit of replication polynucleotides in the cell, capable of replication under its own control. The replication origin may be necessary for replication of the vector in a specific cell host. With defined start points replication expressing vector can be reproduced with a high number of copies in the presence of suitable proteins in the cell. Examples of start points are Autonomous replication can replicate sequences that are effective in yeast, and viral T-antigen, effective in cells COS-7.

The identity between proteins preferably determined using the algorithm of the search for homology Smith-Waterman implemented in the MPSRCH program (OxfordMolecular) using search affine gaps with the parameters of the fine open gap=12 and fine extension gap=1. Usually 50%or higher identity between the two proteins is considered to be an indication of functional equivalence.

In the application here, "allelic variant" nucleic acid molecule or region of a nucleic acid, which provided the sequence of the nucleic acid is a nucleic acid molecule or region of nucleic acid that are found essentially in the same locus in the genome of another or second isolate, and a molecule that due to natural variability due to, for example, mutation or recombination, has a similar but not identical sequence of nucleic acid. Allelic variant of the coding region is typically encodes a protein having similar activity to the activity of the proteins encoded by the genome with which it was compared. Allelic variant can include changes in 5'- or 3'-untranslated regions of the gene, for example, regulatory regions (for example, see U.S. patent US 5753235).

Expression systems

The nucleotide sequence of Neisseria can be expressed in various expression systems; for example, the expression systems that are used by mammalian cells, baculo viruses, plants, bacteria and yeast.

i. System of mammals

Expression systems mammalian known in the Noi area. A mammalian promoter is any DNA sequence that can bind RNA polymerase mammals and initiate in the direction of transcription (3') transcription of the coding sequence (e.g., structural gene) into mRNA. The promoter will have the area of transcription initiation, which is usually located proximally relative to the 5'-end of the coding sequence, and a TATA-block, usually located 25-30 base pairs (BP) above (progress against transcription) from the site of transcription initiation. It is expected that TATA-block specifies the start of RNA synthesis by RNA-polymerase II in the right place. A mammalian promoter also contains left promotor element, usually located at 100-200 BP above TATA-box. Left promotor element determines the rate at which transcription is initiated and can operate in any orientation [Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In Molecular Cloning: A Laboratory Manual, 2ndEd. (1989)].

Genes of mammalian viruses are often vysokoagressivnyh and have a wide range of hosts; thus, sequences encoding genes of mammalian viruses, are especially applicable to the promoter sequence. Examples include the early SV40 Promoter, LTR promoter, murine virus, tumors of the mammary gland, the major late promoter and is of rovirosa (Ad MLP), and the promoter of the herpes simplex virus. In addition, the sequence produced from non-viral genes such as the gene mouse metallothionein also provide the applicable promoter sequence. The expression can be either constitutive or regulated (inducible), depending on whether the promoter to the induction by glucocorticoid in hormone-sensitive cells.

The presence of the enhancer element (enhancer), combined with the above-described promoter elements will typically increase the levels of transcription. Enhancer is a regulatory DNA sequence that can stimulate the transcription of up to 1000-fold level when linking with homologous or heterologous promoters, the synthesis begins at the normal starting (triggering) the website RNA. Enhancers are also active when placed against the course of transcription (above) or in the middle of transcription (below) from the site of transcription initiation, either in normal or in reverse 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, 2nded.]. Enhancer elements are made from viruses that may be particularly applicable, because they usually have a wide range of hosts. Examples include enhancer early gene, SV40 [Dijkema et al. (1985) EMBO J. 4:761] and enhancer/Pro is otori, made of long terminal repeat (LTR) of rous sarcoma virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1989) Cell 41:521]. In addition, some enhancers are adjustable and become active only in the presence of inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

The DNA molecule may be expressed intracellularly in mammalian cells. The promoter sequence may be directly linked with this DNA molecule, and in this case the first amino acid at the N end of the recombinant protein will always be a methionine, which is encoded by an initiating ATG codon. If desirable, this N-end can be derived from protein by incubation in vitro with cyanogenmod.

Alternatively, foreign proteins can also be secreted from the cell into the culture medium by creating chimeric DNA molecules that encode a protein consisting of a fragment of a leader sequence that provides secretion of a foreign protein in mammalian cells. Preferably there are processing sites encoded between the leader fragment and the alien genome, which can be derived in vivo or in vitro. The fragment leader sequence typically encodes a signal peptide comprised of hydrophobin the x amino acids, that control the secretion of the protein from the cell. Adenovirus leader, consisting of three parts, is an example of a leader sequence that provides secretion of a foreign protein in mammalian cells.

Usually the sequence termination and polyadenylation, recognizable by the mammalian cells are regulatory regions located 3' (right) relative to the stop codon broadcast and, therefore, together with the promoter elements that flank the coding sequence. the 3'end of the Mature mRNA is formed site-specific post-translational cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukariotic RNA. In reduced and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of mRNA that can be translated into the polypeptide encoded by this DNA. Examples of signals transcription terminator/polyadenylation include signals produced from SV40 [Sambrook et al. (1989) "Expression of Cloned Genes in Cultured Mammalian Cells." In Molecular Cloning: A Laboratory Manual, 2ndEd. (1989)].

Usually the above-described components, containing the promoter, the polyadenylation signal and the sequence termination of transcription, put together in the expression construct. Enhancers, introns with functional donor and acceptor SIG the mi splicing and leader sequence can also be included in the expression construct, if desirable. Expression design is often stored in a replicon, such as an extrachromosomal element (e.g., plasmids)capable of stable maintenance in a host, such as mammalian cells or bacteria. The replication system of mammals include system derived from animal viruses, which require TRANS-acting factors for replication. For example, a plasmid containing a replication system 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 replicons mammals include replicons derived from bovine papillomavirus and Epstein-Barr. Additionally, the replicon may have two replication system that allows it to persist, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such Shuttle (mammal-bacteria) vectors include pmt2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The process of transformation depends on the subject of transformation of the host. Methods of introduction of heterologous polynucleotides in mammalian cells are known in this field and include indirect dextr the nom transfection, the calcium-phosphate precipitation, indirect polybrene transfection, fusion of protoplasts, electroporation, encapsulation of polynucleotide (polynucleotides) in liposomes, and direct microinjection of the DNA in the nucleus.

Lines of mammalian cells, available as hosts for expression are known in this area and include many immortalized cell lines available from the American type culture Collection (ATSC), including, but not limited to, cells of the Chinese hamster ovary (Cho), HeLa cells, kidney cells baby hamster (KSS)cells, monkey kidney (COS)cells, hepatocellular carcinoma person (for example, ner G2.) and a number of other cell lines.

ii. Baculovirus system

Polynucleotide encoding the protein may also be embedded in suitable expressing the vector insect and functional (operational) associated with regulatory elements in this vector. Construction of vectors uses the methods known in this field. Usually the components of the expression system include vector-vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for integration of the heterologous gene or heterologous genes that must be expressed; a wild type baculovirus with posledovatelno is d, homologous to the baculovirus-specific fragment in the vector-vector (this allows for the homologous recombination of heterologous gene into the genome of the baculovirus); and the appropriate cells of the host insect and environment for growing.

After embedding the DNA sequence that encodes the protein, vector-vector, this vector and the genome of wild-type virus transferout in a cage-the owner of the insect, where this vector and the viral genome give recombine. Packed recombinant virus is expressed and recombinant plaques identify and clean. Materials and methods for baculovirus/insect cell are commercially available in the form set out, inter alia, Invitrogen, San Diego, CA (set "MaxBac"). These methods generally known to experts in the field and in full as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter called "Summers and Smith").

Before embedding the DNA sequence encoding this protein in the baculovirus genome, the above-described components, containing the promoter, leader (if desired)of interest coding sequence and the sequence termination of transcription are usually harvested in the intermediate transferring design (vector-vector). This design may contain a single gene and functionally related R is untranslated elements; multiple genes, each with its own set of functionally related regulatory elements; or multiple genes regulated by one and the same set of regulatory elements. The intermediate transferring designs are often stored in a replicon, such as an extrachromosomal element (e.g., plasmids)capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system that allows it to persist in a suitable host for cloning and amplification.

Currently, the most commonly used vector-vector for introducing foreign genes into AcNPV is RS. Many other vectors known to the experts in this field, were also constructed. They include, for example, pVL985 (which changes the initiating codon polyhedrin from ATG to ATT, and which introduces cloning the BamHI site in the 32 BP downstream of transcription (right) from the ATT; see Luckow and Summers, Virology (1989) 17:31.

This plasmid is usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Environ., 42:177) and prokaryotic gene for resistance to ampicillin (APR) and the origin of replication for selection and propagation in E. coli.

Baculovirus vectors-vectors usually contain a promoter of baculovirus. Promoter of baculovirus is any DNA sequence capable of ativate baculovirus RNA polymerase and initiating the downstream transcription (5'→ 3') transcription of the coding sequence (e.g., structural gene) into mRNA. The promoter will have the area of transcription initiation, which is usually placed proximally relative to the 5'-end of the coding sequence. The area of transcription initiation usually involves the binding site of RNA polymerase and the site of transcription initiation. Baculovirus vector-vector may also have a second domain called an enhancer, in which case its presence is usually located distal from the structural gene. The expression can be either regulated or constitutive.

Structural genes are abundantly transcribed in the later periods of the cycle of viral infection, are especially applicable to the promoter sequence. Examples include sequences derived from the gene encoding a viral protein poliedro, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression" in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127839 and 155476; and the gene encoding protein P10, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted proteins insects or baculovirus, such as the polyhedrin gene of baculovirus (Carbonell et al., (1988) Gene, 73:409). Alternatively, since the signals for posttranslational modificati of mammalian cells (such as OTS the attack of the signal peptide, proteolytic cleavage, and phosphorylation) recognized, apparently, insect cells, and the signals required for secretion and nuclear accumulation also appear to be conservative between the invertebrate cells and vertebrate cells, leaders, comes from insects, such as obtained from genes encoding α-interferon person, Maeda et al., (1985)Nature 315:592; gastrin-releasing peptide person, Lebacq-Verheyden et al., (1988) Molec. Cell. Biol. 8:3129; IL-2 people. Smith et al., (1985) Proc. Natl. Acad. Sci. USA, 82:8404; murine IL-3 (Miyajima et al., (1987) Gene 58:273; and glucocerebrosidase man, Martin et al. (1988) DNA, 7:99, can also be used to ensure secretion in insects.

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 Nikitich foreign proteins usually requires heterologous genes, which ideally have a short leader sequence containing the appropriate signals initiation of translation in front of the starting (triggering) signal ATG. If desired, methionine at the N-end can be derived from the Mature protein by incubation in vitro with cyanogenmod.

Alternatively, recombinant polyprotein or proteins that nature is the bottom are secreted, can be secreted from insect cells by creating chimeric DNA molecules that encode the fused (hybrid) protein consisting of a fragment of a leader sequence that provides secretion of a foreign protein in insects. The fragment leader sequence typically encodes a signal peptide comprised of hydrophobic amino acids that control the translocation of this protein in the endoplasmic reticulum.

After embedding the DNA sequence and/or the gene encoding the expression product precursor of this protein, the cell host insect cotransformation heterologous DNA vector-vector and the genomic DNA of wild-type baculovirus - usually through cotransfection. The promoter and the termination sequence of the transcription of this design will typically contain a section 2-5 TPN genome of the baculovirus. Ways of introducing heterologous DNA into the desired site in the baculovirus known in this field (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, interturbine can be performed in a gene, such as the polyhedrin gene, homologous recombination double crossover; interturbine can also be carried out at the site of restriction enzyme (restrictase)introduced genetic engineering in the desired gene of baculovirus. Miller et al.,(1989), Bioessays 4:91. This DNA sequence cloning instead of the polyhedrin gene in expressing vector flanked from both the 5' (left)and 3' (right) poliakin-specific sequences and below (in the direction of transcription from the polyhedrin promoter.

