WO2003033515A1 - Compositions et methodes pour le traitement et le diagnostic de l'acne vulgaire - Google Patents

Compositions et methodes pour le traitement et le diagnostic de l'acne vulgaire Download PDF

Info

Publication number
WO2003033515A1
WO2003033515A1 PCT/US2002/032727 US0232727W WO03033515A1 WO 2003033515 A1 WO2003033515 A1 WO 2003033515A1 US 0232727 W US0232727 W US 0232727W WO 03033515 A1 WO03033515 A1 WO 03033515A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
group
nos
acnes
further provides
Prior art date
Application number
PCT/US2002/032727
Other languages
English (en)
Inventor
Jennifer L. Mitcham
Yasir A. W. Skeiky
David H. Persing
Ajay Bhatia
Jean-François L. MAISONNEUVE
Yanni Zhang
Siqing Wang
Shyian Jen
Michael J. Lodes
Darin R. Benson
Robert Jones
Darrick Carter
Brenda Barth
John Vallieve-Douglass
Original Assignee
Corixa Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Corixa Corporation filed Critical Corixa Corporation
Publication of WO2003033515A1 publication Critical patent/WO2003033515A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • CD-R No. 1 is labeled Copy 1 - Sequence Listing Part, contains the file 51410PC.txt which is 34,662 KB and created on October 8, 2002
  • CD-R No. 2 is labeled Copy 2 - Sequence Listing Part, contains the file 51410PC.txt which is 34,662 KB and created on October 8, 2002
  • CD-R No. 3 is labeled Copy 3 - Sequence Listing Part, contains the file 51410PC.txt which is 34,662 KB and created on October 8, 2002
  • CD-R No. 4 is labeled CRF, contains the file 51410PC.txt which is 34,662 KB and created on October 8, 2002.
  • the present invention relates generally to therapy and diagnosis of acne, particularly those forms of acne that are caused in whole or in part by the organism
  • Propiombacterium acnes or a related organism is more specifically related to Propiombacterium acnes polynucleotides, polypeptides, antibodies and other compositions for the prevention, treatment and/or diagnosis of acne. Description of the Related Art
  • Propiombacterium comprise a group of anaerobic non-spore- forming gram-positive rods that are the predominant member of anaerobic flora from conjunctiveal cultures of adults and children.
  • Propionibacteria have been implicated in a number of human conditions, including S APHO syndrome (synovitis, acne, pustulosis, hypertosis, and osteomyelitis), uveitis and endophthalamitis. The organism has also been shown to be pathogenic in infections of bone, joints and the central nervous system.
  • Propiombacterium acnes In addition to its ability to stimulate both the classical and alternative complement pathways, Propiombacterium acnes also has been reported to produce small molecular weight, serum-independent neutrophil chemotactic factors that can attract neutrophils through the epithelial wall to Propiombacterium acnes within the sebaceous follicle lumen. Ingestion of Propiombacterium acnes by neutrophils in the presence of antibody specific for Propiombacterium acnes results in the release of hydrolases that may contribute to the disruption of the follicular wall. Along with neutrophil infiltration of the sebaceous follicular epithelium, lymphocyte accumulation and infiltration have been described as early events in pathophysiology of inflammatory acne.
  • the present invention provides compositions and methods for the diagnosis and therapy of skin and other disorders, such as acne vulgaris, that are at least partly caused or contributed to in mammals by Propiombacterium acnes and other related organisms.
  • the present invention provides polypeptides comprising at least a portion of a Propiombacterium acnes protein, or a fragment or variant thereof. Certain portions and other variants are immunogenic, such that the ability of the variant to react with antigen-specific antisera is not substantially diminished.
  • the polypeptide comprises a sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOs: 1-299; (b) variants and biological functional equivalents of a sequence recited in SEQ ID NOs: 1-299; and (c) complements of a sequence of (a) or (b).
  • the polypeptides of the present invention comprise at least a portion of a Propiombacterium acnes protein that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 300-29,212 and variants and derivatives thereof.
  • the present invention provides polynucleotides that encode a polypeptide as described above, or portions thereof (such as a portion encoding at least 15, 25, 35, 45, 55, etc., contiguous amino acid residues of a Propiombacterium acnes protein), as well as expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
  • P. acnes polypeptides have been identified herein by a variety of experimental and bioinformatic approaches.
  • the present invention provides P. acnes transferase-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 927, 1051, 2084, 2525, 2767, 2891, 3255, 3443, 4287, 5495, 5751, 6133, 6186, 10674, 12113, 12316, 12334, 12347, 12610, 12868, 14234, 14307, 14769, 16001, 16403, 17869, 18690, 20108, 20783, 21258, 22948, 24326, 24787, 25803, 26333, 27012, 27019, 27138, 27141, 27307, 28925, 29085 and 29204.
  • the invention further provides P. acnes enterotoxin polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23419.
  • the invention further provides P. acnes lipoprotein-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6740, 12271, 15138, 16073, 19399, 19447, 21711, 24622, 26161 and 28123.
  • the invention further provides P.
  • acnes membrane-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1469, 1909, 2482, 2676, 2905, 3081, 3151, 3773, 4114, 4725, 5864, 6515, 7380, 7425, 7576, 7710, 8042, 8324, 8370, 9100, 9782, 10658, 13122, 13897, 15890, 16337, 16713, 17093, 17631, 19021, 19085, 19108, 19154, 19407, 20132, 20207, 20382, 20395, 20512, 21074, 22771, 23809, 24355, 24770, 24800, 24826, 26504, 26798, 27079, 27470, 27475 and 29132.
  • the invention further provides P. acnes permease-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9383, 9430, 11564, 12052, 12110, 12261, 14484, 14820, 15476, 21154, 21180, 21983, 22019, 22456, 24300, 26784 and 27769.
  • the invention further provides P. acnes protease/proteinase-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10003, 10107, 10197, 17403, 18436, 21368,1585, 2909, 3071, 8768, 10789, 11032, 11033 and 12785.
  • the invention further provides P. acnes protein A-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23820.
  • the invention further provides P. acnes secreted polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3609, 4118, 6124, 8003, 10273, 11816, 14351, 14514, 16142, 17629, 21155, 22996, 23418 and 24185.
  • the invention further provides P. acnes dismutase-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3608.
  • the invention further provides P. acnes adhesin-r elated polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17237.
  • the invention further provides P. acnes transporter-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3979, 4157, 4686, 5329, 6589, 6709, 6896, 7028, 7564, 8125, 8126, 8170, 8541, 9004, 9383, 9430, 11564, 11565, 11624, 11682, 12346, 12429, 12563, 12718, 14103, 14484, 14742, 15884, 16279, 16327, 17306, 17307, 18134, 18177, 18245, 19157, 19191, 19359, 19566, 19581, 20311, 20479, 20923, 20926, 21153, 21172, 22819, 23880, 23891, 25720, 26021, 26025, 26281, 26490, 26700, 26784, 26977, 27523, 28116, 28250 and 28385.
  • the invention further provides P. acnes haemolysin-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7017.
  • the invention further provides P. acnes penicillin binding-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10657, 16595 and 25267.
  • the invention further provides P. acnes sialidase-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6710, 16380, 26503 and 27212.
  • the invention further provides P. acnes siderophore-r elated polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6887.
  • the invention further provides P. acnes lipase-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13402.
  • the invention further provides P. acnes enterobactin receptor-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5334 and 11075.
  • the invention further provides isolated cohemolysin-related polypeptides comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15127, 25540, 6706, 23076, 8751 and 27533.
  • the invention further provides isolated polypeptides, such as those identified herein as described in Examples 6 and 7, comprising at least an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20036, 19872, 6373, 8769, 5991, 19299, 5011, 12317, 11062, 4958, 6923, 11989, 24369, 21491, 7887, 10447, 3070, 7878, 25103, 6868, 9121, 24474, 12029, 20955, 16555, 15237, 20973, 20475, 15259, 6427, 4967, 20100, 10141, 15657, 3608, 16608, 21710, 25380, 4853, 25957, 9071, 3659, 13512, 3697, 7384, 24722, 10674, 1282, 1209, 6501, 3691, 12347, 21762, 15197, 1165, 23091, 6428, 5429, 24758, 3590, 3723, 9106
  • the invention further provides, for example, isolated polypeptides comprising at least an immunogenic fragment of a polypeptide encoded by a DNA molecule identified by serological expression cloning, as described herein, such as a polypeptide sequence selected from the group consisting of SEQ ID NOs: 25131, 25133, 25135, 25163, 25164, 25165, 25166, 25167, 25168, 25169, 25192, 25193, 25215, 25216, 25217, 25218, 7729, 25957, 6884, 7653, 7668, 20966, 20984, 21002, 905, 906, 1008, 5541, 25957, 5527, 5541, 25086, 25088, 25089, 25090, 25103, 25130, 25131, 25133, 25135, 25163, 25164, 25165, 25166, 25167, 25168, 25169, 25192, 25193, 25215, 25216, 25217, 25218, 25922, 25938, 25957, 5527, 5541,
  • the invention further provides polypeptide compositions encoded by any one of the DNA sequences set forth in SEQ ID NOs: 1-299.
  • the invention provides polypeptides encoded by SEQ ID NO: 1 such as those selected from the group consisting of SEQ ID NOs: 318, 319, 337, 340, 348, 351, 364, 365, 366, 369, 373, 374, 382, 383, 384, 398, 418, 427, 428, 429, 431, 443, 444, 470, 486 and 28650.
  • the invention further provides polypeptides encoded by SEQ ID NO: 2 such as those selected from the group consisting of SEQ ID NOs: 493, 504, 506, 513, 518, 524, 547, 559, 572, 578, 589, 593, 600, 617, 620, 624, 632, 642; 644, 665, 666, 692, 701, 709, 710, 716, 727, 736, 737, 771, 786, 787, 789, 790, 791, 792, 805, 807, 808 and 812.
  • SEQ ID NO: 2 such as those selected from the group consisting of SEQ ID NOs: 493, 504, 506, 513, 518, 524, 547, 559, 572, 578, 589, 593, 600, 617, 620, 624, 632, 642; 644, 665, 666, 692, 701, 709, 710, 716, 727, 736, 737, 771
  • the invention further provides polypeptides encoded by SEQ ID NO: 3 such as those selected from the group consisting of SEQ ID NOs: 814, 853, 873, 878, 885, 898, 899, 905, 906, 917, 921, 927, 928, 930, 970, 975, 993, 995, 997, 1007, 1008, 1012, 1026 and 28654.
  • SEQ ID NO: 3 such as those selected from the group consisting of SEQ ID NOs: 814, 853, 873, 878, 885, 898, 899, 905, 906, 917, 921, 927, 928, 930, 970, 975, 993, 995, 997, 1007, 1008, 1012, 1026 and 28654.
  • the invention further provides polypeptides encoded by SEQ ID NO: 4 such as those selected from the group consisting of SEQ ID NOs: 1049, 1051, 1054, 1056, 1081, 1123 and 28657.
  • the invention further provides polypeptides encoded by SEQ ID NO: 5 such as those selected from the group consisting of SEQ ID NOs: 1127, 1133, 1137, 1138, 1153, 1159, 1161, 1162, 1163, 1165, 1167, 1168, 1171, 1172, 1181, 1187, 1196, 1203, 1209, 1212, 1215, 1225, 1229, 1230, 1231, 1235, 1251, 1252, 1269, 1270, 1274, 1275, 1277, 1278, 1282, 1284, 1285, 1289, 1290, 1292, 1293, 1294, 1295, 1297, 1300, 1318, 1323, 1324, 1332, 1334, 1335, 1338, 1339, 1340, 1341,
  • the invention further provides polypeptides encoded by SEQ ID NO: 6 such as those selected from the group consisting of SEQ ID NOs: 1434, 1437, 1442, 1444, 1445, 1446, 1447, 1449, 1452, 1460, 1463, 1469, 1483, 1488, 1489, 1490, 1491, 1508, 1522, 1523, 1524, 1535, 1550, 1567, 1575, 1585, 1586, 1588, 1595, 1596, 1599, 1603, 1607, 1608, 1611, 1616, 1624, 1626, 1627, 1632, 1635, 1636, 1637, 1645, 1646, 1652 and 28661.
  • the invention further provides polypeptides encoded by SEQ ID NO: 7 such as those selected from the group consisting of SEQ ID NOs: 1670, 1674, 1708, 1714, 1716, 1731, 1734, 1744, 1754, 1756, 1767, 1776, 1779, 1780, 1795, 1800, 1827, 1832, 1841 and 1845.
  • the invention further provides polypeptides encoded by SEQ ID NO: 8 such as those selected from the group consisting ofSEQ ID NOs: 1871, 1884, 1895, 1906, 1907, 1909, 1938, 1941, 1952, 1955, 1957, 1968, 1971, 1973, 1979, 1980, 1998, 2004, 2005, 2020, 2025, 2028, 2035, 2036, 2040, 2044, 2084, 2088, 2114, 2127, 2137, 2150, 2153, 2158, 2164, 2180, 2190, 2192, 2198, 2203, 2215, 2218, 2228, 2230, 2231, 2240, 2243, 2246, 2258, 2259, 2260, 2265 and 2278.
  • SEQ ID NO: 8 such as those selected from the group consisting ofSEQ ID NOs: 1871, 1884, 1895, 1906, 1907, 1909, 1938, 1941, 1952, 1955, 1957, 1968, 1971, 1973, 1979, 1980, 1998, 2004, 2005, 2020, 2025, 2028, 2035, 20
  • the invention further provides polypeptides encoded by SEQ ID NO: 9 such as those selected from the group consisting ofSEQ ID NOs: 2288, 2292, 2299, 2300, 2303, 2305, 2306, 2310, 2311, 2315, 2316, 2324, 2326, 2329, 2344, 2346, 2357, 2378, 2379, 2388, 2398, 2402, 2403, 2405, 2410, 2412, 2414, 2417, 2420, 2425, 2427, 2447, 2453, 2457, 2458, 2460, 2461, 2462, 2469, 2474, 2482, 2483, 2487, 2492, 2494, 2497, 2506, 2507 and 28666.
  • SEQ ID NO: 9 such as those selected from the group consisting ofSEQ ID NOs: 2288, 2292, 2299, 2300, 2303, 2305, 2306, 2310, 2311, 2315, 2316, 2324, 2326, 2329, 2344, 2346, 2357
  • the invention further provides polypeptides encoded by SEQ ID NO: 10 such as those selected from the group consisting of SEQ ID NOs: 2512, 2518, 2522, 2525, 2535, 2544, 2548, 2549, 2553, 2576, 2577, 2578, 2586, 2604, 2617, 2618, 2632, 2648, 2657, 2666, 2672, 2676, 2682, 2685, 2703, 2713, 2724, 2742, 2743, 2745, 2748, 2752, 2755, 2760, 2767, 2769, 2784, 2790, 2798, 2799, 2800, 2801, 2802, 2813, 2824, 2825, 2826, 2827, 2828, 2830, 2845, 2850, 2880, 2891, 2894, 2905, 2907, 2909, 2913, 2925, 2926, 2927, 2937, 2940, 2942, 2952, 2956, 2978, 2984, 2986, 2987, 2988, 2989, 2997
  • the invention further provides polypeptides encoded by SEQ ID NO: 11 such as those selected from the group consisting of SEQ. ID NOs: 3217, 3222, 3235, 3255, 3261, 3267, 3272, 3287, 3289, 3296, 3297, 3301, 3302, 3313, 3314, 3315, 3317, 3319, 3320, 3327, 3331, 3334, 3342, 3349, 3369, 3370, 3371, 3373, 3375, 3381, 3382, 3390, 3402, 3403, 3418, 3422, 3425, 3429, 3437, 3441, 3442, 3443, 3449, 3451, 3454, 3455, 3457, 3458, 3471, 3483, 3489, 3499, 3522, 3523, 3525, 3528 and 3536.
  • SEQ ID NO: 11 such as those selected from the group consisting of SEQ. ID NOs: 3217, 3222, 3235, 3255, 3261, 3267, 3272, 3287,
  • the invention further provides polypeptides encoded by SEQ ID NO: 12 such as those selected from the group consisting of SEQ ID NOs: 3539, 3544, 3549, 3551, 3552, 3555, 3558, 3559, 3560, 3572, 3584, 3585, 3586, 3587, 3589, 3591, 3593, 3594, 3608, 3609, 3625, 3626, 3627, 3632, 3635, 3652, 3658, 3659, 3661, 3666, 3682, 3683, 3687, 3690, 3691, 3697, 3701, 3708, 3721, 3722, 3723, 3731, 3736 and 28674.
  • the invention further provides polypeptides encoded by SEQ ID NO: 13 such as those selected from the group consisting of SEQ ID NOs: 3755, 3759, 3763, 3764, 3767, 3773, 3776, 3783, 3799, 3807, 3808, 3809, 3815, 3816, 3817, 3821, 3828, 3831, 3835, 3837, 3838, 3849, 3866, 3875, 3889, 3904, 3909, 3916, 3917, 3918, 3923, 3925, 3945, 3947, 3955, 3956, 3969, 3979, 3988, 4004, 4023, 4024, 4060, 4073, 4080, 4096, 4101, 4102, 4103, 4111, 4114, 4118, 4122, 4134, 4135, 4143, 4149 and 4152.
  • the invention further provides polypeptides encoded by SEQ ID NO: 14 such as those selected from the group consisting of SEQ ID NOs: 4157, 4169, 4171, 4174, 4179, 4188, 4189, 4198, 4207, 4211, 4213, 4220, 4224, 4227, 4240, 4247, 4249, 4258, 4261, 4262, 4265, 4274, 4275, 4278, 4287, 4306, 4307, 4308, 4309, 4310, 4311, 4328, 4333, 4336, 4339, 4345, 4346, 4350 and 4351.
  • SEQ ID NO: 14 such as those selected from the group consisting of SEQ ID NOs: 4157, 4169, 4171, 4174, 4179, 4188, 4189, 4198, 4207, 4211, 4213, 4220, 4224, 4227, 4240, 4247, 4249, 4258, 4261, 4262, 4265, 4274, 4275, 4278, 4287, 4306, 4307
  • the invention further provides polypeptides encoded by SEQ ID NO: 15 such as those selected from the group consisting of SEQ ID NOs: 4362, 4365, 4370, 4373, 4378, 4380, 4386, 4389, 4393, 4394, 4407, 4408, 4417, 4418, 4428, 4435, 4437, 4438, 4439, 4442, 4450, 4454, 4458, 4460, 4462, 4466, 4474, 4495, 4496, 4510, 4511, 4522, 4529, 4531, 4533, 4536, 4539, 4545, 4552, 4553, 4570, 4577, 4581, 4587, 4591, 4594, 4599, 4605, 4612, 4616, 4617, 4618, 4620, 4622, 4630, 4634, 4640, 4643, 4646, 4651, 4659, 4666, 4673, 4678 and 28680.
  • the invention further provides polypeptides encoded by SEQ ID NO: 16 such as those selected from the group consisting of SEQ ID NOs: 4686, 4687, 4694, 4723, 4725, 4742, 4745, 4749, 4754, 4757, 4763, 4766, 4767, 4771, 4772, 4789, 4792, 4825, 4832, 4834, 4847, 4853, 4857, 4866, 4871, 4873, 4875, 4877, 4879, 4887, 4888, 4890, 4897, 4907, 4938, 4945, 4947, 4948, 4950, 4951, 4952, 4958, 4966, 4967, 4968, 4975, 4977, 4978, 4986, 4988, 4996, 5001, 5006, 5008, 5009, 5011, 5017, 5021, 5022, 5025, 5035, 5037, 5038, 5040, 5053, 5054, 5056, 5057, 5062, 5068, 5079, 5111, 5120, 5124, 5125
  • the invention further provides polypeptides encoded by SEQ ID NO: 17 such as those selected from the group consisting of SEQ ID NOs: 5354, 5356, 5358, 5373, 5379, 5389, 5396, 5397, 5399, 5417, 5429, 5437, 5438, 5440, 5441, 5446, 5452, 5463, 5478, 5493, 5495, 5503, 5511, 5512, 5514, 5519, 5520, 5523, 5527, 5541, 5544, 5559, 5568, 5577, 5585, 5586, 5593, 5606, 5607, 5613, 5614, 5639, 5648, 5655, 5662, 5664, 5671, 5673, 5675, 5678, 5716, 5737, 5738, 5747, 5751, 5756, 5758, 5759 and 28685.
  • SEQ ID NO: 17 such as those selected from the group consisting of SEQ ID NOs: 5354, 5356, 5358, 5373, 53
  • the invention further provides polypeptides encoded by SEQ ID NO: 18 such as those selected from the group consisting of SEQ ID NOs: 5770, 5773, 5774, 5776, 5783, 5789, 5810, 5811, 5823, 5834, 5844, 5847, 5852, 5855, 5858, 5860, 5861, 5863, 5864, 5865, 5867, 5868, 5872, 5884, 5886, 5895, 5900 and 28687.
  • SEQ ID NO: 18 such as those selected from the group consisting of SEQ ID NOs: 5770, 5773, 5774, 5776, 5783, 5789, 5810, 5811, 5823, 5834, 5844, 5847, 5852, 5855, 5858, 5860, 5861, 5863, 5864, 5865, 5867, 5868,
  • the invention further provides polypeptides encoded by SEQ ID NO: 19 such as those selected from the group consisting ofSEQ ID NOs: 5922, 5928, 5932, 5975, 5991, 5993, 6001, 6032, 6034, 6042, 6055, 6061, 6084, 6089, 6104, 6108, 6109, 6122, 6124, 6129, 6132, 6133, 6137, 6140, 6152, 6153, 6154, 6164, 6174, 6185, 6186, 6198, 6202 and 6213.
  • SEQ ID NO: 19 such as those selected from the group consisting ofSEQ ID NOs: 5922, 5928, 5932, 5975, 5991, 5993, 6001, 6032, 6034, 6042, 6055, 6061, 6084, 6089, 6104, 6108, 6109, 6122, 6124, 6129, 6132, 6133, 6137, 6140, 6152, 6153, 6154, 6164,
  • the invention further provides polypeptides encoded by SEQ ID NO: 20 such as those selected from the group consisting of SEQ ED NOs: 6225, 6245, 6248, 6252, 6259, 6268, 6270, 6278, 6279, 6280, 6288, 6301, 6302, 6304, 6318, 6321, 6322, 6327, 6331, 6337, 6338, 6342, 6346, 6360, 6361, 6362, 6367, 6373, 6404, 6417, 6427, 6428, 6429, 6432, 6448, 6455, 6482, 6483, 6484, 6496, 6500, 6501, 6505, 6507, 6508, 6511, 6515, 6573, 6576, 6580, 6589, 6597, 6598, 6599, 6607, 6610, 6618, 6639, 6643, 6658, 6666, 6684, 6695, 66
  • the invention further provides polypeptides encoded by SEQ ID NO: 21 such as those selected from the group consisting of SEQ ID NOs: 6711, 6712, 6722, 6731, 6732, 6740, 6741, 6756, 6773, 6782, 6784, 6794, 6795, 6796, 6849, 6852, 6868, 6870, 6875, 6877, 6879, 6881, 6884, 6887, 6889, 6890, 6892, 6893, 6894, 6896, 6908, 6916, 6923 and 28692.
  • the invention further provides polypeptides encoded by SEQ ID NO: 22 such as those selected from the group consisting of SEQ ID NOs: 6927, 6929, 6930, 6931, 6955, 6956, 6957, 6958, 6961, 6962, 6970, 6985, 6996, 6998, 7010, 7016, 7017, 7018, 7026, 7028, 7035 and 7037.
  • the invention further provides polypeptides encoded by SEQ ID NO: 23 such as those selected from the group consisting of SEQ ID NOs: 7040, 7054, 7060, 7066, 7071, 7075, 7084, 7085, 7091, 7092, 7093, 7100, 7102, 7108, 7112 and 7117.
  • the invention further provides polypeptides encoded by SEQ ID NO: 24 such as those selected from the group consisting of SEQ ID NOs: 7125, 7130, 7131, 7132, 7152, 7163, 7164, 7168, 7174, 7176, 7177, 7197, 7209, 7210, 7211, 7212, 7224, 7225, 7258, 7259, 7260, 7261, 7270, 7272, 7274, 7277, 7287, 7292, 7294, 7297, 7309, 7310, 7316, 7318, 7338, 7345, 7352, 7356 and 28697.
  • SEQ ID NO: 24 such as those selected from the group consisting of SEQ ID NOs: 7125, 7130, 7131, 7132, 7152, 7163, 7164, 7168, 7174, 7176, 7177, 7197, 7209, 7210, 7211, 7212, 7224, 7225, 7258, 7259, 7260, 7261, 7270, 7272
  • the invention further provides polypeptides encoded by SEQ ID NO: 25 such as those selected from the group consisting of SEQ ID NOs: 7380, 7381, 7383, 7384, 7386, 7388, 7390, 7394, 7397, 7406, 7412, 7419, 7421, 7422, 7425, 7426, 7438, 7448, 7452, 7466, 7468, 7469, 7474, 7478, 7482, 7488, 7489, 7490, 7491, 7502, 7504 and 28698.