Then the newly formed baculovirus expressing vector packaged into infectious recombinant baculovirus. Homologous recombination occurs with a low probability (between about 1% and about 5%); thus, most of the virus produced after cotransfection, is still the wild-type virus. Thus, you need a way to identify recombinant viruses. The advantage of this expression system is a visual screening to differentiate recombinant viruses. Protein poliakin, which is produced by the native virus is produced at very high levels in the nuclei of infected cells in later periods after viral infection. Accumulated protein poliakin forms the body of inclusion, which also contain immersed particles in them. These bodies include up to 15 μm are with high refraction of light, which gives them a bright shiny appearance, which is easily visualized under a light microscope. Cells infected with recombinant viruses do not have the phone turn on. For from the Iceni recombinant virus from wild-type virus supernatant after transfection applied as spots on a monolayer of cells insects ways, well-known experts in this field. Namely, the plaques are screened under the light microscope for the presence (which is indicative of wild-type virus) or absence (which is a sign of recombinant virus) tel inclusion. "Current Protocols in Microbiology" Vol.2 (Ausubel et al. eds) at 16.8 (Suppl. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expressing vectors were developed for the infection of some insect cells. For example, recombinant baculoviruses have been developed, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, 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 medium for cell cultures are commercially available for both direct and merged the expression of heterologous polypeptides in the baculovirus expression system; the technology of cell culture in General known to specialists with expertise in this area. See, for example, Summers and Smith, supra.

Then modified based on insect cells can be cultured in a suitable nutrient medium, which allows stable maintenance of plasmids (plasmids)that are present in a modified insect host. If a gene product expression is induced under the control of, the owner may be grown to high the Oh density and then induce the expression. Alternatively, if the expression is constitutive, the product will be continuously expressed in the environment and the culture medium must be continuously circulated with the removal of interest product and quantity replenishment depleted of nutrients. The product can be purified by means of chromatography, for example, HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; centrifugation in density gradient; solvent extraction, or the like, if necessary, the product may be further purified, if required, in order to remove essentially any protein of the insect, which is also secreted into the environment, or occur due to lysis of insect cells in order to provide a product that essentially does not contain at least the cellular residues (debris) of the owner, such as proteins, lipids and polysaccharides.

To obtain expression of the protein of the recombinant cell host, obtained from the transformants, incubated under conditions that allow expression of the coding recombinant protein sequence. These conditions will vary depending on the selected host cell. However, these conditions can be easily identified by the specialists of ordinary skill in this field on the basis of what is known in this field.

ii. Herbal system

There are many genetic expression systems using culture of plant cells and whole plants known in this field. Examples of genetic expression systems using plant cells include the system described in patents such as: U.S. patent US 5693506; U.S. patent 5659122 and U.S. patent US 5608143. Additional examples of genetic expression in culture of plant cells have been described in the work Zenk, Phytochemistry 30:3861-3863 (1991). Description of the signal peptides of plant proteins can be found, in addition to the links described above, 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). Description of gene expression regulation of plant hormone, gibberellic acid and sekretiruemyi enzymes induced by gibberellic acid, can be found in the work of R.L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant discrimination, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp.21-52. References that describe other metabolically regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987).

Usually using methods known in this field, the desired polynucleotide sequence is inserted into the Express the traditional cassette, containing genetic regulatory elements, designed for operation in plants. The expression cassette is inserted into the desired expressing vector with auxiliary sequences against the course of transcription and in the course of transcription from this gene-expression cassette suitable for the expression in the plant host. These auxiliary sequences have a plasmid or viral origin and provide necessary characteristics to the vector to create opportunities for these vectors to move DNA from the original clone of the host, such as bacteria, in the desired plant host. The main structure of bacterial/plant vector will preferably provide a prokaryotic replication origin, a broad host range; prokaryotic breeding marker for transformation using Agrobacterium T-DNA sequences for Agrobacterium-mediated transfer of chromosome plants. When the heterologous gene can not be easy to detect, this design would be preferable to have the gene of breeding marker suitable for determining whether the transformed plant. Overview of suitable markers, for example, for family members of cereals can be found in the work Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Recommended that the same sequence, suitable for the integration of heterologous sequences into the genome of plants. They can contain a sequence of transposons, etc. for homologous recombination, and Ti-sequences, which make possible a random interturbine heterologous expression cassette into the genome of plants. Suitable prokaryotic breeding markers include genes for resistance to antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions, as is well known in this field may also be present in this vector.

The nucleic acid molecule of the present invention can be included in the expression cassette for expression of interest protein of interest protein). Usually there will be only one expression cassette, although there are two or more cassettes. Recombinant expression cassette will contain in addition to the sequence that encodes a heterologous protein, the following elements: a promoter region, 5'-noncoding sequences plants, initiating codon, depending on the fitted structural gene that area or not provided, and the sequence termination of transcription and translation. Unique sites of restriction enzymes to the 5'- and 3'-ends of the tape on the bark could easily interturbine in preexisting vector.

Heterologous coding sequence may be any protein related to the present invention. Sequence encoding a protein of interest, will encode the signal peptide, which makes possible the processing and translocation of this protein, as appropriate, and usually will not contain sequences that can lead to binding of the desired protein of the present invention with a membrane. Mainly because the area of transcription initiation will function for a gene that is expressed and translocases during germination, using a signal peptide, which provides translocation can also provide translocation protein of interest. Thus, the protein of interest (protein) will transloziruetsa from the cells in which they are expressed, and can be effectively collected. Usually the secretion of seeds occurs through the aleurone layer and Scutellaria epithelium in the endosperm of the seed. Although it is not mandatory that this protein secretarials from cells in which the protein is produced, it facilitates the isolation and purification of the recombinant protein.

As the final expression of the desired gene product will occur in eukaryotic cells, it is desirable to determine whether both the I-or part of the cloned gene sequence, who will processionals as introns apparatus by splicecom owner. If it is, can be carried out site-directed mutagenesis "intron" area to prevent the loss of part of the genetic matrix in the form of false intron code, see Reed and Maniatis, Cell 41:95-105, 1985.

The vector may be microinjection directly into plant cells using micropipettes to mechanically transfer the recombinant DNA. Cm. Crossway, Mol. Gen. Genet, 202:179-185, 1985. Genetic material may be transferred into the plant cell using polyethylene glycol, see Krens, et al., Nature 296, 72-74, 1982. Another way of introducing segments of nucleic acids is a high-speed ballistic penetration by small particles with the nucleic acid either within the matrix or on the surface of small granules or particles, see Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336, describing the bombing of the particles of the endosperm of barley to create transgenic barley. Another way of introduction could be the fusion of protoplasts with other components or minicircle, cells, lysosomes or other able to merge cells with lipids on the surface, see Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824, 1985). This is the procedure the protoplasts of plants electroporator in the presence of plasmids containing the gene construct. Electric field pulses with high tension do biomembranes reversible permeable, which allows you to enter these plasmids. Elektrooborudovanie plant protoplasts regenerate the cell wall, share and form of plant callus.

All plants from which protoplasts can be isolated and cultured to obtain the whole regenerated plants can be transformed according to this invention in such a way that form whole plants 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 types of sugar cane, 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.

Methods of regeneration vary from species to species of plants, but usually first provide a suspension of transformed protoplasts containing copies heterologic the th gene. Formed callus tissue and the seedlings can be induced from callus and then they take root. The alternative of a suspension of protoplasts can be induced formation of embryos. These embryos germinate as natural germs with the formation of plants. Culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Preferably also adding to the environment of glutamic acid and Proline, particularly for species such as corn and alfalfa. Seedlings and roots usually develop at the same time. Effective regeneration will depend on the environment, genotype and cultural history. If these three variables are controlled, the regeneration will be fully reproducible and repeatable.

In some systems of plant cell cultures the desired protein of the present invention can be excreted or, alternatively, the protein can be extracted from the whole plant. If the desired protein of the present invention is secreted into the environment, it can be assembled. Alternatively, the embryos do not contain germ halves of a seed or other plant tissue can be mechanically destroyed to release all secreted protein between cells and tissues. This mixture can be suspended in a buffer solution for extraction of soluble proteins. Then IP is result conventional methods of isolation and purification for purification of recombinant protein. The parameters of time, temperature, pH, oxygen and quantities adjusted by routine methods for optimizing expression and allocation of the heterologous protein.

iv. Bacterial systems

Methods bacterial expression known in this field. Bacterial promoter is any DNA sequence that is capable of binding bacterial RNA polymerase and initiate in the direction of transcription (3') transcription of the coding sequence (e.g., structural gene) into mRNA. The promoter will have the area of transcription initiation, which is usually located proximally relative to the 5'-end of the coding sequence. The area of transcription initiation usually involves the binding site of RNA polymerase and the site of transcription initiation. Bacterial promoter may also have a second domain called an operator, which may overlap with neighboring binding site of RNA polymerase, which begins the synthesis of RNA. The operator makes it possible to negatively regulated (inducible) transcription, as protein-repressor gene can bind to the operator and thereby to inhibit the transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be the ü achieved through sequence, binding protein-activator gene, which, if present, is usually proximal (5') relative binding RNA polymerase sequence. An example of protein-activator gene is catabolic activator protein (CAP), which sposobstvuet initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Thus, regulated expression can be either positive or negative, by either enhancing or reducing transcription.

The sequence encoding the enzymes of metabolic pathways, are especially applicable to the promoter sequence. Examples include promoter sequences derived from the enzymes metabolizing sugars 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 4738921; EP-A-0036776 and EP-A-0121775]. Promotor system g-lactamase (Na) [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Grosser)], promotor system of bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] promoter and the T5 system [U.S. patent US 4689406] also provide the applicable promoter sequence.

In addition, syntheti the ski promoters, which are not found in nature, also function as bacterial promoters. For example, the sequence of activation of transcription of a single bacterial promoter or promoter of bacteriophage can be connected to the sequences of the operon other bacterial promoter or promoter of bacteriophage with the formation of a synthetic hybrid promoter [U.S. patent 4551433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and sequences of the lac operon, which is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. In addition, the bacterial promoter may include naturally occurring promoters of non-bacterial origin, which have affinity for binding bacterial RNA polymerase and initiation of transcription. The naturally occurring promoter non-bacterial origin may also be connected with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The system of the RNA polymerase of bacteriophage T7/promoter is an example of a conjugate 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 composed of a promoter of bacteriophage and operator of area E. coli (EPO-And-0267851).

Chromeantivirus promoter sequence effective binding site of the ribosome is also suitable for the expression of foreign genes in prokaryotes. In E. coli the binding site of ribosomes is called the sequence of the Shine-Dalgarno (SD), and it includes the initiating codon (ATG) and a sequence with a length of 3 to 9 nucleotides, located 3-11 nucleotides to the left (against the course of transcription) from the initiator codon [Shine et al. (1975) Nature 254:34]. It is believed that the SD sequence stimulates binding of mRNA to the ribosome by the pairing of bases between the SD posledovatelnostyu and 3'-end of the 16S rRNA of E. coli [Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R.F.Goldberger)]. In relation to the expression of eukaryotic genes and prokaryotic genes with weak binding site of the ribosome, see Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual.

The DNA molecule can be expressed intracellularly. The promoter sequence may be directly linked to the DNA molecule, and in this case the first amino acid at the N-end will always be a methionine, which is encoded by the start codon ATG. If desired, methionine at the N-end can be derived from protein by incubation in vitro with cyanogenmod or incubation in vivo or in vitro with bacterial methionine-N-terminal-peptidases (EPO-And-0219237).