  • the invention further provides polypeptides encoded by SEQ ID NO: 26 such as those selected from the group consisting of SEQ ID NOs: 7511, 7564, 7576, 7578, 7601 and 28701.
  • the invention further provides polypeptides encoded by SEQ ID NO: 27 such as those selected from the group consisting of SEQ ED NOs: 7605, 7606, 7607, 7622, 7623, 7632, 7635, 7636, 7641, 7648, 7653, 7661, 7662, 7663, 7668, 7673, 7674, 7683 and 7689.
  • the invention further provides polypeptides encoded by SEQ ID NO: 28 such as those selected from the group consisting of SEQ ID NOs: 7699 and 7710.
  • the invention further provides polypeptides encoded by SEQ ID NO: 29 such as those selected from the group consisting of SEQ ID NOs: 7721, 7725, 7729, 7731, 7739, 7741, 7742, 7752, 7764, 7765, 7767, 7772, 7781, 7787, 7788, 7789, 7793, 7794, 7801, 7803, 7806, 7807, 7822, 7828, 7829, 7831, 7832, 7839, 7841, 7859, 7865, 7878, 7879, 7882, 7883, 7887, 7889, 7891, 7894, 7895, 7897 and 7898.
  • the invention further provides polypeptides encoded by SEQ ID NO: 30 such as those selected from the group consisting of SEQ ID NOs: 7900, 7914, 7915, 7920, 7922, 7925, 7929, 7932, 7942, 7967, 7968, 7970, 7981, 7990, 7991, 7996, 8003, 8014, 8015, 8023, 8024, 8036, 8037, 8039, 8040, 8041, 8042, 8047, 8055, 8066, 8067, 8075, 8104, 8108, 8118, 8120, 8121, 8122, 8125, 8126, 8127, 8153, 8163 and 8170.
  • SEQ ID NO: 30 such as those selected from the group consisting of SEQ ID NOs: 7900, 7914, 7915, 7920, 7922, 7925, 7929, 7932, 7942, 7967, 7968, 7970, 7981, 7990,
  • the invention further provides polypeptides encoded by SEQ ID NO: 31 such as those selected from the group consisting of SEQ ID NOs: 8188, 8208, 8214, 8215, 8218, 8229, 8239, 8242, 8244, 8246, 8249, 8253, 8263, 8279, 8280, 8282, 8289, 8290, 8292, 8298 and 8302.
  • the invention further provides polypeptides encoded by SEQ ID NO: 32 such as those selected from the group consisting of SEQ ID NOs: 8312, 8318, 8321, 8322, 8323, 8324, 8326, 8329, 8333, 8336, 8340, 8343, 8344, 8370, 8371, 8401, 8402, 8406, 8411, 8417, 8424, 8425, 8429, 8434 and 8435.
  • SEQ ID NO: 32 such as those selected from the group consisting of SEQ ID NOs: 8312, 8318, 8321, 8322, 8323, 8324, 8326, 8329, 8333, 8336, 8340, 8343, 8344, 8370, 8371, 8401, 8402, 8406, 8411, 8417, 8424, 8425, 8429, 8434 and 8435.
  • the invention further provides polypeptides encoded by SEQ ID NO: 33 such as those selected from the group consisting of SEQ ID NOs: 8448, 8460, 8461, 8488, 8507, 8509, 8527, 8541, 8546, 8556, 8567, 8575, 8589, 8605, 8627, 8628, 8647 and 8658.
  • the invention further provides polypeptides encoded by SEQ ID NO: 34 such as those selected from the group consisting of SEQ ID NOs: 8685, 8686, 8705, 8731, 8736, 8745, 8747, 8748, 8749, 8751, 8757, 8764, 8768, 8780, 8782, 8783, 8787, 8793, 8794, 8795, 8796, 8805, 8806, 8813, 8818, 8820, 8828, 8861, 8876, 8877, 8878, 8879, 8880, 8883, 8890, 8908, 8912, 8933, 8938, 8949, 8968, 8969, 8976, 8993, 8994, 8995, 8996 and 9004
  • the invention further provides polypeptides encoded by SEQ ID NO: 35 such as those selected from the group consisting of SEQ ID NOs: 9017, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9032, 9033, 9034, 9035, 9036, 9037, 9038, 9040 and 9041.
  • the invention further provides polypeptides encoded by SEQ ID NO: 36 such as those selected from the group consisting of SEQ ID NOs: 9044, 9048, 9052, 9061, 9065, 9069, 9071, 9076, 9077, 9092, 9095, 9097, 9100, 9101, 9105, 9106, 9113, 9114, 9116, 9121, 9122, 9123, 9124, 9129, 9152, 9155, 9169, 9172, 9173, 9174, 9182, 9186, 9187, 9188, 9189, 9190, 9192, 9193, 9194, 9195, 9196, 9197, 9198, 9200, 9201, 9203, 9205, 9207, 9232, 9246, 9247, 9248, 9251, 9254, 9255, 9267, 9272, 9288, 9296, 9298, 9299, 9301, 9311 and 9312.
  • the invention further provides polypeptides encoded by SEQ ID NO: 37 such as those selected from the group consisting of SEQ ID NOs: 9337, 9338, 9340, 9342, 9353, 9356, 9364, 9365, 9367, 9383, 9385, 9392, 9409, 9411, 9414, 9415, 9420, 9430, 9434, 9438, 9451, 9452, 9464, 9471, 9472, 9476, 9483, 9484, 9485, 9506, 9536, 9537, 9548, 9549, 9556, 9557, 9560, 9587, 9589, 9590, 9611, 9612, 9621, 9639, 9652, 9654, 9657, 9664, 9667, 9674 and 9679.
  • SEQ ID NO: 37 such as those selected from the group consisting of SEQ ID NOs: 9337, 9338,
  • the invention further provides polypeptides encoded by SEQ ID NO: 38 such as those selected from the group consisting of SEQ ID NOs: 9685, 9689, 9691, 9698, 9715, 9716, 9717, 9718, 9722, 9725, 9755, 9770, 9772, 9773, 9776, 9780, 9781, 9782, 9787, 9795, 9798, 9803, 9814, 9824, 9842, 9851, 9855, 9856, 9858, 9861, 9862, 9866, 9867, 9883, 9897, 9902, 9906, 9941, 9949, 9955, 9956, 9957, 9960, 9994, 9997, 9998, 10002, 10003, 10021, 10038, 10105, 10107, 10110, 10123, 10129, 10131, 10141, 10161, 10192, 10197, 10199, 10202, 10218, 102
  • the invention further provides polypeptides encoded by SEQ ID NO: 39 such as those selected from the group consisting of SEQ ID NOs: 10226, 10229, 10250, 10254, 10258, 10261, 10273, 10306, 10309, 10315, 10336, 10339, 10341, 10350 and 28715.
  • the invention further provides polypeptides encoded by SEQ ID NO: 40 such as those selected from the group consisting of SEQ ID NOs: 10356, 10358, 10377, 10389, 10396, 10437, 10439, 10447, 10459, 10467, 10473, 10487, 10489, 10534, 10535, 10540, 10541, 10547, 10548, 10552, 10558, 10564, 10581, 10600, 10601, 10616, 10629, 10637, 10645, 10657, 10658, 10674, 10678, 10681, 10683, 10684, 10686, 10687, 10692, 10698, 10700, 10708, 10723, 10741, 10744, 10747, 10785, 10789, 10799, 10802, 10803, 10807, 10808, 10809, 10812, 10825,
  • the invention further provides polypeptides encoded by SEQ ID NO: 41 such as those selected from the group consisting of SEQ ID NOs: 11091, 11092, 11093, 11094, 11098, 11099, 11107, 11108, 11110, 11113, 11122, 11138, 11145, 11146, 11148, 11154, 11168, 11172, 11174, 11195, 11199, 11202, 11212, 11218, 11222, 11223, 11224, 11225, 11236, 11249, 11250, 11258, 11261, 11273, 11275, 11277, 11284, 11285, 11286, 11303, 11314, 11319 and 11342.
  • SEQ ID NO: 41 such as those selected from the group consisting of SEQ ID NOs: 11091, 11092, 11093, 11094, 11098, 11099, 11107, 11108, 11110, 11113, 11122, 11138, 11145, 11146, 11148, 11154, 11168, 11172, 11174, 11195, 11199,
  • the invention further provides polypeptides encoded by SEQ ID NO: 42 such as those selected from the group consisting of SEQ ID NOs: 11391 and 28723.
  • the invention further provides polypeptides encoded by SEQ ID NO: 43 such as those selected from the group consisting of SEQ ID NOs: 11399, 11401, 11417, 11427, 11430, 11439, 11441, 11453, 11457, 11460, 11462, 11463, 11464, 11469, 11470, 11475, 11476, 11490, 11491, 11503, 11526, 11530, 11537, 11550, 11560, 11564, 11565, 11571, 11572, 11577, 11580, 11582, 11583, 11585, 11599, 11613, 11624, 11641, 11646, 11648, 11655, 11658, 11661, 11667, 11682 and 28724.
  • the invention further provides polypeptides encoded by SEQ ID NO: 44 such as those selected from the group consisting of SEQ ID NOs: 11707, 11716, 11719, 11724, 11747, 11749, 11756, 11771, 11776, 11782, 11783, 11788, 11796, 11800, 11806, 11809, 11811, 11814, 11816, 11825, 11834, 11838, 11849, 11855, 11856, 11858, 11863, 11864, 11878, 11883, 11890, 11899, 11900, 11907, 11914, 11920, 11930, 11931, 11935, 11937, 11938, 11959, 11960, 11961, 11962, 11973, 11974, 11976, 11979, 11984, 11989, 11991, 11997, 11998, 12002, 12004, 12006,
  • the invention further provides polypeptides encoded by SEQ ID NO: 45 such as those selected from the group consisting of SEQ ID NOs: 12310, 12314, 12316, 12317, 12319, 12320, 12322, 12324, 12330, 12331, 12334, 12341, 12342, 12343, 12346, 12347, 12357, 12374, 12377, 12378, 12379, 12385, 12395, 12405, 12410, 12411, 12413, 12414,28726 and 28727.
  • SEQ ID NO: 45 such as those selected from the group consisting of SEQ ID NOs: 12310, 12314, 12316, 12317, 12319, 12320, 12322, 12324, 12330, 12331, 12334, 12341, 12342, 12343, 12346, 12347, 12357, 12374, 12377, 12378, 12379, 123
  • the invention further provides polypeptides encoded by SEQ ID NO: 46 such as those selected from the group consisting of SEQ ID NOs: 12418, 12420, 12429, 12440, 12441, 12442, 12447, 12448, 12453, 12456, 12460, 12481, 12487, 12501, 12503, 12524, 12528, 12533, 12534, 12535, 12546, 12558, 12563, 12574, 12576, 12586, 12587, 12588, 12589, 12590, 12600, 12609, 12610, 12611, 12626, 12645, 12647, 12649, 12650, 12654, 12659, 12667, 12668, 12669, 12670, 12674, 12675, 12686, 12718, 12739, 12740, 12741, 12749, 12750, 12751, 12752, 12755,
  • the invention further provides polypeptides encoded by SEQ ID NO: 47 such as those selected from the group consisting of SEQ ID NOs: 12861, 12862, 12866, 12868, 12871, 12875, 12879, 12887, 12890, 12897, 12902, 12904, 12907, 12913, 12914, 12916, 12917, 12920, 12921, 12926, 12941, 12945, 12948, 12962, 12976, 12978, 12979, 12981, 12983, 12992, 13002, 13006, 13011, 13016, 13023, 13026, 13029, 13031, 13032, 13034, 13036, 13038, 13039, 13040, 13054, 13056, 13062, 13063, 13066, 13069, 13085, 13086 and 13088.
  • SEQ ID NO: 47 such as those selected from the group consisting of SEQ ID NOs: 12861, 12862, 12866, 12868, 12871, 12
  • the invention further provides polypeptides encoded by SEQ ID NO: 48 such as those selected from the group consisting of SEQ ID NOs: 13093, 13097, 13115, 13119, 13122, 13124, 13130, 13131, 13134, 13139, 13152, 13156, 13161, 13165, 13171, 13173, 13184, 13185, 13187, 13200, 13201, 13206, 13214, 13232, 13243, 13248, 13273, 13277, 13284, 13297, 13301, 13312, 13318, 13321, 13324, 13326, 13328, 13332, 13340, 13342, 13348, 13349, 13352, 13356, 13358, 13383, 13385, 13386, 13394, 13396, 13402, 13413, 13417, 13424, 13428, 13430, 13437, 13438, 13440, 13451, 13455, 13475,
  • the invention further provides polypeptides encoded by SEQ ID NO: 49 such as those selected from the group consisting of SEQ ID NOs: 13494, 13496, 13500, 13502, 13505, 13506, 13507, 13512, 13517, 13524, 13526, 13527, 13528, 13529, 13538, 13547, 13550, 13552, 13557, 13580, 13593, 13594, 13609, 13617, 13618, 13641, 13646 and 13647.
  • SEQ ID NO: 49 such as those selected from the group consisting of SEQ ID NOs: 13494, 13496, 13500, 13502, 13505, 13506, 13507, 13512, 13517, 13524, 13526, 13527, 13528, 13529, 13538, 13547, 13550, 13552, 13557, 13580, 13593, 13594, 13609, 13617, 13618, 13641,
  • the invention further provides polypeptides encoded by SEQ ID NO: 50 such as those selected from the group consisting of SEQ ID NOs: 13661, 13667, 13673, 13684, 13690, 13691, 13700, 13718, 13732 and 13797.
  • the invention further provides polypeptides encoded by SEQ ID NO: 51 such as those selected from the group consisting of SEQ ID NOs: 13830, 13841, 13861, 13863, 13865, 13866, 13877, 13878, 13879, 13880 and 13893.
  • the invention further provides polypeptides encoded by SEQ ID NO: 52 such as those selected from the group consisting of SEQ ID NOs: 13897, 13905, 13915, 13952, 13961, 13967, 14001, 14031, 14041, 14042, 14062, 14064, 14068, 14094, 14101 and 14103.
  • the invention further provides polypeptides encoded by SEQ ID NO: 53 such as those selected from the group consisting of SEQ ID NOs: 14120, 14124, 14126, 14143, 14151, 14152, 14168, 14169, 14173, 14181, 14188, 14189, 14195, 14209, 14214, 14215, 14216, 14222, 14229, 14233, 14234, 14236, 14242, 14247, 14249, 14261, 14268, 14277, 14279, 14287, 14301, 14306, 14307, 14308, 14312, 14313, 14321, 14322, 14323, 14324, 14331, 14336, 14338, 14339, 14346, 14349, 14351, 14353, 14365, 14368, 14369, 14370, 14374, 14376, 14378, 14379 and 14380.
  • SEQ ID NO: 53 such as those selected
  • the invention further provides polypeptides encoded by SEQ ID NO: 54 such as those selected from the group consisting of SEQ ID NOs: 14398, 14408, 14411, 14429, 14456, 14458, 14464, 14471, 14480, 14482, 14484, 14485, 14503, 14509, 14514, 14518, 14520, 14521, 14545 and 28741.
  • the invention further provides polypeptides encoded by SEQ ID NO: 55 such as those selected from the group consisting of SEQ ID NOs: 14552, 14557, 14558, 14575, 14577, 14579, 14580, 14589, 14592, 14593, 14595, 14602, 14603, 14606, 14608, 14618, 14625, 14637, 14638, 14641, 14644, 14647, 14652, 14653, 14656, 14659, 14661, 14671, 14672, 14675, 14676, 14699, 14702, 14707, 14708, 14709, 14712, 14720, 14721 and 28744.
  • SEQ ID NO: 55 such as those selected from the group consisting of SEQ ID NOs: 14552, 14557, 14558, 14575, 14577, 14579, 14580, 14589, 14592, 14593
  • the invention further provides polypeptides encoded by SEQ ID NO: 56 such as those selected from the group consisting of SEQ ID NOs: 14742, 14745, 14751, 14752, 14764, 14769, 14789, 14796 and 14798.
  • the invention further provides polypeptides encoded by SEQ ID NO: 57 such as those selected from the group consisting of SEQ ID NOs: 14819, 14820, 14833, 14834, 14836, 14837, 14838, 14849, 14870, 14872, 14876, 14877, 14885, 14898, 14912, 14913, 14923, 14924, 14933, 14935, 14972, 14973, 14991, 14992, 14993, 14994, 14998, 15002, 15036, 15043, 15047, 15049, 15050, 15065, 15080, 15085, 15090, 15094, 15095, 15096, 15101, 15106, 15113, 15127, 15138, 15147, 15149, 15170, 15172, 15192, 15193, 15197, 15198, 15215, 15217, 15218 and 28748.
  • the invention further provides polypeptides encoded by SEQ ID NO: 58 such as those selected from the group consisting of SEQ ID NOs: 15237, 15250, 15255, 15259, 15265, 15272, 15278, 15286, 15288, 15298, 15302, 15303, 15305, 15311, 15312, 15318, 15319, 15322, 15323, 15324, 15325, 15335, 15336, 15338, 15339, 15350 and 15371.
  • the invention further provides polypeptides encoded by SEQ ID NO: 59 such as those selected from the group consisting of SEQ ID NOs: 15385, 15386, 15410, 15411, 15413, 15421, 15422, 15423, 15424, 15425, 15437, 15441, 15455, 15458, 15470, 15472, 15476, 15479, 15486, 15487, 15488, 15490, 15496, 15497, 15500, 15502, 15507, 15508, 15518, 15519, 15525, 15531, 15541, 15544, 15549, 15560, 15561, 15563, 15567, 15570, 15573, 15598, 15614, 15620, 15622, 15624, 15627, 15629, 15633, 15635, 15643, 15646, 15647, 15652, 15653, 15657,
  • the invention further provides polypeptides encoded by SEQ ID NO: 60 such as those selected from the group consisting of SEQ ID NOs: 15695, 15697, 15706, 15724, 15728 and 15742
  • the invention further provides polypeptides encoded by SEQ ID NO: 61 such as those selected from the group consisting of SEQ ID NOs: 15757, 15769, 15795, 15796, 15808, 15816, 15819, 15821, 15837, 15844, 15845, 15849, 15851, 15853, 15875, 15876, 15884, 15888, 15890, 15896, 15915, 15916, 15917, 15918, 15931, 15935, 15938, 15948, 15949, 15960, 15964, 15965, 15966, 15970, 15982, 15987, 15989, 15990, 15991, 15992, 15993, 15994, 15997, 16001, 16002, 16003, 16015, 16018, 16026, 16029, 16045, 16050, 16052, 16053, 16068, 16069, 16073, 16078, 16079, 16080, 16088,
  • the invention further provides polypeptides encoded by SEQ ID NO: 62 such as those selected from the group consisting of SEQ ID NOs: 16131, 16134, 16138, 16141, 16142, 16145, 16146, 16155, 16168, 16176, 16187, 16189, 16190, 16192, 16195, 16210, 16219, 16225, 16232, 16234, 16238, 16241, 16242, 16248, 16256 and 16258.
  • the invention further provides polypeptides encoded by SEQ ID NO: 63 such as those selected from the group consisting of SEQ ID NOs: 16265, 16272, 16274, 16279, 16285, 16297, 16299, 16312, 16315, 16316, 16327, 16328, 16337, 16352, 16353, 16354, 16355, 16356, 16357, 16371, 16372, 16378, 16379, 16380, 16392, 16399, 16400, 16403, 16424, 16426, 16439, 16442, 16447, 16448, 16453, 16471, 16483, 16492 and 16498.
  • SEQ ID NO: 63 such as those selected from the group consisting of SEQ ID NOs: 16265, 16272, 16274, 16279, 16285, 16297, 16299, 16312, 16315, 16316, 16327, 16328, 16337, 16352, 16353, 16354, 16355, 1635
  • the invention further provides polypeptides encoded by SEQ ID NO: 64 such as those selected from the group consisting of SEQ ID NOs: 16501, 16502, 16504,
  • the invention further provides polypeptides encoded by SEQ ID NO: 65 such as those selected from the group consisting of SEQ ID NOs: 16805, 16808, 16811, 16812, 16816, 16817, 16842, 16843, 16845, 16850 and 16865.
  • the invention further provides polypeptides encoded by SEQ ID NO: 66 such as those selected from the group consisting of SEQ ID NOs: 16881, 16888, 16891, 16901, 16902, 16910, 16920, 16924, 16932, 16935, 16962, 16967, 16971, 16982, 16989, 16990, 16992, 16993 and 28767.
  • the invention further provides polypeptides encoded by SEQ ID NO: 67 such as those selected from the group consisting of SEQ ID NOs: 17001, 17006, 17011, 17020, 17022, 17029, 17037, 17039, 17044, 17049, 17050, 17055, 17057, 17058, 17064, 17073, 17080, 17093, 17097, 17098, 17099, 17102, 17113 and 17116.
  • the invention further provides polypeptides encoded by SEQ ID NO: 68 such as those selected from the group consisting of SEQ ID NOs: 17120, 17121, 17134, 17135, 17145, 17148, 17163, 17169, 17170, 17171, 17189, 17190, 17191, 17192, 17193, 17194, 17200, 17210, 17212, 17226, 17229, 17237, 17257, 17259, 17284, 17287, 17294, 17295, 17298, 17306, 17307, 17311, 17317, 17319, 17328, 17329, 17330, 17331, 17332, 17348, 17351, 17372, 17377, 17380, 17388, 17395, 17396, 17403, 17407, 17430, 17436, 17440, 17454, 17455, 17463, 17469, 17480, 17482, 17497, 17514 and 17521.
  • the invention further provides polypeptides encoded by SEQ ID NO: 69 such as those selected from the group consisting of SEQ ID NOs: 17526, 17527, 17537, 17560, 17561 and 28771.
  • the invention further provides polypeptides encoded by SEQ ID NO: 70 such as those selected from the group consisting of SEQ ID NOs: 17584, 17597, 17613, 17629 and 17631.
  • the invention further provides polypeptides encoded by SEQ ID NO: 71 such as those selected from the group consisting of SEQ ID NOs: 17665, 17681, 17685, 17691, 17703 and 17706.
  • the invention further provides polypeptides encoded by SEQ ID NO: 72 such as those selected from the group consisting of SEQ ID NOs: 17711, 17713, 17734, 17738, 17739, 17744, 17745, 17748, 17749, 17754, 17757, 17759, 17776, 17778, 17782, 17798, 17806, 17814, 17815, 17819, 17820, 17821, 17822, 17823, 17841, 17850, 17858, 28773 and 28774.
  • SEQ ID NO: 72 such as those selected from the group consisting of SEQ ID NOs: 17711, 17713, 17734, 17738, 17739, 17744, 17745, 17748, 17749, 17754, 17757, 17759, 17776, 17778, 17782, 17798, 17806, 17814, 17815, 17819, 17820
  • the invention further provides polypeptides encoded by SEQ ID NO: 73 such as those selected from the group consisting of SEQ ID NOs: 17860, 17869, 17870, 17874, 17883, 17884, 17887, 17888, 17919, 17931, 17935, 17940, 17946, 17959, 17960,
  • the invention further provides polypeptides encoded by SEQ ID NO: 74 such as those selected from the group consisting of SEQ ID NOs: 18080, 18083, 18086, 18089, 18123, 18124, 18134, 18142, 18149, 18160, 18161, 18164, 18166, 18177, 18189, 18199, 18200, 18202, 18220, 18221, 18222, 18235, 18245, 18248 and 18249.
  • SEQ ID NO: 74 such as those selected from the group consisting of SEQ ID NOs: 18080, 18083, 18086, 18089, 18123, 18124, 18134, 18142, 18149, 18160, 18161, 18164, 18166, 18177, 18189, 18199, 18200, 18202, 18220, 18221, 18222, 18235, 18245, 18248 and 18249.
  • the invention further provides polypeptides encoded by SEQ ID NO: 75 such as those selected from the group consisting of SEQ ID NOs: 18270, 18276, 18284,
  • the invention further provides polypeptides encoded by SEQ ID NO: 76 such as those selected from the group consisting of SEQ ID NOs: 18422, 18423, 18430, 18432, 18433, 18434, 18436, 18437, 18438, 18439, 18440, 18446, 18454, 18460, 18471, 18472, 18473, 18474, 18480, 18483 and 18484.
  • the invention further provides polypeptides encoded by SEQ ID NO: 77 such as those selected from the group consisting of SEQ ID NOs: 18491, 18492, 18493, 18495, 18497, 18500, 18513, 18514, 18517, 18521, 18525, 18526 and 18532.