Slit proteins provide an alternative to direct expression. Usually a DNA sequence, encoding the N-terminal portion of an endogenous bacterial protein Il the other stable protein, merge with the 5'end of heterologous coding sequences. When the expression of this design will ensure that the merger of two amino acid sequences. For example, gene cells bacteriophage lambda can be linked at the 5'end of the foreign gene and expressed in bacteria. The obtained protein preferably retains a site for processimage enzyme (factor XA) for removal of protein from bacteriophage foreign gene [Nagai et al. (1984) Nature 309:810]. Fused (hybrid) protein can also be made with the sequences of the genes lacZ [Jia et al. (1987) Gene 60:197], trpE [Alien et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Environ. 135:11] and Chey [EP-A-0324647]. The DNA sequence at the junction of two amino acid sequences can encode or may not encode a cleavage site. Another example is the protein of ubiquitin. This protein is prepared with the district of ubiquitin, which preferably retains a site for processimage enzyme (e.g., specific for ubiquitin processorsa protease for cleavage of ubiquitin from a foreign protein. Using this method, the native alien protein can be identified [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secretariats from the cell by creating chimeric DNA molecules that encode if the first protein, consisting of a fragment of a signal peptide sequence that provides for secretion of the foreign protein in bacteria [US patent US 4336336]. The fragment of the signal sequence is typically encodes a peptide comprised of hydrophobic amino acids that control the secretion of this protein from the cell. This protein is secreted or in the culture medium (in the case of gram-positive bacteria)or periplasmatic the space between the inner and outer cell membranes (in the case of gram-negative bacteria). Preferably there are processing sites, which can be converted in vivo or in vitro, encoded between the fragment of the signal sequence and alien genome.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the gene of the protein of the outer membrane of E. coli (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the signal sequence of alkaline phosphatase E. coli (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the gene alpha-amylase from various strains of Bacillus can be used for secretion of heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0244042].

Usually the sequence termination of transcription at Nehemia bacteria, are regulatory regions located at position 3' relative to the stop codon of translation, and thus they together with the promoter flank the coding sequence. These sequences direct the transcription of mRNA that can be translated into the polypeptide encoded by this DNA. Sequence termination of transcription often include a DNA sequence of approximately 50 nucleotides, capable of forming structures such as "stem-loop"that contribute to the termination of transcription. Examples include sequences termination of transcription derived from genes with strong promoters such as the trp gene in E. coli, as well as other biosynthetic genes.

Typically, the above described components, comprising a promoter, signal sequence (if desirable)of interest coding sequence and a termination sequence transcription, put together in the expression construct. Expression design is often stored in a replicon, such as an extrachromosomal element (e.g., plasmids)capable of stable maintenance in a host, for example, in bacteria. The replicon may have a replication system that allows it to persist in a prokaryotic host for either expression or cloning and and the plification. In addition, the replicon can be a plasmid or with high or low kopiosto. A plasmid with a high number of copies will usually have a number of copies in the range of from about 5 to about 200, and usually about 10 to about 150. The owner, containing the plasmid of high copy number, will contain at least 10 and more preferably at least 20 plasmids. Can be selected vector with a high or low number of copies depending on the actions of this vector and a foreign protein on the host.

An alternative expression constructs can be integrated into the bacterial genome integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome, allowing the vector to integrate. Integration seems to occur due to recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various strains of Bacillus, are integrated into the chromosome of Bacillus (EP-A-0127328). Integrating vectors can consist of sequences of bacteriophage or transposons.

Usually extrachromosomal and integrated expression constructs may contain breeding markers for selection of bacterial strains, whichwere transformed. Breeding markers can be expressed in bacterial host and may include genes that make bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Ann. Rev. Environ. 32:469]. Breeding markers can also include biosynthetic genes, such as genes involved in the pathways of the biosynthesis of histidine, tryptophan and leucine.

Alternative some of the above components can be placed together in transforming vectors. Transforming vectors usually contain a breeding marker that is either stored in the replicon, or it can develop into an integrating vector, as described above.

Expressing and transforming the vectors or extrachromosomal replicons, or integrating vectors have been developed for transformation of many bacteria. For example, expressing vectors were developed, inter alia, for the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0036259 and EP-A-0063953; 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-0036776, EP-A-0136829 and EP-A-0136907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Micribiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Environ. 54:655], Streptomyces lividans [US patent US 474556056].

Methods of introducing exogenous DNA into bacterial hosts, horoscopist in this area and usually include the transformation of bacteria, processed or CaCl2or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary depending on the bacterial species that should be transformed. See, for example, [Masson et al. (1989) FEMS Environ. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0036259 and EP-A-0063953; 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; Which Deletes An Object (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Environ. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Environ. 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. Environ. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Expression in yeast

Expression systems, yeast is also known to person of ordinary skill in this field. The yeast promoter is any DNA sequence capable of connecting the yeast RNA polymerase and initiate in the direction of transcription (3') transcription of the coding pic is ecovalence (for example, structural gene) into mRNA. The promoter will have the area of transcription initiation, which is usually located proximally relative to the 5'-end of the coding sequence. The area of transcription initiation usually involves the binding site of RNA polymerase ("TATA box") and the site of transcription initiation. Yeast promoter may also have a second domain called right (3')-activator sequence (UAS), which, if present, usually is distal with respect to the structural gene. UAS enables regulated (inducible) expression. Constitutive expression occurs in the absence of UAS. Regulated expression can be either positive or negative, as a consequence, either enhancing or reducing transcription.

Yeast is fermenters organism with an active pathway, so the sequence encoding the enzymes in this metabolic pathway, are especially applicable to the promoter sequence. Examples include alcoholdehydrogenase (ADH) (EP-A-0284044), enolase, glucokinase, glucose-6-fortismere, glyceraldehyde-3-phosphatedehydrogenase (GAP or GAPDH), geksokinazou, phosphofructokinase, 3-phosphoglycerate and piruwatkinaza (Hands) (EPO-And-0329203). Gene RNA yeast, acid encoding fosfate also provides applications to the appropriate promoter sequence [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters that do not occur in nature also function as yeast promoters. For example, the sequence UAS one yeast promoter can be connected to the district activate transcription of another yeast promoter with the formation of a synthetic hybrid promoter. Examples of hybrid promoters include regulatory sequence of the ADH associated with the area of activation of transcription of GAP (U.S. patent US 4876197 and 4880734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of the gene ADH2, GAL4, GAL10, or RNA, combined with the area of activation of transcription of glycolytic enzyme, such as GAP or Hands (EP-A-0164556). In addition, yeast promoter may include naturally occurring promoters of non-yeast origin, which are able 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 Environ. Immunol. 196:119; 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].

The DNA molecule can expressional the Xia intracellular yeasts. The promoter sequence may be directly linked to the DNA molecule, and in this case the first amino acid at the N end of the recombinant protein will always be a methionine, which is encoded by the start codon ATG. If desired, methionine at the N-end can be derived from protein by incubation in vitro with cyanogenmod.

Slit proteins provide an alternative to yeast expression systems, as well as in the case of gene-expression systems, mammalian, baculovirus and bacterial systems. Usually a DNA sequence, encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is drained from the 5'end of heterologous coding sequences. When the expression of this design will ensure that the merger of two amino acid sequences. For example, the gene superoxide dismutase (SOD) yeast or humans can be linked at the 5'end of the foreign gene and expressed in yeast. The DNA sequence at the junction of two amino acid sequences can encode or may not encode a cleavage site. See, for example, EP-A-0196056. Another example is the protein of ubiquitin. Such a protein is obtained in the district of ubiquitin, which preferably retains a site for processimage enzyme (e.g., specific for ubiquitin process is the dominant protease for cleavage of ubiquitin from a foreign protein. Thus, using this method, the native alien protein can be selected (for example, WO 88/024066).

Alternatively, foreign proteins can also be secretariats of cells in the culture medium by creating chimeric DNA molecules that encode a protein consisting of a fragment of a leader sequence that provides secretion of a foreign protein in yeast. Preferably there are processing sites between the leader fragment and the alien genome, which can be converted in vivo or in vitro. The fragment leader sequence typically encodes a signal peptide comprised of hydrophobic amino acids that control the secretion of this protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as invertase gene of yeast. (European patent EP-A-0012873; JPO Japan patent 62096086) gene and the A-factor. (U.S. patent US 4588684). Alternatively, there are leaders of non-yeast origin, such as the leader of interferon, which also provide for secretion in yeast (EP-A-0060057).

A preferred class secreting leaders are the leader sequence, using the fragment of the gene alpha factor for yeast, which contains both "pre"signal sequence, and "por the district. Types of fragments of alpha-factor, which can be used include a full-sized pre-Pro-leader, alpha factor (approximately 83 amino acid residue), and shortened the leaders of the alpha-factor (typically about 25 to about 50 amino acid residues) (U.S. patent US 4546083 and 4870008; EP-A-0324274). Additional leaders, using the fragment leader, alpha factor, which ensures secretion include hybrid leaders of alpha-factor, obtained from pre-posledovatelnostyu first yeast, but with the Pro-region of the alpha-factor other yeast (for example, see WO 89/02463).

Usually the sequence termination of transcription, recognizable by yeast are regulatory regions located from 3' relative to the stop codon of translation, and thus they together with the promoter flank the coding sequence. These sequences direct the transcription of mRNA that can be translated into the polypeptide encoded by this DNA. Examples of the sequence termination of transcription and other recognizable yeast sequences termination of transcription are sequence encoding glycolytic enzymes.

Typically, the above described components, comprising a promoter, leader (if desired)of interest encoding a serial is inost and the sequence termination of transcription, put together in the expression construct. Expression design is often stored in a replicon, such as an extrachromosomal element (e.g., plasmids)capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication system, which therefore allows it to be persisted, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such Shuttle vectors, yeast-bacteria include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [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, the replicon can be a plasmid or with high or low kopiosto. A plasmid with a high number of copies will usually have a number of copies in the range of from about 5 to about 200, and usually about 10 to about 150. The owner, containing the plasmid of high copy number, will contain at least 10 and more preferably at least 20 plasmids. Can be selected vector with a high or low number of copies depending on the actions of this vector and a foreign protein on the host. See, for example. Brake et al., supra.

Alternatively, expression constructs can be integrated into the yeast genome integrating vector. Integrating vectors usually contain at least one placentas is required, homologous to yeast chromosome, allowing the vector to integrate into and preferably contain two homologous sequences flanking this expression construction. Integration seems to occur due to recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. Integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. Cm. Orr-Weaver et al., supra. Can integrate one or more expression constructs, possibly affecting the levels produced recombinant protein [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. Chromosomal sequences included in the vector can contain either a single segment in the vector that leads to the integration of the entire vector, or in the form of two segments homologous to adjacent segments in the chromosome and flanking the expression construct this vector, which can lead to stable integration of this expression construction.

Usually extrachromosomal and integrated expression constructs may contain breeding markers for selection of yeast strains that have been transformed. Breeding markers may include biosint the political genes, which can be expressed in yeast cells, hosts, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and G418 resistance gene, which give the yeast cells resistant to tunicamycin and G418, respectively. In addition, suitable breeding marker can also provide the yeast with the ability to grow in the presence of toxic compounds such as metals. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Environ. Rev. 51:351].

Alternatively, some of the above components can be placed together in transforming vectors. Transforming vectors usually contain a breeding marker that is either stored in the replicon, or it can develop into an integrating vector, as described above.

Expressing and transforming the vectors or extrachromosomal replicons, or integrating vectors have been developed for transformation of many yeast. For example, expressing vectors were developed, inter alia, for the following yeasts: [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Environ. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Environ. 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 Environ. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; US Patent Nos. 4837148 and 4929555], Saccharomyces crevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) A Bacteriol. 153-163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 500:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, el al. (1985) Curr. Genet. 10-49].

Methods of introducing exogenous DNA into yeast hosts are well known in this area and usually include either the transformation of spheroplasts or transformation of intact yeast cells treated with cations of alkali metals. Transformation procedures usually vary depending on the species of yeast, which should be transformed. See, for example, [Kurtz et a2. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Environ. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Environ. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacleriol. 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 Environ. 25:141; US Patent Nos. 4837148 and 4929555; 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

In the application here, the term "antibody" refers to a polypeptide or group of polypeptides consisting "of at least one antigennegative centre antibodies (antidetonant). "Antigennegative center is a three-dimensional connecting the space with the inner surface configuration and charge distribution complementary p is iznakam epitope of the antigen, that provides the binding of antibody to the antigen. "Antibody" refers to, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, altered antibodies, incomplete (monovalent) antibodies, Fab proteins and antibodies with a single domain.