  • the invention further provides polypeptides encoded by SEQ ID NO: 78 such as those selected from the group consisting of SEQ ID NOs: 18544, 18553, 18557,
  • the invention further provides polypeptides encoded by SEQ ID NO: 79 such as those selected from the group consisting of SEQ ID NOs: 18716, 18735, 18737, 18749 and 18757.
  • the invention further provides polypeptides encoded by SEQ ID NO: 80 such as those selected from the group consisting of SEQ ID NOs: 18778, 18790, 18799, 18803, 18816, 18829, 18833, 18834, 18837, 18847, 18868, 18871, 18879, 18888, 18893, 18895, 18897, 18909, 18921, 18927, 18928, 18931 and 18938.
  • SEQ ID NO: 80 such as those selected from the group consisting of SEQ ID NOs: 18778, 18790, 18799, 18803, 18816, 18829, 18833, 18834, 18837, 18847, 18868, 18871, 18879, 18888, 18893, 18895, 18897, 18909, 18921, 18927, 18928, 18931 and 18938.
  • the invention further provides polypeptides encoded by SEQ ID NO: 81 such as those selected from the group consisting of SEQ ID NOs: 18979, 18986, 19021, 19023, 19026, 19028, 19030, 19034 and 19036.
  • the invention further provides polypeptides encoded by SEQ ID NO: 82 such as those selected from the group consisting of SEQ ID NOs: 19053, 19060, 19063, 19067, 19072, 19080, 19085, 19090, 19091, 19105 and 19108.
  • the invention further provides polypeptides encoded by SEQ ID NO: 83 such as those selected from the group consisting of SEQ ID NOs: 19119, 19133, 19134, 19135, 19136, 19137, 19138, 19139, 19141, 19142, 19143, 19144, 19149, 19154, 19157, 19158, 19159, 19177, 19178, 19179, 19180, 19183, 19184, 19191, 19199, 19200, 19204, 19205, 19208, 19212, 19213, 19218, 19244, 19245, 19246, 19248, 19249, 19258, 19261, 19273, 19277, 19287, 19288, 19298 and 19299.
  • SEQ ID NO: 83 such as those selected from the group consisting of SEQ ID NOs: 19119, 19133, 19134, 19135, 19136, 19137, 19138, 19139, 19141, 19142, 19143, 19144, 19149, 19154, 19157, 19158, 19159, 19177, 19178, 19179, 19180, 19183, 19184, 19191,
  • the invention further provides polypeptides encoded by SEQ ID NO: 84 such as those selected from the group consisting of SEQ ID NOs: 19300, 19330, 19331, 19344, 19345, 19350, 19353, 19359, 19367, 19368, 19370, 19391, 19399, 19400, 19401, 19407, 19422, 19424, 19427, 19447, 19448, 19449 and 28794.
  • the invention further provides polypeptides encoded by SEQ ID NO: 85 such as those selected from the group consisting of SEQ ID NOs: 19456, 19460, 19487, 19499, 19500, 19501, 19519, 19521, 19526, 19533, 19535, 19542, 19553, 19566, 19567, 19573, 19577, 19581, 19586, 19589, 19595 and 28796.
  • the invention further provides polypeptides encoded by SEQ ID NO: 86 such as those selected from the group consisting of SEQ ID NOs: 19600, 19605, 19607, 19608, 19609, 19610, 19611, 19612, 19631, 19634, 19641, 19642, 19644, 19646, 19648, 19649, 19651, 19660, 19663, 19668, 19670, 19676, 19682, 19683, 19684, 19687 and 19705.
  • the invention further provides polypeptides encoded by SEQ ID NO: 87 such as those selected from the group consisting of SEQ ID NOs: 19734, 19743 and 19757.
  • the invention further provides polypeptides encoded by SEQ ID NO: 88 such as those selected from the group consisting of SEQ ID NOs: 19771, 19772, 19773, 19775, 19780, 19782, 19788, 19805, 19809, 19810, 19813, 19820, 19843, 19844, 19854, 19855, 19856, 19863, 19864, 19865, 19872, 19883, 28800 and 28801.
  • the invention further provides polypeptides encoded by SEQ ID NO: 89 such as those selected from the group consisting of SEQ ID NOs: 19886, 19891, 19896, 19898, 19903, 19926, 19930, 19933, 19940, 19941, 19945, 19961, 19965, 19965, 19967, 19968, 19970, 19972, 19977, 19979 and 19981.
  • the invention further provides polypeptides encoded by SEQ ID NO: 90 such as those selected from the group consisting of SEQ ID NOs: 20005, 20013 and 28806.
  • the invention further provides polypeptides encoded by SEQ ID NO: 91 such as those selected from the group consisting of SEQ ID NOs: 20029, 20036, 20041, 20046, 20048, 20052, 20057, 20068, 20069, 20070, 20092, 20100, 20106, 20108, 20113, 20116, 20119, 20129, 20132, 20133, 20147, 20163, 20164, 20165, 20166, 20174, 20177, 20186, 20193, 20194, 20197, 20198, 20199, 20201, 20202, 20204, 20207, 20208, 20213, 20214, 20221, 20229, 28807 and 28809.
  • SEQ ID NO: 91 such as those selected from the group consisting of SEQ ID NOs: 20029, 20036, 20041, 20046, 20048, 20052, 20057, 20068, 20069, 20070, 20092, 20100, 20106, 20108, 20113, 20116, 20119, 20129, 20132, 20133, 20147, 20163, 20164, 20165, 20166, 20174, 20177, 20186, 20193, 20194, 20197, 20198, 20199, 20201, 20202, 20204, 20207
  • the invention further provides polypeptides encoded by SEQ ID NO: 92 such as those selected from the group consisting of SEQ ID NOs: 20262.
  • the invention further provides polypeptides encoded by SEQ ID NO: 93 such as those selected from the group consisting of SEQ ID NOs: 28812.
  • the invention further provides polypeptides encoded by SEQ ID NO: 94 such as those selected from the group consisting of SEQ ID NOs: 20284, 20286, 20287, 20288, 20289, 20290, 20292, 20293, 20294, 20296, 20297, 20298, 20299, 20300 and 28814.
  • SEQ ID NO: 95 such as those selected from the group consisting of SEQ ID NOs: 20311.
  • the invention further provides polypeptides encoded by SEQ ID NO: 96 such as those selected from the group consisting of SEQ ID NOs: 20329, 20332, 20333, 20334, 20357, 20360, 20369, 20370, 20372, 20382, 20385, 20386, 20391, 20392, 20393, 20395, 20399, 20400, 20401, 20411, 20412, 20414, 20415, 20422, 20425, 20442, 20458, 20463, 20474, 20475, 20476, 20479, 20480, 20485, 20494, 20510, 20512, 20517, 20525, 20528, 20530 and 20542.
  • SEQ ID NO: 96 such as those selected from the group consisting of SEQ ID NOs: 20329, 20332, 20333, 20334, 20357, 20360, 20369, 20370, 20372, 20382
  • the invention further provides polypeptides encoded by SEQ ID NO: 97 such as those selected from the group consisting of SEQ ID NOs: 20562, 20564, 20565, 20573, 20577, 20585, 20590, 20608, 20609, 20611, 20616, 20617, 20618, 20620, 20630, 20635, 20638, 20658, 20669, 20671, 20683, 20684, 28820 and 28822.
  • SEQ ID NO: 97 such as those selected from the group consisting of SEQ ID NOs: 20562, 20564, 20565, 20573, 20577, 20585, 20590, 20608, 20609, 20611, 20616, 20617, 20618, 20620, 20630, 20635, 20638, 20658, 20669, 20671, 20683, 20684, 28820 and 28822.
  • the invention further provides polypeptides encoded by SEQ ID NO: 98 such as those selected from the group consisting of SEQ ID NOs: 20703, 20704, 20705, 20714, 20715, 20733 and 20734.
  • the invention further provides polypeptides encoded by SEQ ID NO: 99 such as those selected from the group consisting of SEQ ID NOs: 20742, 20756, 20759, 20764, 20766, 20774, 20776, 20783, 20799, 20800, 28826 and 28827.
  • the invention further provides polypeptides encoded by SEQ ID NO: 100 such as those selected from the group consisting of SEQ ID NOs: 20806, 20832, 20836, 20854, 20855, 20856, 20877, 20882, 20897, 20913, 20923, 20925, 20926, 20935, 20943, 20949 and 20951.
  • the invention further provides polypeptides encoded by SEQ ID NO: 101 such as those selected from the group consisting of SEQ ID NOs: 20952, 20955, 20963, 20966, 20973, 20975, 20984, 20987, 21002, 21018, 21049 and 21054.
  • the invention further provides polypeptides encoded by SEQ ID NO: 102 such as those selected from the group consisting of SEQ ID NOs: 21065, 21070, 21074,
  • the invention further provides polypeptides encoded by SEQ ID NO: 103 such as those selected from the group consisting of SEQ ID NOs: 21241, 21250, 21256, 21258, 21259, 21264, 21265, 21266, 21267, 21271, 21287, 21300, 21315, 21333, 21348, 21349, 21350, 21355, 21359, 21363, 21368, 21372, 21387, 21399, 21402, 21405, 21428, 21435, 21446, 21447, 21449, 21453, 21466, 21477, 21483, 21485, 21486, 21489, 21490, 21493 and 21495.
  • the invention further provides polypeptides encoded by SEQ ID NO: 104 such as those selected from the group consisting of SEQ ID NOs: 21504, 21511, 21519, 21522, 21526, 21528, 21529, 21531, 21532, 21533, 21539, 21543, 21544, 21567, 21568, 21570, 21573, 21574, 21575, 21579, 21584, 21587, 21597, 21598, 21608, 21615, 21619, 21620, 21634, 21641, 21643, 21649, 21650, 21655, 21658, 21659, 21663, 21670, 21671, 21674, 21675, 21680, 21691, 21693, 21695, 21703, 21705, 21707, 21708, 21710, 21711, 21740, 21743, 21749, 21762, 21765, 21774, 21778, 21780, 21788, 28834 and
  • the invention further provides polypeptides encoded by SEQ ID NO: 105 such as those selected from the group consisting of SEQ ID NOs: 21789, 21791, 21803, 21806, 21808, 21827 and 21831.
  • the invention further provides polypeptides encoded by SEQ ID NO: 106 such as those selected from the group consisting of SEQ ID NOs: 21845, 21850 and 21856.
  • the invention further provides polypeptides encoded by SEQ ID NO: 107 such as those selected from the group consisting of SEQ ED NOs: 21861, 21872, 21875, 21894, 21897, 21908, 21910, 21913, 21923, 21926, 21978, 21979, 21983, 21997, 22003, 22014, 22019, 22020, 22027, 22042, 22054, 22062, 22063, 22064, 22065, 22071, 22072, 28844 and 28845.
  • the invention further provides polypeptides encoded by SEQ ID NO: 108 such as those selected from the group consisting of SEQ ID NOs: 22084, 22085, 22086, 22090, 22093, 22098, 22107, 22108, 22119, 22124, 22125, 22127, 22139, 22146, 22148, 22152, 22153, 22157, 22163, 22164, 22165, 22167, 22168, 22177, 22185, 22188, 22189, 22196, 22211, 22218, 22219, 22227, 22233, 22239, 22247, 22256, 22265, 22269, 22270, 22289, 22290, 22292, 22298, 22300, 22309, 22311, 22312, 22318, 22323, 22324 and 22328.
  • SEQ ID NO: 108 such as those selected from the group consisting of SEQ ID NOs: 22084, 22085, 22086, 22090, 22093, 22098, 22107,
  • the invention further provides polypeptides encoded by SEQ ID NO: 109 such as those selected from the group consisting of SEQ ID NOs: 22329, 22337, 22342, 22343, 22347, 22350, 22355, 22363 and 28852.
  • the invention further provides polypeptides encoded by SEQ ID NO: 110 such as those selected from the group consisting of SEQ ID NOs: 22369, 22379, 22381, 22393, 22394, 22395, 22396, 22406, 22427, 22444, 22454, 22456 and 22462.
  • the invention further provides polypeptides encoded by SEQ ID NO: 111 such as those selected from the group consisting of SEQ ID NOs: 22481, 22515, 22519, 22523, 22534, 22543, 22547, 22551, 22563, 22581, 22586, 22590, 22595, 22596, 22597, 22615, 22616, 22619, 22622, 22629, 22630 and 28857.
  • polypeptides encoded by SEQ ID NO: 111 such as those selected from the group consisting of SEQ ID NOs: 22481, 22515, 22519, 22523, 22534, 22543, 22547, 22551, 22563, 22581, 22586, 22590, 22595, 22596, 22597, 22615, 22616, 22619, 22622, 22629, 22630 and 28857.