Antibodies against the proteins of the present invention is applicable for affinity chromatography, immunoassays, and distinguish/identify proteins of Neisseria.

Antibodies to proteins of the present invention, both polyclonal and monoclonal, can be obtained 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 obtaining polyclonal sera due to the amount of the resulting serum and availability of labeled anti-rabbit and anti-goat antibodies. Immunization is usually performed by mixing or emulsifying the protein in saline, preferably in Freund, such as full beta-blockers, and by injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). Usually sufficient dose is 50-200 mcg per injection. Immunization is usually repeated after 2-6 weeks with one or more injections of this protein in saline solution, preferably using nepolnoj the adjuvant's adjuvant. Alternatively, to generate antibodies by immunization in vitro using methods known in this field that are considered for the purposes of the present invention is equivalent to immunization in vivo. Polyclonal antisera obtained by sampling blood from the immunized animal in a glass or plastic container with incubating the blood at 25°C for one hour followed by incubation at 4°C for 2-18 hours. The serum is removed by centrifugation (for example, 1000 g for 10 minutes). From rabbits you can get 20-50 ml of blood in one selection.

Monoclonal antibodies obtained using the standard method of Kohler & Millstein [Nature (1975) 256:495-96] or its modifications. Usually mouse or rabbit subjected to immunization, as described above. However, it is preferable to take the blood from the animal for the extraction of serum and remove the spleen (and, if necessary, large lymph nodes) and break it down into separate cells. If desired, the spleen cells can be subjected to screening (after removal of non-specific sticking of cells) applying the cell suspension to the Cup or hole, covered with a protein antigen. B cells expressing membrane-bound immuno globulin-specific antigen associated with this Cup and not washed out with the rest of the suspension. Then the In-KL is TCA or all of the cells are destroyed in the spleen induce for fusion with myeloma cells with the formation of hybridomas, and are cultured in a selective medium (e.g., in a medium containing gipoksantin, aminopterin, thymidine, "HAT"). Received hybridoma seeded according to the method of limiting dilution and examined for the production of antibodies that specifically associated with immunizing antigen (and which is not associated with foreign antigens). Then selected mAb-secreting pituitary hybridoma cultivated in vitro (for example, in the culture flasks for tissue or reactors with hollow fibers) or in vivo (in ascites in mice).

If preferably, antibodies (polyclonal or monoclonal) may be labeled using conventional methods. Suitable labels include fluorophores, chromophores, radioactive atoms (in particular,32P and125I)reagents with high electronic density, enzymes, and ligands having specific binding partners. The enzymes will usually detect their activity. For example, horseradish peroxidase is usually detected by its ability to transform 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, which is determined quantitatively using a spectrophotometer. "Specific binding partner" is called a protein capable of binding the molecule-ligand with high specificity, as for example, in the case of antigen and specific antibodies. Other specific binding partners include Biotin and avidin or strept the Vidin IgG and protein a, and numerous pairs of receptor-ligand known in this field. It should be clear that the above description is not intended to distribute numerous marks on clearly defined classes, as the same label can serve several different mechanisms of action. For example,125I can serve as a radioactive label or as a reagent with increased electron density. HRP (horseradish peroxidase) can serve as an enzyme or as an antigen for mAb. Next, you can combine different labels for the desired effect. For example, mAb and avidin also require labels in the practice of this invention: for example, you can tag mAb Biotin and to detect their presence by Avidya labeled125I, or monoclonal antibodies against Biotin labeled with HRP. Other permutations and possibilities will be obvious to specialists of ordinary skill in this field and are considered equivalents within the scope of this invention.

The pharmaceutical composition

The pharmaceutical compositions may contain or polypeptides, or antibodies, or nucleic acid of the present invention. The pharmaceutical compositions will contain a therapeutically effective amount or polypeptides, or antibodies, or polynucleotides this izopet the deposits.

The term "therapeutically effective amount" as applied here refers to the amount of therapeutic agent to treat, mitigate or prevent the disease or condition or for the manifestation of therapeutic or preventive actions. This action can be detected, for example, through chemical markers or antigens. Therapeutic effects include the reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition and therapeutics or combination of therapeutics selected for administration. Thus, it makes no sense to determine the exact amount in advance.

However, the effective amount for a particular situation can be determined by routine experimentation and is within the capabilities of the evaluation by the treating physician.

For the purposes of this invention, an effective dose will be between about 0.01 mg/kg and 50 mg/kg or 0.05 mg/kg and about 10 mg/kg DNA structures for the individual, which it is entered.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means a carrier clevudine therapeutic agent, such as antibodies or polypeptide, genes and other therapeutic agents. This term refers to any pharmaceutical carrier that does not itself induce the formation of antibodies harmful to the individual receiving the composition, and which can be entered without unwanted toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids and inactive virus particles. Such carriers are well known in the art of ordinary skill in this field.

In them can be used pharmaceutically acceptable salt, for example, mineral salts such as hydrochloride, hydrobromide, phosphates, sulfates and the like; and organic acid salts such as acetates, propionate, malonate, benzoate etc. Detailed discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers for therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol. Such media may optionally be present auxiliary substances, such as moisturizing or emulsifying agents, bufferedio pH substances, etc. Usually, these terapeutiche which such compositions are prepared in the form of injection solutions, in the form of liquid solutions or suspensions; can also be prepared solid forms suitable for solution or suspension in liquid media prior to injection. The definition of a pharmaceutically acceptable carrier are also included liposomes.

Shipping methods

After the preparation of compositions of this invention can be administered directly to a subject. To be treated subjects may be animals, in particular can be treated people.

Direct delivery of the compositions of this invention usually performed via injection, subcutaneously, intraperitoneally, motivando or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered in damage. Other routes of administration include oral and pulmonary introduction, suppositories, and transdermal or percutaneous injection (for example, see WO 98/20734), using needles, gene guns or hyposprays. Dosage treatment may be a scheme of injection of a single dose or scheme with the introduction of multiple doses.

Vaccines

Vaccines contain immunizing antigen (antigens), immunogen (immunogen), polypeptide (a polypeptide), protein (protein) or nucleic acid, usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induces the formation of an is ITIL, harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known in the art of ordinary skill in this field. In addition, these carriers can function as immunostimulating agents ("adjuvants"). In addition, the antigen or immunogen can be conjugated to a bacterial toxin, such as toxoids of diphtheria, tetanus, cholera, N. pylori and other pathogens.

The preferred. adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) the emulsion composition of the type oil-in-water (with other specific immunostimulating agents such as muramylpeptide (see below) or components of the walls of the bacterial cells, or without them), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of the and MTP-PE (see below), although this is not mandatory), prepared in the form of submicron particles using microfluidizer, such as microfluidizer model HOY (Microfluidics, Newton, MA), (b) SAF, containing 10% squalene, 0.4% Tween 80, 5% pluronic-blakolmer L121, and thr-MDP (see below), or microfluidizers to submicron emulsion or processed in a vortex mixer to obtain emulsions with particles of larger size, and (C) the system Ribi adjuvant™ (RAS), (Ribi Immunochem, Hamilton, MT)containing 2% squalene, 0.2% Tween 80, and one or more components of the walls of the bacterial cells from the group consisting of monophosphorylated A (MPL), regulatability (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); (3) saponine adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, MA)may be used or particles derived from them, such as ISCOM (immunostimulating complexes); (4) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc), interferons (e.g. gamma interferon), macrophage colony-stimulating factor (M-GSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Preferred are MF59™ and alum.

As mentioned above, meramipop the IDA include, but not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine (MTP-PE), etc.

Immunogenic compositions (e.g., immunizing antigen/immunogen/polypeptide/protein/nucleic acid, a pharmaceutically acceptable carrier and adjuvant) should normally contain diluents, such as water, saline, glycerol, ethanol, etc. In such media may optionally be present auxiliary substances such as wetting or emulsifying agents, bufferedio pH substances, etc.

Typically, these immunogenic compositions are prepared in the form of injection solutions, either as liquid solutions or suspensions; can also be prepared solid forms suitable for solution or suspension in liquid media prior to injection. The preparation may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above with respect pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines contain immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above components if the need is I. By "immunologically effective amount" mean that the introduction of this quantity to the individual, as a single dose or successively fractional parts doses is effective to treat or prevent diseases. This number varies depending on the health status and physical condition of the subject to treatment of the individual taxonomic groups subject to treatment of the individual (for example, of the primacy of the collective, of the primacy and so on), the ability of the human immune system to synthesize antibodies, the degree of desired protection, vaccine composition, assessment of the medical situation of a doctor and other relevant factors. It is expected that this number will be located in a relatively broad range that can be determined by routine tests.

Immunogenic compositions are typically administered parenterally, e.g. by injection, subcutaneously, intramuscularly or transdermal/percutaneous (for example, WO 98/20734). Additional compositions suitable for other forms of introduction, include oral and pulmonary compositions, suppositories, and transdermal devices. Dosage treatment may be a scheme for the introduction of separate doses or scheme is the introduction of multiple doses. The vaccine may be administered in conjunction with other immunoregulatory agents.

Alternatively, the vaccines on the Nova proteins can be used vaccination with DNA [e.g., Robinson and Torres (1997) Seminars in Immunology 9:271-283; Donelly et al. (1997) Annu Rev Immunol 15:617-648; see below].

Carriers for gene delivery

Gene therapy carriers for delivery of constructs containing the coding sequence of a therapeutic agent of the present invention, which should be delivered in a mammal may be administered either locally or systemically. These designs can use the techniques with viral or non-viral vector methods in vivo or ex vivo. The expression of such coding sequences can be induced using endogenous promoters for mammalian or heterologous promoters. The expression of the coding sequence in vivo may be constitutive or regulated.

This invention includes the native gene delivery, able to Express the considered sequence of nucleic acids. Carrier delivery of genes is preferably a viral vector, and more preferably retroviral, adenoviral, adeno-associated virus (AAV), herpes virus or alphavirus vector. The viral vector can also be Astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus or togaviruses vector. See, in General. Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845852; Connely (1995) Human Gene Therapy 6:185-193 and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in this field, the inventors assume that any retroviral vector of haemotherapy is applicable in this invention, including retroviruses types b, C and D, xenotropic retroviruses (e.g., NZB-X1, NZB-X2 and NZB9-1 (see O'neill (1985) J. Virol. 53:160), polytropic retroviruses, for example, MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. Cm. RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Part of retroviral vectors for gene therapy can be obtained from various retroviruses. For example, retractor LTR can be produced from virus murine sarcoma, tRNA binding site of the rous sarcoma virus, the packaging signal of the virus, murine leukemia and start point of the second synthesis circuit from leukosis virus of birds.

These recombinant retroviral vectors can be used to generate particles transduction-competent retroviral vector by introducing them into appropriate packaging cell lines (see U.S. patent US 5591624). Retroviral vectors can be designed for site-specific integration into the DNA of the host cell activating chimeric enzyme integrase in this retroviral particle (see WO 96/37626). Preferably, the recombinant viral vector was replication-defective (i.e., the Def is ktym for replication of the recombinant virus.

Packaging cell lines suitable for use with the above-described retroviral vectors are well known in this area, they are easy to obtain (see WO 95/30763 and WO 92/05266), and they can be used to create cell lines-producers (also called vector cell lines or "VCL") for the production of recombinant vector particles. Preferred packaging cell lines are prepared from a source of human cells (e.g. cells NT) or the original cell lines mink, which eliminates inactivation in human serum.

Preferred retroviruses for the design of retroviral vectors for gene therapy include leukosis virus of birds, leukosis virus bovine virus murine leukemia virus, inducing lesions in mink cells, the virus murine sarcoma virus reticuloendotheliosis and rous sarcoma virus. Particularly preferred viruses murine leukemia include A and A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), the Kirsten sarcoma virus, Harvey and virus Rausher (virus erythroleucus and lymphatic leukemia mice) (ATCC No. VR-998) and leukosis virus of mice, Malone (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 Izv the local sources using public methods.

Examples of known retroviral gene therapy vectors used in this invention include vectors, described in patent applications GB 2200651, EP 0415731, EP 0345242, EP 0334301, WO 89/02468, WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, WO 91/02825, WO 95/079994, US 5219740, US 4405712, US 4861719, US 4980289, US 4777127, US 5591624. Cm. also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53:83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Nati Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Adenoviral vectors for human gene therapy is also known in this field and can be used in this invention. See, for example, Berkner (1988) Biotechniques 6:616, and Rosenfeld (1991) Science 252:431 and WO 93/07283, WO 93/06223 and WO 93/07282. Examples are known of adenoviral vectors for gene therapy used in this invention include vectors, described in the quotation above documents and in WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO 95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO 95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively, it may be used for the introduction of DNA related to killed adenovirus, as described in Curiel (1992) Hum. Gene Ther. 3:147-154. Examples of carriers for delivery of genes of this invention are also adeno-associated viral (AAV) vectors. The main and preferred examples of such in the Ktorov for use in this invention are vectors based on AAV-2, described Srivastava, WO 93/09239. The most preferred AAV-vectors contain two inverted terminal repeat of AAV, in which native D-sequence modified by the substitution of nucleotides, so that at least 5 of the natural nucleotides and up to 18 natural nucleotides, preferably at least 10 natural nucleotides to 18 natural nucleotides, most preferably 10 natural nucleotides saved, and the remaining nucleotides of the D-sequence deleterows or replaced by non-natural nucleotides. Natural D-sequence of the inverted terminal repeats of AAV sequences are the 20 consecutive nucleotides in each inverted terminal repeat of AAV (i.e. there is one sequence at each end)that are not involved in the formation of HP. Unnatural replacement nucleotide can be any nucleotide other than the nucleotide that is found in natural D-sequence in the same position. Other applied examples of AAV-vectors are pWP-19, pWN-1, both described in Nahreini (1993) Gene 124:257-262. Another example of such AAV-vector is psub201 (see Samulski (198.7) J. Virol. 61:3096). Another example used AAV-vector is a vector of Double-D ITR. Design vector Double-D ITR described in U.S. patent US 5478745. Other vectors are the vectors described in U.S. patent 479738, issued Carter, and U.S. patent 5139941 issued Muzyczka, U.S. patent 5474935 issued Chartejee, and WO 94/288157, Kotin. Another example of AAV-vector applicable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumen promoter and regulates the expression predominantly in the liver. Its structure and design are described in the work of Su (1996) Human Gene Therapy 7:463-470. Additional AAV-vectors in gene therapy are described in U.S. patent US 5354678, US 5173414, US 5139941 and US 5252479.

Vectors for gene therapy of the present invention also include herpes virus vectors. The main and preferred examples are vectors based on herpes simplex virus, containing the sequence encoding the polypeptide timedancing, such as described in U.S. patent US 5288641 and in the European patent EP 0176170 (Roizman). Additional examples of vectors based on herpes simplex virus include HFEM/ICP6-LacZ as described in WO 95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1661-1669 and in WO 90/09441 and WO 92/07945, HSV Us3::pgC-lacZ described by Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in European patent EP 0453242 (Breakefield), and the vectors deposited ATSS under the access numbers of ATS VR-977 and ATSS VR-260.

Also considered gene therapy vectors based alphavirus that can be used in this invention. Preferred alpha virus is cerami are vectors of virus-based fever Sindbis, togavirus, virus, Semliki forest (ATSS VR-67; ATS VR-1247), the virus of Middleburg ATS VR-370), the virus Ross river (ATSS VR-373; ATCC VR-1246), Venezuelan virus equine encephalitis (ATSS VR-923; ATCC VR-1250); ATCC VR 1249; ATCC VR-532), and the vectors described in U.S. patent US 5091309, 5217879 and WO 92/10578. More specifically, can be used vectors based alphavirus described in patent US with the registration number 08/405627 filed March 15, 1995, WO 94/21792, WO 92/10578, WO 95/07994, US 5091309 and US 5217879. Such alpha viruses can be obtained from depositories or collections such as the American type culture Collection ("ATCC") in Rockville, Maryland, or isolated from known sources using public methods. Preferably used alphavirus vectors with reduced cytotoxicity (see US 08/679640).

Vector systems DNA, such as multiple eukaryotic expression systems are also applicable for the expression of nucleic acids of the present invention. Cm. WO 95/07994 in relation to detailed descriptions of multiple eukaryotic expression systems. Preferably multiple eukaryotic expression system of the present invention produced from alphavirus vectors, and most preferably from vector-based virus fever Sindbis.

Other viral vectors suitable for use in this invention, including the act itself viral vectors, derived from poliovirus, for example, ATCC VR-58, and vectors described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example, ATCC VR-1110, and the vectors described in the work of Arnold (1990) J Cell Biochem L401; poxviruses, such as variola virus Canaries or vaccinia virus, for example, ATSC VR-111 and ATSS VR-2010, and the vectors described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; U.S. patent US 4603112 and US 4769330 and WO 89/01973; the SV40 virus, for example, ATSC VR-305 and vectors described by Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example, ATSC VR-797 and recombinant influenza viruses obtained using reverse genetics, as described in U.S. patent US 5166057 and work Enami (1990) Proc. Natl. Acad. Sci. 87:3802-3805; Enami and 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); the human immunodeficiency virus, is described in EP-0386882 and work Buchschacher (1992) J. Virol. 66:2731; measles virus, for example, ATSC VR-67 and VR-1247, and viruses, are described in EP-0440219; Aurelius, for example, ATSC VR-368; virus Bebaru, for example, ATSC VR-600 and ATSS VR-1240; virus Cabasso, for example, ATSC VR-922; Chikungunya virus, for example, ATSC VR-64 and ATSS VR-1241; virus, Fort Morgan, for example, ATSC VR-924; virus geta, for example, ATSC VR-369 and ATSS VR-1243; virus Kizilagac, for example, ATSC VR-927; virus Mayaro, for example, ATSC VR-66; virus Mucambo, for example, ATSC VR-580 and ATSS VR-1244; virus Ndumu,for example, ATS VR-371; virus Pixuna, for example, ATSC VR-372 and ATSS VR-1245; virus Tonite, for example, ATSC VR-925; virus Trinity, for example, ATSC VR-469; virus una, for example, ATSC VR-374; virus Mataroa, for example, ATSC VR-926; virus Y-62-33, for example, ATSC VR-375; virus O-Gong-Gong, virus, Eastern encephalitis, for example, ATSC VR-65 and ATSS VR-1242; virus Western encephalitis, for example ATS VR-70, ATSS VR-1251, ATSS VR-622 and ATSS VR-1252; and coronavirus, for example, ATSC VR-740 and viruses described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

The delivery compositions of the present invention into cells is not limited to the above-mentioned viral vectors. Other methods and delivery environment can be used, such as, for example, expression vectors, nucleic acids, polycation-condensed DNA linked or not linked to killed adenovirus, for example, see application U.S. number 08/366787, filed December 30, 1994 and the work Curiel (1992) Hum Gene Ther 3:147-154 associated ligand DNA, for example, see Wu (1989) J Biol Chem 264:16985-16987, cell carriers for delivery to eukaryotic cells, for example, at the request of the U.S. the number 08/240030, filed may 9, 1994, and the application USA room 08/404796, application of the polymerized under the action of light hydrogenrich materials, portable gun for particles-carriers of genes, as described in U.S. patent US 5149655, ionizing radiation, as described in U.S. patent US 5206152 and in WO 92/11033, neutralization of the charge well is Lanovoy acid or fusion with cell membranes. Additional approaches are described in the works of Philip (1994) Mol Cell Biol 14:2411-2418 and Woffendin (1994) Proc Nati Acad Sci 91:1581-1585.

Can be used indirectly particles gene transfer, for example, see application U.S. number 60/023867. Briefly, the sequence can be integrated into conventional vectors that contain conventional regulatory sequence for expression of a high level, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations, such as polylysine, Protamine, and albumin, linked with the target cells by ligands, such as Belorusskoe, as described by Wu and Wu (1987) J. Biol. Chem. 262:4429-4432, insulin, as described in the work Hucked (1990) Biochem Pharmacol 40:253-263, galactose, as described in the work Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Can also be used "naked" DNA. Examples of methods of introducing naked DNA described in WO 90/11092 and U.S. patent US 5580859. The absorption efficiency can be improved with the use of biodegradable latex beads. Covered DNA latex granules efficiently transported into the cells after the initiation of endocytosis these granules. This method can be improved by additional processing of the granules to increase the hydrophobicity and thereby facilitate the destruction of endosome and release of DNA into the cytoplasm.

Liposomes, which mo is ut to act as carriers for gene delivery, described in U.S. patent US 5422120, WO 95/13796, WO 94/23697, WO 91/14445 and EP 0524968. As described in US 60/023867, in the case of non-viral delivery of nucleic acid sequence encoding the polypeptide, can be introduced into conventional vectors that contain conventional regulatory sequence for expression of a high level, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations, such as polylysine, Protamine, and albumin, linked with the target cells by ligands, such as Belorusskoe, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA containing a gene under control of different tissue-specific or ubiquito-active promoters. Additional non-viral delivery, suitable for use, includes a mechanical system of delivery, such as the approach described in Woffendin et al. (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. In addition, the coding sequence and its expression product can be delivered through the polymerized under the action of light hydrogenrich materials. Other traditional methods of gene delivery that can be used to deliver this coding sequence include, for example, the use of portable gun for ASTIC vectors of genes as described in U.S. patent US 5149655, the use of ionizing radiation as described in U.S. patent US 5206152 and in WO 92/11033.

Examples of liposomal and poly carriers for gene delivery carriers are described in U.S. patent US 5422120 and 4762915; WO 95/13796, WO 94/23697, WO 91/14445 and ER-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 Nati Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

Polynucleotide composition may contain a therapeutically effective amount of a carrier for gene therapy, as that term is defined above. For the purposes of this invention, an effective dose will be equal to from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg DNA structures for the individual to whom it is administered.

Shipping methods

After preparation of polynucleotide compositions of this invention can be introduced (1) directly to the subject; (2) delivered ex vivo to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. Entities that should be treated, can be mammals or birds. Can also be treated subjects-people.

Direct delivery of the compositions usually performed by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered in vnutritelostnoe space fabric. The composition may is also be entered in damage. Other routes of administration include oral and pulmonary introduction, suppositories, and transdermal or percutaneous injection (for example, see WO 98/20734), using needles, gene guns or hyposprays. Dosage treatment may be a scheme of injection of a single dose or scheme with the introduction of multiple doses.

Methods for delivery ex vivo and re-implantation of the transformed cells to a subject are known in this field and are described, for example, in WO 93/14778. Examples of cells that can be used in ex vivo applications include, for example, stem cells, in particular hematopoietic, lymphatic cells, macrophages, dendritic cells or tumor cells.

Typically, the delivery of nucleic acids in applications such as ex vivo and in vitro can be performed using the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, indirect polybrene transfection, fusion of protoplasts, electroporation, encapsulation of polynucleotide (polynucleotides) in liposomes, and direct microinjection of the DNA in the nucleus, as is well known in this field.

Polynucleotide and polypeptide pharmaceutical compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynuclear innymi and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides, which include, without limitation: Belorusskoe (ASOR); transferrin; asialoglycoprotein; antibodies; antibody fragments; ferritin; interleukins; interferons; granulocyte-marathahalli colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor, and erythropoietin. Can be also used viral antigens such as envelope protein, and proteins from other invasive organisms, such as the peptide of 17 amino acids from circumsporozoite protein of Plasmodium falciparum, known as RII.

Century, Hormones, vitamins, etc.

Other groups that may be included are, for example, hormones, steroids, androgens, estrogens, thyroid hormone, or vitamin, folic acid.

C. Polyalkylene, polysaccharides, etc.

Polyalkyleneglycol can be enabled with the desired polynucleotides/polypeptides. In a preferred embodiment, polyalkyleneglycol is polyethylene glycol. Additionally, there may be included mono-, di - or polysaccharides. In the preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran, and chitosan and polymerized copolymer of lactide and glycolide.