  • the invention further provides polypeptides encoded by SEQ ID NO: 112 such as those selected from the group consisting of SEQ ID NOs: 22659, 22660, 22669, 22670, 22675, 22676, 22677, 22678 and 22684.
  • the invention further provides polypeptides encoded by SEQ ID NO: 113 such as those selected from the group consisting of SEQ ID NOs: 22693, 22706, 22712, 22719, 22726, 22738, 22747, 22748, 22752, 22771, 22775, 22784, 22790, 22804 and 28861.
  • the invention further provides polypeptides encoded by SEQ ID NO: 114 such as those selected from the group consisting of SEQ ID NOs: 22809, 22819, 22821, 22837, 22838, 22842, 22846 and 22872.
  • the invention further provides polypeptides encoded by SEQ ID NO: 115 such as those selected from the group consisting of SEQ ID NOs: 22879, 22882, 22886, 22906, 22907, 22910, 22920, 22922, 22937, 22938, 22939, 22948, 22958, 22976, 22977, 22984, 22987, 22988, 22989, 22990, 22991, 22996, 23001, 23013, 23017, 23021, 23034, 23038, 23039, 23044, 23046, 23063, 23065, 23074, 23075, 23076, 23079, 23082, 23084, 23090, 23091, 23092 and 23093.
  • SEQ ID NO: 115 such as those selected from the group consisting of SEQ ID NOs: 22879, 22882, 22886, 22906, 22907, 22910, 22920, 22922, 22937, 22938, 22939,
  • the invention further provides polypeptides encoded by SEQ ID NO: 116 such as those selected from the group consisting of SEQ ID NOs: 23096, 23098, 23101, 23106, 23112, 23114, 23115, 23116, 23118, 23125, 23127, 23130, 23136, 23138, 23140, 23144, 23151, 23164, 23168, 23170 and 28871.
  • the invention further provides polypeptides encoded by SEQ ID NO: 117 such as those selected from the group consisting of SEQ ID NOs: 23186, 23187, 23189, 23190, 23194, 23195, 23202, 23203, 23209, 23213 and 28873.
  • the invention further provides polypeptides encoded by SEQ ID NO: 118 such as those selected from the group consisting of SEQ ID NOs: 23215, 23219, 23222,
  • the invention further provides polypeptides encoded by SEQ ID NO: 119 such as those selected from the group consisting of SEQ ID NOs: 23418, 23419, 23420, 23437, 23441, 23442, 23443, 23444, 23448, 23449 and 23452.
  • the invention further provides polypeptides encoded by SEQ ID NO: 120 such as those selected from the group consisting of SEQ ID NOs: 23481, 23484, 23485, 23489, 23513, 23518, 23532, 23533, 23535, 23538, 23543, 23549, 23558, 23561, 23567, 23571, 23577, 23582, 23586, 23587 and 23591.
  • the invention further provides polypeptides encoded by SEQ ID NO: 121 such as those selected from the group consisting of SEQ ID NOs: 23610, 23613, 23614, 23616, 23618, 23624, 23635, 23647, 23651, 23657, 23658, 23664, 23682, 23683, 23685, 23687, 23706, 23707, 23708, 23709, 23711, 23719, 23722 and 28882.
  • SEQ ID NO: 121 such as those selected from the group consisting of SEQ ID NOs: 23610, 23613, 23614, 23616, 23618, 23624, 23635, 23647, 23651, 23657, 23658, 23664, 23682, 23683, 23685, 23687, 23706, 23707, 23708, 23709, 23711, 23719, 23722 and 28882.
  • the invention further provides polypeptides encoded by SEQ ID NO: 122 such as those selected from the group consisting of SEQ ID NOs: 23729, 23741, 23745, 23750, 23763, 23768, 23770, 23790, 23791, 23792, 23796, 23799, 23805, 23806, 23809 and 23817.
  • the invention further provides polypeptides encoded by SEQ ID NO: 123 such as those selected from the group consisting of SEQ ID NOs: 23847, 23857, 23864, 23871, 23880, 23886, 23891 and 23907.
  • the invention further provides polypeptides encoded by SEQ ID NO: 124 such as those selected from the group consisting of SEQ ID NOs: 23911, 23913, 23925, 23926, 23927, 23928, 23931, 23932, 23934, 23937, 23938, 23941, 23943, 23944, 23945, 23950, 23951, 23954, 23956, 23957, 23959, 23972, 23999, 24011, 24018, 24022, 24024, 24027, 24032, 24034 and 28887.
  • SEQ ID NO: 124 such as those selected from the group consisting of SEQ ID NOs: 23911, 23913, 23925, 23926, 23927, 23928, 23931, 23932, 23934, 23937, 23938, 23941, 23943, 23944, 23945, 23950, 23951, 23954, 23956, 23957, 23959, 23972, 23999, 24011, 24018, 24022
  • the invention further provides polypeptides encoded by SEQ ID NO: 125 such as those selected from the group consisting of SEQ ID NOs: 24047, 24051, 24055, 24097, 24099, 24107, 24113, 24114, 24118, 24128, 24146, 24154, 24180, 24181, 24185 and 24211.
  • the invention further provides polypeptides encoded by SEQ ID NO: 126 such as those selected from the group consisting of SEQ ID NOs: 24227, 24231, 24244, 24246, 24248, 24262, 24271, 24273, 24275, 24279, 24284 and 28892.
  • the invention further provides polypeptides encoded by SEQ ID NO: 127 such as those selected from the group consisting of SEQ ID NOs: 24293, 24294, 24300, 24303, 24317, 24319, 24326, 24327, 24331, 24341, 24342, 24351, 24352, 24355, 24361, 24398, 24399, 24406, 24420, 24431, 24433, 24434, 24435, 24442, 24450, 24456, 24459, 24463, 24465, 24473, 24474, 24482, 24487, 24499, 24514 and 24515.
  • SEQ ID NO: 127 such as those selected from the group consisting of SEQ ID NOs: 24293, 24294, 24300, 24303, 24317, 24319, 24326, 24327, 24331, 24341, 24342, 24351, 24352, 24355, 24361, 24398, 24399, 24406, 24420
  • the invention further provides polypeptides encoded by SEQ ID NO: 128 such as those selected from the group consisting of SEQ ID NOs: 24528, 24534, 24543, 24545, 24566, 24587, 24588, 24589, 24590, 24597, 24602, 24605, 24606, 24609, ,24612, 24619, 24620, 24621, 24622, 24623, 24626, 24629, 24630, 24639, 24640, 24641, 24652, 24663, 24665, 24667, 24673, 24676, 24679, 24684, 24685, 24687, 24696, 24698, 24704, 24705, 24709, 24715, 24716, 24721, 24722, 24728, 24730, 24739, 24742, 24743, 24745, 24749, 28897 and 28900.
  • SEQ ID NO: 128 such as those selected from the group consisting of SEQ ID NOs: 24528, 24534, 24543, 24545
  • the invention further provides polypeptides encoded by SEQ ID NO: 129 such as those selected from the group consisting of SEQ ID NOs: 24755, 24758, 24761, 24770, 24778, 24779, 24780, 24787, 24788, 24791, 24800, 24801, 24821, 24824, 24825, 24826, 24829, 24840, 24850, 24851, 24861, 24879, 24895, 24903, 24907, 24913, 24936, 24937, 24950, 24952, 24955 and 24957.
  • the invention further provides polypeptides encoded by SEQ ID NO: 130 such as those selected from the group consisting of SEQ ID NOs: 24961, 24967, 24968, 24969, 24973, 24981, 24984, 24986, 24987, 24992, 24993, 24995, 25004, 28903 and 28904.
  • the invention further provides polypeptides encoded by SEQ ID NO: 131 such as those selected from the group consisting of SEQ ID NOs: 25024, 25029, 25038, 25045, 25067 and 25076.
  • the invention further provides polypeptides encoded by SEQ ID NO: 132 such as those selected from the group consisting of SEQ ID NOs: 25082, 25085, 25086, 25088, 25089, 25090, 25094, 25096, 25102, 25103, 25112, 25129, 25130, 25131, 25133, 25135, 25139, 25150, 25160, 25163, 25164, 25165, 25166, 25167, 25168, 25169, 25174, 25178, 25187, 25192, 25193, 25194, 25195, 25197, 25208, 25215, 25216, 25217 and 25218.
  • SEQ ID NO: 132 such as those selected from the group consisting of SEQ ID NOs: 25082, 25085, 25086, 25088, 25089, 25090, 25094, 25096, 25102, 25103, 25112, 25129, 25130, 25131, 25133, 25135, 25139, 25150, 25160, 25163, 25164, 25165, 25166, 25167, 25168,
  • the invention further provides polypeptides encoded by SEQ ID NO: 133 such as those selected from the group consisting of SEQ ID NOs: 25229, 25237, 25239, 25246, 25267, 25271, 25274, 25275, 25283 and 28908.
  • the invention further provides polypeptides encoded by SEQ ID NO: 134 such as those selected from the group consisting of SEQ ID NOs: 25288, 25304, 25309, 25313, 25315, 25320, 25339, 25340, 25346, 25354, 25355, 25356, 28911 and 28912.
  • the invention further provides polypeptides encoded by SEQ ID NO: 135 such as those selected from the group consisting of SEQ ID NOs: 25365, 25367, 25378, 25379, 25380, 25385, 25387, 25404, 25410 and 25417.
  • the invention further provides polypeptides encoded by SEQ ID NO: 136 such as those selected from the group consisting of SEQ ID NOs: 25442, 25445, 25456, 25458, 25474, 25475, 25479, 25480, 25490 and 25516.
  • the invention further provides polypeptides encoded by SEQ ID NO: 137 such as those selected from the group consisting of SEQ ID NOs: 25535, 25539, 25540, 25546, 25550, 25553, 25562, 25578, 25584 and 25590.
  • the invention further provides polypeptides encoded by SEQ ID NO: 138 such as those selected from the group consisting of SEQ ID NOs: 25602, 25603, 25611, 25613, 25620, 25621, 25622, 25629 and 28920.
  • the invention further provides polypeptides encoded by SEQ ID NO: 139 such as those selected from the group consisting of SEQ ID NOs: 25644 and 25671.
  • the invention further provides polypeptides encoded by SEQ ID NO: 140 such as those selected from the group consisting of SEQ ID NOs: 25684, 25685, 25687, 25692, 25700, 25706, 25715, 25720, 25737, 25738, 25742, 25745, 25746, 25760, 25764, 25766, 25777 and 25780.
  • the invention further provides polypeptides encoded by SEQ ID NO: 141 such as those selected from the group consisting of SEQ ID NOs: 25784, 25788, 25791, 25803, 25804, 25807, 25812, 25815, 25828, 25830 and 25832.
  • the invention further provides polypeptides encoded by SEQ ID NO: 142 such as those selected from the group consisting of SEQ DD NOs: 25853.
  • the invention further provides polypeptides encoded by SEQ ID NO: 143 such as those selected from the group consisting of SEQ ID NOs: 25862, 25873, 25874, 25877, 25884, 25896 and 28925.
  • the invention further provides polypeptides encoded by SEQ ID NO: 144 such as those selected from the group consisting of SEQ DD NOs: 25906, 25911, 25913 and 28927.
  • the invention further provides polypeptides encoded by SEQ ID NO: 145 such as those selected from the group consisting of SEQ ID NOs: 25916, 25917, 25918, 25922, 25929, 25938, 25952, 25957, 25960 and 25963.
  • the invention further provides polypeptides encoded by SEQ ID NO: 147 such as those selected from the group consisting of SEQ ID NOs: 25987, 25990, 25994, 25997, 25998, 26005, 26006, 26011, 26012, 26016, 28932 and 28933.
  • the invention further provides polypeptides encoded by SEQ ID NO: 148 such as those selected from the group consisting of SEQ ID NOs: 26021, 26025, 26036 and 26037.
  • the invention further provides polypeptides encoded by SEQ ID NO: 149 such as those selected from the group consisting of SEQ ID NOs: 26044, 26061, 26067, 26071 , 26090, 26099 and 28940.
  • the invention further provides polypeptides encoded by SEQ ID NO: 150 such as those selected from the group consisting of SEQ ID NOs: 26122, 26140, 26141, 26143, 26149, 26156, 26157, 26161, 26163, 26171, 26175 and 28941.
  • the invention further provides polypeptides encoded by SEQ ID NO: 151 such as those selected from the group consisting of SEQ ID NOs: 26181, 26195, 26196, 26200, 26207, 26214, 26216, 26221, 26232, 28943 and 28945.
  • the invention further provides polypeptides encoded by SEQ ID NO: 152 such as those selected from the group consisting of SEQ ID NOs: 26247.
  • the invention further provides polypeptides encoded by SEQ ID NO: 153 such as those selected from the group consisting of SEQ ID NOs: 26281, 26284 and 28950.
  • the invention further provides polypeptides encoded by SEQ ID NO: 154 such as those selected from the group consisting of SEQ ID NOs: 26287 and 28953.
  • the invention further provides polypeptides encoded by SEQ ID NO: 155 such as those selected from the group consisting of SEQ DD NOs: 26321, 26333, 26337 and 28955.
  • the invention further provides polypeptides encoded by SEQ ID NO: 156 such as those selected from the group consisting of SEQ ID NOs: 26370, 26380, 26388, 26390, 26392, 26397 and 28956.
  • the invention further provides polypeptides encoded by SEQ ID NO: 157 such as those selected from the group consisting of SEQ ED NOs: 26436, 26437 and 26442.
  • the invention further provides polypeptides encoded by SEQ ID NO: 158 such as those selected from the group consisting of SEQ ID NOs: 26490, 26503 and 26504.
  • the invention further provides polypeptides encoded by SEQ ID NO: 159 such as those selected from the group consisting of SEQ ID NOs: 28962.
  • the invention further provides polypeptides encoded by SEQ ID NO: 160 such as those selected from the group consisting of SEQ ID NOs: 26532.
  • the invention further provides polypeptides encoded by SEQ ID NO: 161 such as those selected from the group consisting of SEQ ID NOs: 26549, 26555 and 28965.
  • the invention further provides polypeptides encoded by SEQ ID NO: 162 such as those selected from the group consisting of SEQ ID NOs: 26567 and 26586.
  • the invention further provides polypeptides encoded by SEQ ID NO: 163 such as those selected from the group consisting of SEQ ID NOs: 26598, 26605 and 28969.
  • the invention further provides polypeptides encoded by SEQ ID NO: 164 such as those selected from the group consisting of SEQ ID NOs: 26618, 26619, 26621, 26625, 26629 and 28970.
  • the invention further provides polypeptides encoded by SEQ ID NO: 165 such as those selected from the group consisting of SEQ DD NOs: 26658 and 26659.
  • the invention further provides polypeptides encoded by SEQ ID NO: 166 such as those selected from the group consisting of SEQ ID NOs: 26691, 26692 and 28976.
  • the invention further provides polypeptides encoded by SEQ ID NO: 167 such as those selected from the group consisting of SEQ ID NOs: 26693, 26700 and 26703.
  • the invention further provides polypeptides encoded by SEQ ID NO: 168 such as those selected from the group consisting of SEQ ID NOs: 26726.
  • the invention further provides polypeptides encoded by SEQ ID NO: 169 such as those selected from the group consisting of SEQ ID NOs: 26738.
  • the invention further provides polypeptides encoded by SEQ ID NO: 171 such as those selected from the group consisting of SEQ ID NOs: 28986.
  • the invention further provides polypeptides encoded by SEQ ID NO: 172 such as those selected from the group consisting of SEQ ID NOs: 26770 and 26773.
  • the invention further provides polypeptides encoded by SEQ ID NO: 173 such as those selected from the group consisting of SEQ ID NOs: 26776 and 26784.
  • the invention further provides polypeptides encoded by SEQ ID NO: 174 such as those selected from the group consisting of SEQ DD NOs: 26793.
  • the invention further provides polypeptides encoded by SEQ ID NO: 175 such as those selected from the group consisting of SEQ ID NOs: 26798 and 28991.
  • the invention further provides polypeptides encoded by SEQ ID NO: 176 such as those selected from the group consisting of SEQ ID NOs: 26816, 26817, 26819 and 28992.
  • the invention further provides polypeptides encoded by SEQ ID NO: 178 such as those selected from the group consisting of SEQ ID NOs: 26852.
  • the invention further provides polypeptides encoded by SEQ ID NO: 179 such as those selected from the group consisting of SEQ ID NOs: 26880, 28997 and 28998.
  • the invention further provides polypeptides encoded by SEQ ID NO: 180 such as those selected from the group consisting of SEQ ID NOs: 26894.
  • the invention further provides polypeptides encoded by SEQ ID NO: 181 such as those selected from the group consisting of SEQ ID NOs: 26900 and 26909.
  • the invention further provides polypeptides encoded by SEQ ID NO: 182 such as those selected from the group consisting of SEQ ID NOs: 26915.
  • the invention further provides polypeptides encoded by SEQ ID NO: 183 such as those selected from the group consisting of SEQ DD NOs: 26937 and 29004.
  • the invention further provides polypeptides encoded by SEQ ID NO: 184 such as those selected from the group consisting of SEQ DD NOs: 26945.
  • the invention further provides polypeptides encoded by SEQ ID NO: 185 such as those selected from the group consisting of SEQ DD NOs: 26949 and 26962.
  • the invention further provides polypeptides encoded by SEQ ID NO: 186 such as those selected from the group consisting of SEQ DD NOs: 26966, 26971 and 26975.
  • the invention further provides polypeptides encoded by SEQ ID NO: 187 such as those selected from the group consisting of SEQ ID NOs: 26977, 26980, 26984,
  • the invention further provides polypeptides encoded by SEQ ID NO: 188 such as those selected from the group consisting of SEQ ID NOs: 26995, 26996, 26998 and 29012.
  • the invention further provides polypeptides encoded by SEQ ID NO: 189 such as those selected from the group consisting of SEQ ID NOs: 27001, 27005, 27009, 27012 and 27019.
  • the invention further provides polypeptides encoded by SEQ ID NO: 190 such as those selected from the group consisting of SEQ ID NOs: 27023, 27029, 27030, 27031, 27036, 27037, 27050, 27051 and 29014.
  • the invention further provides polypeptides encoded by SEQ ID NO: 192 such as those selected from the group consisting of SEQ ID NOs: 27070 and 27072.
  • the invention further provides polypeptides encoded by SEQ ID NO: 193 such as those selected from the group consisting of SEQ DD NOs: 27079 and 27084.
  • the invention further provides polypeptides encoded by SEQ ID NO: 194 such as those selected from the group consisting of SEQ DD NOs: 27090 and 29024.
  • the invention further provides polypeptides encoded by SEQ ID NO: 196 such as those selected from the group consisting of SEQ ID NOs: 27116, 27118, 27122, 27124, 27126, 27127, 27131, 27134, 27135, 27137, 27138, 27141 and 29027.
  • the invention further provides polypeptides encoded by SEQ ID NO: 197 such as those selected from the group consisting of SEQ DD NOs: 27152, 27153, 27154 and 29030.
  • the invention further provides polypeptides encoded by SEQ ID NO: 198 such as those selected from the group consisting of SEQ ID NOs: 27155, 27163 and 27165.
  • the invention further provides polypeptides encoded by SEQ ID NO: 199 such as those selected from the group consisting of SEQ ID NOs: 27167, 27168, 27171, 27172, 27175 and 29035.
  • the invention further provides polypeptides encoded by SEQ ID NO: 200 such as those selected from the group consisting of SEQ DD NOs: 27190.
  • the invention further provides polypeptides encoded by SEQ ID NO: 201 such as those selected from the group consisting of SEQ DD NOs: 27199, 27205 and 29040.
  • the invention further provides polypeptides encoded by SEQ ID NO: 202 such as those selected from the group consisting of SEQ ID NOs: 27212 and 27218.
  • the invention further provides polypeptides encoded by SEQ ID NO: 204 such as those selected from the group consisting of SEQ ID NOs: 29041.
  • the invention further provides polypeptides encoded by SEQ ID NO: 205 such as those selected from the group consisting of SEQ ID NOs: 27270 and 27271.
  • the invention further provides polypeptides encoded by SEQ ID NO: 206 such as those selected from the group consisting of SEQ ID NOs: 27283 and 29043.
  • the invention further provides polypeptides encoded by SEQ ID NO: 207 such as those selected from the group consisting of SEQ DD NOs: 27307 and 29044.
  • the invention further provides polypeptides encoded by SEQ ID NO: 208 such as those selected from the group consisting of SEQ ID NOs: 27315 and 27319.
  • the invention further provides polypeptides encoded by SEQ ID NO: 209 such as those selected from the group consisting of SEQ ID NOs: 27328 and 29047.
  • the invention further provides polypeptides encoded by SEQ ID NO: 210 such as those selected from the group consisting of SEQ DD NOs: 29048 and 29049.
  • the invention further provides polypeptides encoded by SEQ ID NO: 212 such as those selected from the group consisting of SEQ ID NOs: 27366.
  • the invention further provides polypeptides encoded by SEQ ID NO: 213 such as those selected from the group consisting of SEQ ID NOs: 27375 and 29053.
  • the invention further provides polypeptides encoded by SEQ ID NO: 214 such as those selected from the group consisting of SEQ DD NOs: 27398 and 29056.
  • the invention further provides polypeptides encoded by SEQ ID NO: 215 such as those selected from the group consisting of SEQ ID NOs: 27399.
  • the invention further provides polypeptides encoded by SEQ ID NO: 217 such as those selected from the group consisting of SEQ ID NOs: 27419, 27431, 27434, 27438, 29060 and 29062.
  • the invention further provides polypeptides encoded by SEQ ID NO: 218 such as those selected from the group consisting of SEQ ID NOs: 27446.
  • the invention further provides polypeptides encoded by SEQ ID NO: 219 such as those selected from the group consisting of SEQ ID NOs: 27449 and 27461.
  • the invention further provides polypeptides encoded by SEQ ID NO: 220 such as those selected from the group consisting of SEQ ID NOs: 27470, 27475 and 29069.
  • the invention further provides polypeptides encoded by SEQ ID NO: 221 such as those selected from the group consisting of SEQ ID NOs: 27485, 27486 and 29071.
  • the invention further provides polypeptides encoded by SEQ ID NO: 222 such as those selected from the group consisting of SEQ ID NOs: 27497 and 29076.
  • the invention further provides polypeptides encoded by SEQ ID NO: 223 such as those selected from the group consisting of SEQ ID NOs: 27503, 27508, 27523 and 27525.
  • the invention further provides polypeptides encoded by SEQ ID NO: 224 such as those selected from the group consisting of SEQ ID NOs: 27527, 27533 and 27541.
  • the invention further provides polypeptides encoded by SEQ ID NO: 225 such as those selected from the group consisting of SEQ ID NOs: 27550, 27558, 27561, 29082 and 29083.
  • the invention further provides polypeptides encoded by SEQ ID NO: 226 such as those selected from the group consisting of SEQ ID NOs: 27578, 27580, 27582, 27584, 27585, 27586 and 27590.
  • the invention further provides polypeptides encoded by SEQ ID NO: 227 such as those selected from the group consisting of SEQ ID NOs: 27598, 27601, 27602,
  • the invention further provides polypeptides encoded by SEQ ID NO: 228 such as those selected from the group consisting of SEQ ID NOs: 27607, 27610 and 29087.
  • the invention further provides polypeptides encoded by SEQ ID NO: 229 such as those selected from the group consisting of SEQ ID NOs: 27640 and 29089.
  • the invention further provides polypeptides encoded by SEQ ID NO: 230 such as those selected from the group consisting of SEQ ID NOs: 27656 and 27657.
  • the invention further provides polypeptides encoded by SEQ ID NO: 231 such as those selected from the group consisting of SEQ DD NOs: 27665 and 29092.
  • the invention further provides polypeptides encoded by SEQ ID NO: 232 such as those selected from the group consisting of SEQ DD NOs: 27687.
  • the invention further provides polypeptides encoded by SEQ ID NO: 233 such as those selected from the group consisting of SEQ ID NOs: 27692, 27698, 27701, 27704 and 27709.
  • the invention further provides polypeptides encoded by SEQ ID NO: 234 such as those selected from the group consisting of SEQ ID NOs: 27712 and 27720.
  • the invention further provides polypeptides encoded by SEQ ID NO: 235 such as those selected from the group consisting of SEQ DD NOs: 27738, 27746 and 29098.
  • the invention further provides polypeptides encoded by SEQ ID NO: 236 such as those selected from the group consisting of SEQ ID NOs: 27759 and 29099.
  • the invention further provides polypeptides encoded by SEQ ID NO: 237 such as those selected from the group consisting of SEQ ID NOs: 27767, 27769, 27770 and 29101.
  • the invention further provides polypeptides encoded by SEQ ID NO: 238 such as those selected from the group consisting of SEQ ID NOs: 29102.