D. Lipids and liposomes

Desired by nucleotid/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or its derived cells.

Lipid encapsulation is carried out using liposomes, which can stably bind or enclose in yourself and keep nucleic acid. The ratio of condensed polynucleotide lipid drug may vary, but will typically be about 1:1 (mg DNA:micromol lipid) or with a large number of lipid. In the review of the application 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 this invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. It was shown that cationic liposomes mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1980) 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[1-2,3-dialerace)propyl]-N,N,N-triethylammonium (DOTMA)liposomes are available under the trade name Lipofectin from GIBCO BRL, Grand Island, NY. (See also, Felgner supra). Other commercially available liposomes include Transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be obtained from readily available materials using methods well known in this about the Asti. See, for example, Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO 90/11092 in the description of the synthesis of DOTAP (1,2-bis(oliloqui)-3-(trimethylammonio)propane)liposomes.

Similarly, anionic and neutral liposomes are readily available, for example, from Avanti Polar Lipids (Birmingham, AL), or can be obtained using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPG), dioleoylphosphatidylcholine (DOPE). These materials can also be mixed with the source material DOTMA and DOTAP in suitable proportions. The methods of preparation of liposomes with the use of these materials is known in this field.

Liposomes can contain multilayer vesicles (MLV) and single-layer vesicles (SUV) or large single-layer vesicles

(LUV). Various complexes of liposome-nucleic acid prepared using methods known in this field. See, for example, 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 U982) Science 215:166.

E. Lipoprotein

In addition, lipoproteins, which may include subject delivery polynucleotide/polypeptide. Examples of lipoproteins, which can be used include: chylomicrons, HDL, IDL, LDL and VLDL. Can also be used mutants, fragments or fusion (hybrid) of these proteins. Can also be used modifications of the naturally occurring lipoproteins, such as acetylated LDL. These lipoproteins can be targeted delivery of polynucleotides in cells expressing receptors for lipoproteins. Preferably, if the lipoproteins include polynucleotide, which should be delivered in this song do not include any other targeting ligand.

The naturally occurring lipoproteins contain lipid and protein part. The protein part is called apoproteins. Currently, were isolated and identified apoprotein a, b, C, D and E. At least two of them contain several proteins, denoted by Roman numerals, AI, AII, AIV; CI, CII, CIII.

LP may contain more than one apoprotein. For example, the naturally occurring chylomicrons contain a, b, C and E, these lipoproteins lose and gain apoprotein C and E. VLDL contains apoprotein a, b, C and E, LDL contains apoprotein; HDL contains apoprotein a, C and E.

Amino acids these apoproteins known and described, for example, in the work of Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp. Med. Biol. 151:162; Chen (1986). J Biol Chem 261:12928; Kane (1980) Proc Natl Acad Sci USA77:2465 and Uterman (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids, including triglycerides, cholesterol (free and esters and phospholipids. In the naturally occurring lipoproteins composition of lipids varies. For example, chylomicrons contain mostly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The lipid composition is chosen in such a way that they contributed to the creation of the desired conformation of apoprotein for receptor-binding activity. The lipid composition may also be selected to facilitate hydrophobic interactions and communications with polynucleotide-binding molecule.

The naturally occurring lipoproteins can be separated from the serum, for example, by ultracentrifugation. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochim. 255:5454-5460 and Mahey (1979) J Clin. Invest 64:743-750. Lipoproteins can also be obtained in vitro or by recombinant methods gene expression of apoprotein in the desired cell host. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 39:443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Technologies, Inc., Stoughton, Massachusetts, USA. Additional description of lipoproteins can be found in W098/06437 (Zuckermann et al.).

F. Poly-agents

Poly agents, with lipoproteina the or without lipoprotein, can be included in the composition with the desired polynucleotide/polypeptide.

Poly agents usually have a positive charge at physiological pH and are able to neutralize the electrical charge of nucleic acids to facilitate delivery to the desired location. These agents have applications both in vitro, ex vivo and in vivo. Poly agents can be used to deliver nucleic acids to the living subject intramuscularly, subcutaneously, etc.

The following are examples of applicable polypeptides as poly-agent: polylysin, polyalanine, poliorcetes and Protamine. Other examples include histones, Protamine, human serum albumin, DNA-binding proteins, nonhistone proteins, chromosomes, proteins, membranes, DNA viruses, such as X174, transcription factors, which also contain domains that bind DNA and, therefore, can be used as condensing nucleic acid agents. Briefly, transcription factors such as C/SEVRES, c-jun, c-fos, AP-1, AR-2, AR-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP and TFIID, contain basic domains, which bind DNA sequences.

Organic poly agents include: spermine, spermidine and putrescine.

Dimensions and physical properties of poly agent can be extrapolated from the bring the frame above list to construct other poly agents or synthetic poly-agents.

Synthetic poly-agents, which are applicable include, for example, DEAE-dextran, polybrene. Lipofectin™ and lipofectamine™ are the monomers that form the poly-complexes when combined with polynucleotide/polypeptide.

Immunodiagnostics tests

Antigens of Neisseria this invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria antibodies can be used to detect levels of antigens). The immunoassays based on well-defined recombinant antigens can be designed to replace invasive diagnostic methods. Antibodies to proteins of Neisseria can be detected in a biological sample, including, for example, samples of blood or serum. The creation of immunoassays is the subject of a large number of variations, and many of them are well-known in this field. Protocols for immunoassay can be based, for example, by competition or by direct reaction or analysis type of sandwich assay. Protocols can also be used, for example, solid carriers, or may include immunoprecipitation. Most analyses include the use of labeled antibodies or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactively in the form of molecules of dyes. Assays that amplify the signals from the probe are also known; examples are the analyses that utilize Biotin and avidin, and labeled and mediated by enzyme immunoassays, such as ELISA assays.

Kits suitable for immunodiagnostic and containing the appropriate labeled reagents are packaging the appropriate materials, including compositions of the present invention, in suitable containers, along with the remaining reagents and materials (e.g., appropriate buffers, saline solutions, and so on)required to conduct this analysis, as well as the appropriate set of instructions to the analysis.

Hybridization of nucleic acids

The term "hybridization" refers to the binding of two sequences of nucleic acids to each other through hydrogen bonds. Usually one sequence is fixed on a solid carrier, and the other is free in solution. Then these two sequences are placed in contact with each other under conditions which favor the formation of hydrogen bonds. Factors that affect the formation of hydrogen bonds, include: the type and amount of solvent; reaction temperature; hybridization; mixing; agents for blocking nonspecific accession sequence from the liquid phase to the solid carrier (Rea the enta Denhardt or BLOTTO); the concentration of sequences; the use of compounds that increase the rate of binding sequences (doctranslate or polyethylene glycol); and the stringency of the washing conditions after hybridization. Cm. Sambrook et al. [supra], Volume 2, chapter 9, pages 9.47-9.57.

The term "stringency" refers to conditions in the reaction of hybridization that favor the preferential communication is very similar sequences in comparison with sequences that differ. For example, should be selected combination of temperature and salt concentration at which the temperature is approximately 120-200°C below the calculated Tm (melting temperature) of the investigated hybrid. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on the filters, hybridized with interest the sequence and then washed under conditions of different severity. Cm. Sambrook et al., page 9.50.

Variables to consider, for example, when performing a southern-blot analysis are: (1) the complexity of the DNA to be blotting, and (2) the homology between the probe and detectivesyme sequences. The total number of fragments (fragments)to be investigated, may vary with the amplitude of 10, from 0.1-1 µg for product splitting PLA the foreign ministries or phage to 10 -9-10-8g for malambing gene in complex eukaryotic genome. For polynucleotides low complexity can be used for significantly shorter periods blotting, hybridization and exposure, fewer source polynucleotides and lower specific activity probes. For example, melakopides gene can be detected with an exposure time of only 1 hour from the source 1 ág of yeast DNA, blotting for two hours and hybridization within 4-8 hours with the probe with 108pulse/min/µg. For malambing gene of the mammal conservative approach starts with 10 μg DNA as starting material, blotting carried out during the night and hybridization overnight in the presence of 10% doctranslate using a probe with more than 108pulse/min/g, which leads to the exposure time ˜24 hours.

Several factors can affect the melting temperature (Tm) of the hybrid DNA-DNA between the probe and interest fragment and, therefore, suitable conditions for hybridization and washing. In many cases, the probe is not 100% homologous to the fragment. Other common variables include the length and the total G+C content of hybridization sequences and ionic strength and the content of formamide in the hybridization buffer. The action is all of these factors can be approximated by a single equation:

Tm=81+16,6(logioCi)+0,4 [%(G+C)]and-0.6(%formamide)-600/n-1,5(%erroneous pairing)

where Ci denotes the concentration of salt (monovalent ions), and n denotes the length of the hybrid in P.N. (slightly modified in comparison with the work of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284).

In the planning of the experiment, the hybridization of some of the factors influencing hybridisation of nucleic acids can be modified appropriately. It is easier to adjust the temperature of the hybridization and washing and salt concentration during leaching. Increasing the temperature of hybridization (i.e. severity) hybridization between chains that are non-homologous, it becomes more difficult to occur, and this consequently reduces the background. If radioactively labeled probe is not completely homologous to the immobilized fragment (as is often the case for experiments with hybridization in the family genes and interspecific hybridization, hybridization temperature should be lowered and the background will increase. The temperature of the leaching affects the intensity of the band hybridization and the degree of background in a similar manner. The severity of leaching also increases with decreasing salt concentrations.

Usually suitable hybridization temperatures in the presence of 50% formamide are 42°for the probe, which is 95%-100% homologous to a fragment of the target 37°for God is the biology of 90%-95% and 32° For the homology of 85%-90%. For lower homology content of formamide should be lowered, and the temperature is adjusted using the above equations. If the homology between the probe and the fragment-target is unknown, the simplest approach is to start with the conditions of hybridization and washing, which are lax. If after autoradiography observed nonspecific bands or high background, the filter can be washed at high stringency and re-exposed on the film. If the time required for the display, makes this approach impractical to test in parallel several degrees of stringency of hybridization and/or washing.

Identification of meningococcal protein 80-85 kDa

Observed that different drugs bubbles (vesicles) of the outer membrane of N. meningitidis sero-groups contained In component approximately 80-85 kDa. This protein was purified by gel-electrophoresis in the LTO-page and N-terminal sequenced (SEQ ID NO:1).

Antibodies induced against purified by electrophoresis in LTO-SDS page protein cross-reacted with equivalent proteins in more than 50 strains of N. meningitidis different serological groups and serotypes. Also observed cross-reactivity with N. gonorrhoeae, N. polysaccharia and N. lactamica. Pastamania Siva ODI from vaccinated patients also reacted with this protein.

The complete gene was cloned from N. meningitidis serotype B (SEQ ID NO:2), and was transcribed sequence of the encoded protein (SEQ ID NO:3). By comparison with N-terminal sequencing, described above, were decoded signal peptide (SEQ ID NO:4} and the Mature sequence (SEQ ID NO:5).

Identification of corresponding genes in N. meningitidis sero-groups a and N. gonorrhoeae

Based on a sequence of N. meningitidis serological group b were cloned and sequenced genes from N. meningitidis sero-groups a and N. gonorrhoeae.

Full gene from N. meningitidis sero-group a is shown as SEQ ID NO: 6, encoded protein as SEQ ID NO: 7. The signal peptide and the Mature sequence shown as SEQ ID NO:8 and SEQ ID NO:9.

Full gene from N. gonorrhoeae shown as SEQ ID NO: 10 encoded protein as SEQ ID NO:11. The signal peptide and the Mature sequence shown as SEQ ID NO:12 and SEQ ID NO:13.

Comparison of the sequences

Protein sequences were compared, and they are vysokomolochnye.

The sequence of the N. meningitidis sero-groups and the sequence of N. gonorrhoeae discover the identity of 95.4% in the overlap 797 amino acids:

Sequences of N. meningitidis sero-groups a and b find the Ute identity of 99.9% overlap 797 amino acids:

A high degree of conservatism dictates that the only protein may be able to induce immune responses against many species of Neisseria.

Vaccines

Three proteins identified, as described above, expressed and used for immunization. Watched a good immune response against these proteins.

Combined vaccines

In addition, each of these proteins was combined with antigens against other pathogenic organisms (e.g., polysaccharide vaccine Chiron against meningitis serological groups) and used for immunization. Watched a good immune response.

Additional components NmB

Although it is effective, the protection induced by the Norwegian OMV vaccine is limited by the strain used to prepare the vaccine. Clinical trials of this vaccine only got 57,2% efficiency after 29 months in adolescents, although IgG response was observed in almost 100% of patients [see, for example, Rosenqvist et al. (1995) Infect. Immun. 63:4642-4652].

It has been unexpectedly discovered that the addition addition of certain components to the Norwegian OMV vaccine was significantly expanded its effectiveness.

The Norwegian vaccine induces protection against strain 2996 NmB. Certain proteins from strain 2996) was added to the Norwegian vaccine, and it was shown that the effectiveness of this vaccine is peliculas to an amazing degree. In addition, adding a polysaccharide conjugate antigen NmB [see, for example, Constantino et al. (1992) Vaccine 10:691-698] gave excellent results.

Bactericidal activity of these combinations are shown in the following table:

GroupNorwegian OMVAntigen NmB*Antigen NmCBactericidal activity against strain 2996 NmB
1+--<4
2+#1-512
3+#2->2048
4+#3-1024
5+#3+256
6-#3-2048
7-#3+2048
* Used three different antigen NmB:

No. 1: ORF1 for example, example 77 from WO 99/24578 (see also WO 99/55873)

No. 2: protein '287' for example, figure 21 of WO 99/57280 (also SEQ ID NO:3103-3108)

No. 3: a mixture of Al(OH)3#1, #2 and protein '919' (SEQ ID NO:14, as shown here; see also figure 23 of WO 99/57280 and SEQ ID NO:3069-3074 there).

It is easy to see that the inefficiency of the Norwegian vaccine strain 2996 (group 1) can be overcome by the addition of specific antigens from strain 2996. Results using protein '287' NmB are especially good. Thus, the Norwegian vaccine can be improved without the need for OMV from a number of different strains.

This vaccine also provides protection against heterologous strains Mepv. The same vaccine, obtained using proteins of strain 2996, was tested against five other strains. The titles were as follows:

Group2996BZ133BZ2321000MSNGH38
1<4-1024<4>2048>204832
2512512<4>2048>2048256
3409640962561024>20481024
410242048<42048>204864
5256>32000<4>2048> 2048128
6204820484<4644
72048>3200041281024128
Control*327684096819216384163848192
* Controls: strains 2996, BZ133 and 1000=OMV prepared from the homologous strain; strain BZ232=OMV prepared from 2996; S & NGH38=SEAM3.

The second test "Norwegian" OMV, supplemented with protein from strain 2996 NmB:

protein 919, expressed in E. coli without the slit partner

- ORF1, expressed in E. coli as His-fused protein

protein 287, expressed as GST-fused protein

the mixture of these three proteins, with optional NmC-conjugate

The drugs were combined with the adjuvant Al(OH)C and tested against the homologous strain using bactericidal analysis. The results were as follows:

NmBNmC
Antigen2996NGH38394/98C11BZ133
WMD<432 1024*<41024
+919<4<44<4512
+ORF151225620484096512
+2874096102410245124096
+mix1024644642048
+mix +NmC256128204864000>32000
* antibodies were bacteriostatic, not bactericidal

Additional research antigen 287

The combination of the Norwegian OMV antigen 287 investigated additionally. 20 μg of antigen 287 combined with the Norwegian vaccine MRA (15 µg MRA + Al(OH)3) and used for immunization of mice. These antibodies were tested in bactericidal analysis, and they were effective against all the tested strains. The results were as follows:

NmBNmANmC
Antigen2996BZ133BZ23A1000MSNGH38NZ F6124C11
CMV<41024<4>20483276832<4--
28780004096256<45122048102410242048
OMV+ 287409640962561024409610241024--

Thus, in almost all cases, the combination of OMV + protein 287 unexpectedly gives better results than one OMV.

Recombinant WMD

E. coli transformed to the expression of ORF1, ORF40 and ORF46. OMV derived from recombinant E. coli, were able to induce bactericidal antibodies against N. meningitidis.

ORF1, ORF40 and ORF46 (strain 2996) expressed as His-tagged fusions in E. coli and received either in the form of pure proteins or in the form of OMV. Bactericidal titers against both drugs were tested using strain 2996 as antigenic stimulation:

AntigenORF1ORF40ORF46
Cleared64204816000
WMD124 256128000

Was measured bactericidal titers using heterologous strains for antigenic stimulation. ORF46 gives the titer of 4096 against strain MS in pure form, but the titer increased to 32000 in the provision in the form of OMV. ORF1 gives a titer of 128 against the strain F6124 NmA in its pure form, but it increases up to 512 when used in the form of OMV. ORF40 gives a titer of 512 against the strain MS in pure form, but this result is doubled when used in the form of OMV.

These data show that the antigens NmB retain immunogenicity upon receipt in E. coli as OMV and, moreover, that immunogenicity can be really improved.

It should be clear that this application describes an invention only by way of example, and may be made of modifications, which will remain within the claims and the essence of this invention.

1. The composition for inducing an immune response that contains (a) outer membrane vesicles of N. meningitidis sero-group and (b) immunogenic component containing the

(i) an amino acid sequence selected from the group consisting of:

MFKRSVIAMACIFPLSACGGGGCGSPDVKSADTPSKPAAPVVAENAGEGVLPKEKKDEEAAGGAPQADTQDATAGEGSQDMAAVSAENTGNGGAATTDNPKNEDAGAQNDMPQNAAESANQTGNNQPAGSSDSAPASNPAPANGGSDFGRTNVGNSVVIDGPSQNTLTHCKGDSCNGDNLLDEEAPSKSEFEKLSDEEKIKRYKKDEQRENFVGLVADRVKKDGTNKYIIFYTDKPPTRSARSRRSLPAEIPLLPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPAKGEMLVGTAVYNGEVLHFHMENGRPYPSGGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKDRD,

MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTADNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGGQNAGNTAAQGANQAGNNQAAGSSDPIPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDKFVGLVADSVQMKGINQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD

and

MFKRSVLAMACIVALSACGGGGGGSPDVKSADtlskpaapvvtedvgeevlpkekkdeeavsgapqadtqdatagkggqdmaavsaentgnggaattdnpenkdegpqndmpqnaadtdsstpnhtpapnmptrdmgnqapdagesaqpanqpdmanaadgmqgddpsagenagntadqaanqaennqvggsqnpasstnpnatnggsdfgrinvangikldsgsenvtlthckdkvcdrdfldeeappksefeklsdeekinkykkdeqrenfvglvadrvekngtnkyviiykdksassssarfrrsarsrrslpaemplipvnqadtlivdgeavsltghsgnlfapegnyryltygaeklsggsyalsvqgepakgemlagtavyngelhfhmengrpspsggrfaakvdfgsksvdgiidsgddlhmgtqkfkavidgngfkgtwtengggdvsgrfygpageevagkysyrptdaekggfgvfagkkeqd.

(ii) an immunogenic fragment of (i) 10 or more amino acids and/or

(iii) amino acid sequence having identity with respect to (i)equal to 70% or higher.

2. The composition according to claim 1, where component (b) includes protein from N. meningitidis strain that is different from the strain from which you obtained component (a).

3. Composition according to any one of the preceding paragraphs, where the outer membrane vesicles (OMV) are desoxycholate extract of N. meningitidis.

4. Composition according to any one of the preceding paragraphs, where one or more of its components adsorbed on Al(OH)3.

5. The composition according to claim 4, where component (a) adsorbed on Al(OH)3.

6. The composition according to any preceding paragraph, further containing one or more of the following components:

protective antigen against Neisseria meningitidis sero-group A;

protective antigen against Neisseria meningitidis sero-group;

protective antigen against Neisseria meningitidis sero-group Y;

protective antigen against Neisseria meningitidis sero-group W;

protective antigen against Haemophilus influenzae;

protective antigen against Pneumococcus;

protective antigen against diphtheria;

protective antigen against tetanus;

protective antigen against pertussis;

protective antigen FR is in Helicobacter pylori;

protective antigen against polio and/or

protective antigen against hepatitis C.

7. The composition according to any preceding paragraph, where the composition is a vaccine.

8. The composition according to any preceding paragraph for use as a medicine.

9. The method of treatment of a patient, introducing this patient a therapeutically effective amount of the composition induces an immune response, according to any one of claims 1 to 8.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method involves applying radiation therapy and two regional intra-arterial chemotherapy courses with Cysplatin given at a dose of 90 mg/m2 with hyperglycemia combined with microwave frequency hyperthermia 4-6 weeks later with 4 weeks long pause. Cyclophosphan is additionally introduced concurrently with radiation therapy for 10 weeks once a week at a dose of 200 mg. Roncoleukin is intravesicularly introduced after the second regional intra-arterial chemotherapy course at a dose of 2.0 mg dissolved in 50 ml of 0.9% NaCl solution twice a week during at least 8 weeks.

EFFECT: reduced risk of complications; increased clinical response frequency.

FIELD: medicine, pediatrics, infection diseases.

SUBSTANCE: method involves subcutaneous administration of roncoleukine in the dose 250000 U by the following schedule: course is 4 injections, at two days intervals on the third day, and this course is repeated if necessary up to disappearance of clinical symptoms. Invention promotes to more complete body sanitation due to inhibition of blood virus replication, decrease and in some cases elimination of symptoms of extrapyramid syndrome, diminishing hepatomegalia and normalization of the immune function system. Invention can be used in treatment of early age children with cytomegalovirus (CMV) infection.

EFFECT: improved treatment method.

8 tbl

FIELD: medicine, therapy.

SUBSTANCE: invention relates to a method for prognosis of effectiveness in treatment of infectious and inflammatory diseases with recombinant cytokines. Method involves using assay of functional polymorphism of cytokine gene as a prognostic criterion as the effectiveness degree and by-side effect of therapy with recombinant cytokine analog. The advantage of invention involves the possibility for prognosis of effectiveness of treatment with recombinant cytokine regulating inflammation and depending on genetic specificity. Invention can be used in immunotherapy, immune correction, immune rehabilitation or immune prophylaxis of different diseases, in particular, in diseases of otorhinolaryngological organs, chronic hepatitis C and pulmonary tuberculosis.

EFFECT: improved prognosis method of treatment.

3 cl, 4 dwg, 3 ex

FIELD: medicine, immunology.

SUBSTANCE: invention relates to compositions and method for immunosuppression achievement. Claimed compositions contain two main agents: namely the first agent targeted to interleukin-15 receptor (IL-15R), and the second agent which inhibits costimulating signal transferred between T-cell and antigen-presenting cell (APC).

EFFECT: diagnosis and therapy of immune deceases, in particular autoimmune deceases with improved effectiveness.

45 cl, 3 dwg

FIELD: medicine, infectious diseases, psychotherapy.

SUBSTANCE: method involves antiviral therapy, immune correction with thymus hormones and interferon inductors. Since the first day the relapse symptom method involves prescription of antiox+ (1 capsule per a day) and detox+ (1 capsule, 2 times per a day) for 30 days, profluzak (20 mg, 3 times per a day for 5 days) and then in the dose 20 mg, 1 time per a day for 20 days. Derinate is prescribed topically as installation into urethra in the dose 3-5 ml or with tampon into vagina and with simultaneous prescription of microenemas in the dose 10-40 ml for 10 days. Since 10-14 day in exacerbation period in the proliferative stage of an antiherpetic immune response derinate is prescribed by intramuscular injections in the dose 5 ml, 1 time in a day, 10 injections in total number. Then since 6-th day of exacerbation and intake of profluzak psychotherapy seances are carried out. The first seance of rational psychotherapy involves explanation to a patient in available form mechanism of the disease, the necessity of prolonged treatment and motivation for treatment is enhanced by suggestion. The second psychotherapy seance involves neurolinguistic programming wherein a patient colorful and detailed description of desirable function when he imagines achievement of the desire result, and positive emotional and vegetative symptoms are notes and the conditional-reflect association is formed by tactile contact. Under psychotherapist control a patient imagines "part of person" responsible for achievement of the desire result the patient attention is accented for the desire result and arisen physiological responses are fixed by using tactile contact. Also, new behavior methods are proposed to take for a patient that are directed for achievement of the desire result - avoiding sexual contacts during exacerbation of genital herpes in one of partner and during every month hormonal cycles, avoiding stress situations, and in case of each stress situation significant for patient profluzak has to be intake in a single dose 40 mg, using a condom in sexual contact in the exacerbation period. Patient analyzes the proposed new behavior methods that help avoiding relapses, provide good state of health, promotes to recovery process of genitals recovery and selects at least three the most rationally available for him behavior methods. In the case of the positive response that is controlled by physiological symptoms the result is fixed by tactile contact. The third seance involves the suggestive psychotherapy directed for fixing the attained result. The suggestive therapy seance is carried out once per a week for 6 months. Method provides declining the treatment time, to reduce relapse frequency of genital herpes and to recover the emotional state of patient.

EFFECT: improved treatment method.

2 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves applying basic therapy until stroke type is unclear and differentiated therapy after having determined stroke type. Recombinant interleukine-2-roncoleukine as subcutaneous injections at a dose of 500 000 MU into external surface of intact arm at the fourth-fifth day after stroke beginning. Roncoleukine is administered as a course of 1-3 injections given with two-three days pause on the background of traditional stroke treatment.

EFFECT: enhanced effectiveness of treatment.

2 cl, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves combining vaccine prepared from 107 cells of autologic tumor lysate with 60 mg of betaleukine and introducing it strictly in intracutaneous paravertebral mode in three points arranged 3 cm far from each other. The introduction takes place every 3 weeks so that the first two vaccinations are combined with 470 mg of betaleukine introduced into anterior abdominal wall. Vaccination treatment is continued on receiving positive retarded type hypersensitivity response to injection after every vaccination with tumor lysate without betaleukine.

EFFECT: enhanced effectiveness in inducing and supporting antitumor immune response.

FIELD: medicine, oncology.

SUBSTANCE: after removing malignant cerebral tumor one should conduct successive course of: cytokinotherapy consisting of 3 intramuscular injections of leukineferon at 48-h-long interval, adaptive immunotherapy with lymphokine-activated killer cells (LAKC) generated in the presence of recombinant interleukin-2 (IL-2). Moreover, LAKC should be introduced into the channel of removed tumor in combination with IL-2 as 2 procedures at 24-h-long interval. Then comes the course of adaptive immunotherapy with cytotoxic lymphocytes (CTL) generated due to cultivating patient's mononuclear blood cells together with dendritic cells loaded with a tumor antigen, in the presence of recombinant IL-2 to be introduced in combination with it into the channel of removed tumor as 2 procedures at 48-h-long interval. For obtaining dendritic cells in patients before operation it is necessary to isolate monocytes to cultivate with granulocytic-macrophageal colony-stimulating factor and interferon-α at maturating dendritic cells in the presence of monocytic conditioned medium and incubation of dendritic cells in the presence of tumor antigenic material for their loading with a tumor antigen. After immunotherapy with CTL on should conduct the course of vaccinotherapy with dendritic cells loaded with a tumor antigen in combination with subcutaneous injections of IL-2. The method enables to induce high specific anti-tumor immune response along with improved quality of life and prolonged duration of relapse-free period.

EFFECT: higher efficiency of immunotherapy.

2 cl, 2 ex

FIELD: medicine, gastroenterology, pharmacy.

SUBSTANCE: invention relates to agents used in treatment of ulcerous-erosion injures in gastroduodenal region. Method involves diluting 100 mcg of dry lyophilized powder of immunomodulating agent "Superlimf" in 3-5 ml of 0.9% isotonic solution and irrigation of ulcer or erosion with this solution 1 time per a day by the endoscopy method. The treatment course is 3-4 procedures with break for 4-5 days. Method provides alteration of cytokine pattern of tissues, induction of influx of mononuclear phagocytes to the injure focus that results to localization of inflammation and the complete epithelization of ulcers and erosions.

EFFECT: improved and effective method for treatment.

1 ex

FIELD: cardiovascular disorders.

SUBSTANCE: invention provides methods for modulating tissue antiogenesis using Raf and/or Ras protein, modified Raf and Ras proteins, nucleic acids encoding them. Antiogenesis is inhibited using inactive Raf or Ras proteins or nucleic acids encoding them, and antiogenesis is potentiated using active Raf or Ras proteins or nucleic acids encoding them. Use of gene transportation system to provide nucleic acids encoding Raf or Ras proteins or modified forms thereof.

EFFECT: enlarged choice of tissue antiogenesis control methods and means.

44 cl, 20 dwg

Lantibiotic // 2279481

FIELD: biotechnology, in particular antibacterial protein salivaricine B belonging to lantibiotics.

SUBSTANCE: antibacterial protein is expressed by strain Streptococcus salivarius K12 (DSM N 13084), has molecular weight of about 2733 Da, contains N-terminal amino acid sequence (AS) representing in SEQ ID NO:1, as defined in description, or AS representing in SEQ ID NO:3, and has bactericide activity against S. pyogenes. Both protein and microorganism producing thereof are useful as active ingredients in antibacterial and antibacterial therapeutic compositions, wherein the latter one may additionally contain salivaricine A2 (AS SEQ ID NO:5, as defined in description). Also invention relates to polynucleotide, encoding protein (salivarius B) optionally including nucleotide sequence (NS) (NS SEQ ID NO:2), as defined in description). Polynucleotide optionally includes DNA sequence encoding antibacterial protein, representing genome part of strain K12 (DSM 13084). Strain S. salivarius K12 (DSM N 13084) and S. salivarius K30 (DSM N 13085) being producers of protein salivaricine B also are described.

EFFECT: new protein having both antibacterial and bactericide activity.

29 cl, 3 dwg, 1 tbl

FIELD: biotechnology, in particular utilization of strain Lactobacillus salivarius UCC 118 for prophylaxis and/or treatment of undesired inflammation activity and cancer prophylaxis.

SUBSTANCE: human-original strain Lactobacillus salivarius UCC 118[NCIMB 40829] is isolated from dissected and washed human gastrointestinal tract. Strain application makes it possible to decelerate development of malignant diseases and to reduce undesired inflammation activity such as intestine inflammation or irritable colon.

EFFECT: strain for prophylaxis and/or treatment of undesired inflammation activity and cancer prophylaxis.

42 cl, 29 dwg, 1 tbl, 4 ex

FIELD: medicine, in particular treatment of gastrointestinal inflammations associated with Helicobacter pylori.

SUBSTANCE: claimed method includes monotherapy with "Vitaflor" preparation in exacerbation step for an least four weeks. Daily dosage is determined in accordance to patient age. Method of present invention makes it possible to suppress topical hyperergic reactions, expeditious of superior dyspepsia syndrome, accelerated arresting of stomach and duodenum ulcer without deformations and cicatrices, prophylaxis of helicobacterial infection backsets for one year.

EFFECT: improved method for treatment of inflammatory gastrointestinal diseased associated with helicobacter pylori.

2 ex, 2 tbl

FIELD: medicine, pharmacology, pharmacy.

SUBSTANCE: invention relates to the development of a new medicinal agent representing the complex enzyme preparation with the digestive effect. Agent comprises enzyme pectate lyase providing effective cleavage of vegetable food intercellular substances, in particular, pectin and protopectin that results to formation of the homogenous and easily digestible mass, and promotes to assimilation vegetable food that is not assimilated by human organism that results to the more complete digestion of food, its cleavage and assimilation of nutrient substances. The optimal content of enzyme pectate lyase and pancreatin in a tablet covered by the enterosoluble envelope promotes to the more rapid and complete digestion of food in intestine and shows significant advantages as compared with the known digestive preparations.

EFFECT: improved and valuable properties of agent.

5 cl, 11 tbl

FIELD: veterinary medicine.

SUBSTANCE: method involves per os administering 10% Enrofloxacin solution in 50 ml/100 ml water dilution once a day during 5 days and Immunobac is introduced twice a day during 5 days in three courses depending on hen age in the amount of 0.5 times dose at the age of 6-10 days, 1 dose at the age of 30-34 days and 2 doses at the age of 70-74 days per one hen.

EFFECT: enhanced effectiveness in suppressing mycoplasmosis pathogens; normalized intestinal microbiocenosis.

7 tbl

FIELD: veterinary medicine.

SUBSTANCE: method involves administering Aversect-2 with Cordeon probiotic added at a dose of 50 mg/kg of live weight during 5 days.

EFFECT: enhanced effectiveness of treatment; improved blood biochemical and hematological properties.

3 tbl

FIELD: medicine.

SUBSTANCE: method involves administering antihistamine preparations and antibiotics, applying external treatment of injured skin areas with Hypchlophos, intramuscularly introducing anatoxin vaccine against animal staphylococcosis and immunostimulating agent like Immunoparasitan. Reinfusion session is applied at the treatment beginning after ultrasonic proper blood irradiation having been applied at a dose of 1-3 cm3/kg of body mass. Vitamin B12 is additionally intramuscularly introduced at a dose of 3-7 mcg/kg of body mass at the treatment course end. Hypchlophos is applied as oil emulsion in 1:1 proportion. Reinfusion combined with ultrasonic proper blood irradiation is administered together with anatoxin vaccine introduction at the same day continuing the treatment within 2-3 days.

EFFECT: enhanced effectiveness in normalizing metabolism processes and in increasing immunostimulation; reduced toxic adverse side effects.

4 cl, 1 tbl

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves the complex use of native yeast autolyzate as a component of nutrient and protective media for lyophilization and taken in the amount 5-25% of the bacterial suspension volume. Protective medium comprises defatted milk and native yeast autolyzate taken in the ratio = 1:(0.5-1). Invention provides standardization of raw and process in preparing nutrient and protective media, reducing cost in preparing probiotics and enhances effectiveness of their production. Invention can be used in producing probiotic preparations: lactobacterin, bifidumbacterin, acilact and others.

EFFECT: improved preparing method.

2 cl, 1 tbl, 2 ex

FIELD: medicine, phytotherapy.

SUBSTANCE: invention relates to non-medicinal treatment and prophylaxis of diseases, sanitation and rejuvenation measures. Method involves cleansing intestine at the first stage and that of liver at the second stage. Cleansing intestine is carried out by its irrigation with a solution containing 5-10 doses of bifidobacteria per 10-12 l of filtered water. Cleansing liver is carried out by intaking the homogenous mass consisting of milk thistle oil with defatted kefir that is alternated with intaking infusions of gastroenteric, hepatic and renal phytospecies. Phytospecies infusion is drunk with curative tea prepared from the following components, g: green tea, 25.0-35.0; common balm or peppermint, 25.0-35.0; honey, 25.0-35.0; lemon juice, 45.0-55.0, and 1.1-1.3 l of boiling water. Method provides cleansing detoxification organs - intestine, liver and kidneys, to decrease "biological age" by 4-5 years, to improve cerebral and peripheral circulation and immunity that is suitable for a patient due to reducing cleansing periods and diminishing doses of consumed substances.

EFFECT: improved method for treatment and prophylaxis.

6 cl, 5 ex

FIELD: biotechnology, cosmetic industry, medicine, veterinary, in particular manufacturing of healthy products.

SUBSTANCE: Bifidobacterium lactis 668 strain is isolated from healthy infant bulk. Strain of present invention has increased biological activity, environmental strength, accelerated growth on artificial broth, increased acid-forming and antagonistic activity against pathogenic and opportunistic microflora. Claimed strain is useful in production of healthy products, in particular to normalize microflora of human and animal intestinal and urogenital tracts, skin and mucous membranes.

EFFECT: enhanced probiotic assortment.

2 tbl, 5 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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