  • the invention further provides polypeptides encoded by SEQ ID NO: 239 such as those selected from the group consisting of SEQ ID NOs: 27800, 27807 and 29105.
  • the invention further provides polypeptides encoded by SEQ ID NO: 240 such as those selected from the group consisting of SEQ DD NOs: 27819.
  • the invention further provides polypeptides encoded by SEQ ID NO: 241 such as those selected from the group consisting of SEQ ID NOs: 27836.
  • the invention further provides polypeptides encoded by SEQ ID NO: 242 such as those selected from the group consisting of SEQ ID NOs: 27848.
  • the invention further provides polypeptides encoded by SEQ ID NO: 243 such as those selected from the group consisting of SEQ ID NOs: 27857, 27870, 27879, 29112, 29114 and 29115.
  • the invention further provides polypeptides encoded by SEQ ID NO: 244 such as those selected from the group consisting of SEQ ID NOs: 27885 and 27887.
  • the invention further provides polypeptides encoded by SEQ ID NO: 245 such as those selected from the group consisting of SEQ ID NOs: 27906, 27908 and 29118.
  • the invention further provides polypeptides encoded by SEQ ID NO: 246 such as those selected from the group consisting of SEQ DD NOs: 27911, 27921 and 27926.
  • the invention further provides polypeptides encoded by SEQ ID NO: 247 such as those selected from the group consisting of SEQ ID NOs: 27927, 27928 and 27935.
  • the invention further provides polypeptides encoded by SEQ ID NO: 248 such as those selected from the group consisting of SEQ ID NOs: 27938 and 27940.
  • the invention further provides polypeptides encoded by SEQ ID NO: 249 such as those selected from the group consisting of SEQ ID NOs: 27943, 27944, 27952, 27953 and 29124.
  • the invention further provides polypeptides encoded by SEQ ID NO: 250 such as those selected from the group consisting of SEQ ID NOs: 27957, 27958, 27964 and 27966.
  • the invention further provides polypeptides encoded by SEQ ID NO: 251 such as those selected from the group consisting of SEQ ED NOs: 27980.
  • the invention further provides polypeptides encoded by SEQ ID NO: 252 such as those selected from the group consisting of SEQ ID NOs: 27986 and 27991.
  • the invention further provides polypeptides encoded by SEQ ID NO: 253 such as those selected from the group consisting of SEQ ID NOs: 29129.
  • the invention further provides polypeptides encoded by SEQ ID NO: 254 such as those selected from the group consisting of SEQ DD NOs: 28021 and 29132.
  • the invention further provides polypeptides encoded by SEQ ID NO: 255 such as those selected from the group consisting of SEQ ID NOs: 28032, 28033 and 28042.
  • the invention further provides polypeptides encoded by SEQ ID NO: 256 such as those selected from the group consisting of SEQ ID NOs: 28051.
  • the invention further provides polypeptides encoded by SEQ ID NO: 257 such as those selected from the group consisting of SEQ ID NOs: 28067 and 28079.
  • the invention further provides polypeptides encoded by SEQ ID NO: 258 such as those selected from the group consisting of SEQ ID NOs: 28087.
  • the invention further provides polypeptides encoded by SEQ ID NO: 259 such as those selected from the group consisting of SEQ ID NOs: 28100 and 29139.
  • the invention further provides polypeptides encoded by SEQ ID NO: 260 such as those selected from the group consisting of SEQ DD NOs: 28116, 28119 and 28121.
  • the invention further provides polypeptides encoded by SEQ ID NO: 261 such as those selected from the group consisting of SEQ ID NOs: 28123 and 29144.
  • the invention further provides polypeptides encoded by SEQ ID NO: 262 such as those selected from the group consisting of SEQ ID NOs: 28132.
  • the invention further provides polypeptides encoded by SEQ ID NO: 263 such as those selected from the group consisting of SEQ ID NOs: 28143, 28146 and 29149.
  • the invention further provides polypeptides encoded by SEQ ID NO: 264 such as those selected from the group consisting of SEQ ID NOs: 28161 and 29150.
  • the invention further provides polypeptides encoded by SEQ ID NO: 265 such as those selected from the group consisting of SEQ DD NOs: 28166, 28169 and 28175.
  • the invention further provides polypeptides encoded by SEQ ID NO: 266 such as those selected from the group consisting of SEQ ID NOs: 28177 and 28179.
  • the invention further provides polypeptides encoded by SEQ ID NO: 267 such as those selected from the group consisting of SEQ DD NOs: 28187, 28199 and 28203.
  • the invention further provides polypeptides encoded by SEQ ID NO: 268 such as those selected from the group consisting of SEQ ID NOs: 28207 and 28221.
  • the invention further provides polypeptides encoded by SEQ ID NO: 269 such as those selected from the group consisting of SEQ DD NOs: 28230.
  • the invention further provides polypeptides encoded by SEQ ID NO: 270 such as those selected from the group consisting of SEQ ID NOs: 28248, 28250 and 29166.
  • the invention further provides polypeptides encoded by SEQ ID NO: 271 such as those selected from the group consisting of SEQ ED NOs: 28253, 28263 and 28265.
  • the invention further provides polypeptides encoded by SEQ ID NO: 272 such as those selected from the group consisting of SEQ ID NOs: 28269 and 28274.
  • the invention further provides polypeptides encoded by SEQ ID NO: 273 such as those selected from the group consisting of SEQ ED NOs: 28282, 28290 and 28291.
  • the invention further provides polypeptides encoded by SEQ ID NO: 274 such as those selected from the group consisting of SEQ ID NOs: 28295, 28300, 28302, 28305, 28312 and 28316.
  • the invention further provides polypeptides encoded by SEQ ID NO: 275 such as those selected from the group consisting of SEQ ID NOs: 28328.
  • the invention further provides polypeptides encoded by SEQ ID NO: 276 such as those selected from the group consisting of SEQ ID NOs: 28334, 28339, 28342, 28346, 29174 and 29175.
  • the invention further provides polypeptides encoded by SEQ ID NO: 277 such as those selected from the group consisting of SEQ ID NOs: 28357, 28358 and 28361.
  • the invention further provides polypeptides encoded by SEQ ID NO: 279 such as those selected from the group consisting of SEQ ID NOs: 28385 and 28387.
  • the invention further provides polypeptides encoded by SEQ ID NO: 280 such as those selected from the group consisting of SEQ ID NOs: 28394, 28396, 29182 and 29184.
  • the invention further provides polypeptides encoded by SEQ ID NO: 281 such as those selected from the group consisting of SEQ DD NOs: 28410.
  • the invention further provides polypeptides encoded by SEQ ID NO: 282 such as those selected from the group consisting of SEQ ID NOs: 28424, 28432 and 28434.
  • the invention further provides polypeptides encoded by SEQ ID NO: 283 such as those selected from the group consisting of SEQ ID NOs: 28439, 28440 and 28446.
  • the invention further provides polypeptides encoded by SEQ ID NO: 284 such as those selected from the group consisting of SEQ DD NOs: 28452, 28454 and 28461.
  • the invention further provides polypeptides encoded by SEQ ID NO: 285 such as those selected from the group consisting of SEQ ID NOs: 28474, 28476, 28477 and 29191.
  • the invention further provides polypeptides encoded by SEQ ID NO: 286 such as those selected from the group consisting of SEQ DD NOs: 28490, 29192 and 29193.
  • the invention further provides polypeptides encoded by SEQ ID NO: 287 such as those selected from the group consisting of SEQ DD NOs: 28497.
  • the invention further provides polypeptides encoded by SEQ ID NO: 288 such as those selected from the group consisting of SEQ DD NOs: 28503, 28509, 28512 and 28513.
  • the invention further provides polypeptides encoded by SEQ ID NO: 289 such as those selected from the group consisting of SEQ ID NOs: 28523 and 28526.
  • the invention further provides polypeptides encoded by SEQ ID NO: 290 such as those selected from the group consisting of SEQ ID NOs: 29198.
  • the invention further provides polypeptides encoded by SEQ ID NO: 291 such as those selected from the group consisting of SEQ ID NOs: 28544, 28547 and 28558.
  • the invention further provides polypeptides encoded by SEQ ID NO: 292 such as those selected from the group consisting of SEQ ID NOs: 28560, 28564 and 29201.
  • the invention further provides polypeptides encoded by SEQ ID NO: 294 such as those selected from the group consisting of SEQ DD NOs: 28584 and 28586.
  • the invention further provides polypeptides encoded by SEQ ID NO: 295 such as those selected from the group consisting of SEQ ID NOs: 28588, 28589, 28593, 28594, 28597 and 28598.
  • the invention further provides polypeptides encoded by SEQ ID NO: 296 such as those selected from the group consisting of SEQ ID NOs: 28604.
  • the invention further provides polypeptides encoded by SEQ ID NO: 297 such as those selected from the group consisting of SEQ DD NOs: 28612 and 29204.
  • the invention further provides polypeptides encoded by SEQ ID NO: 298 such as those selected from the group consisting of SEQ ID NOs: 29207.
  • the invention further provides polypeptides encoded by SEQ ID NO: 299 such as those selected from the group consisting of SEQ ID NOs: 28644.
  • compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
  • vaccines for prophylactic or therapeutic use comprise a polypeptide or polynucleotide as described above and an immunostimulant.
  • the present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a Propiombacterium acnes protein; and (b) a physiologically acceptable carrier.
  • the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient.
  • Antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.
  • vaccines are provided that comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) an immunostimulant.
  • the present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins.
  • compositions comprising a fusion protein, or a polynucleotide encoding a fusion protein, in combination with a physiologically acceptable carrier are provided.
  • Vaccines are further provided, within other aspects, that comprise a fusion protein, or a polynucleotide encoding a fusion protein, in combination with an immunostimulant.
  • the present invention provides methods for the treatment of acne in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as recited above.
  • the patient may be afflicted with acne, in which case the methods provide treatment for the disease, or patient considered at risk for acne may be treated prophylactically.
  • Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a Propiombacterium acnes protein, comprising contacting T cells with one or more of: (i) a polypeptide as described above; (ii) a polynucleotide encoding such a polypeptide; and/or (iii) an antigen presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.
  • Isolated T cell populations comprising T cells prepared as described above are also provided.
  • the present invention provides methods for inhibiting the development or progression of acne in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
  • the present invention further provides methods for the treatment of acne in a patient, comprising the steps of: (a) incubating CD4 + and/or CD8 + T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of a Propiombacterium acnes protein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen-presenting cell that expresses such a polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development or progression of acne in the patient.
  • Proliferated cells may, but need not, be cloned prior to administration to the patient.
  • the present invention provides methods for determining the presence or absence of Propiombacterium acnes in a patient, comprising: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cut-off value, and therefrom determining the presence or absence of the organism in the patient.
  • the binding agent is an antibody, more preferably a monoclonal antibody.
  • the present invention also provides, within other aspects, methods for monitoring the progression of acne in a patient.
  • Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b).
  • the present invention further provides, within other aspects, methods for determining the presence or absence of a Propiombacterium acnes in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a Propiombacterium acnes protein; (b) detecting in the sample a level of a polynucleotide, preferably mRNA, that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of the organism in the patient.
  • the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as recited above, or a complement of such a polynucleotide.
  • the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.
  • methods for monitoring the progression of acne in a patient comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a Propiombacterium acnes protein; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b).
  • the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
  • Figure 1 is an immunoblot demonstrating that sera from individuals with severe cases of facial acnes react with several dominant species having sizes about 18 -28 kDa; 35-40 kDa; -50 kDa; -70 kDa; -lOOkDa, as well as several low molecular weight species below 18kDa.
  • Figure 2 is a gel showing the molecular weights of two proteins found in clinical isolates, referred to as ZI and ZII.
  • Figure 3 shows the amino sequences of the predicted ORF of P. acnes sequence ID#15127.
  • the underlined residue (M) at position +29 (start), indicate the probable initiator methionine of the full length protein.
  • the remaining underlined sequences indicate residues identified by N-terminal (non-italicized) and mass spec (MS) (italicized) sequencing of ZI & II.
  • the predicted full length protein is 30.4 kDa (313 amino acid residues) while those of ZI & II are -27.7- (257 amino acid residues) and 24-kDa (222 amino acid residues) respectively in agreememt with their observed mobilities.
  • compositions and methods for using the compositions for example in the therapy and diagnosis of acne, particularly those forms of acne in whole or in part caused by the organism Propiombacterium acnes.
  • Certain illustrative compositions described herein include Propiombacterium acnes polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APCs) and/or immune system cells (e.g., T cells).
  • Certain preferred Propiombacterium acnes polypeptides include those that react detectably (within an immunoassay, such as an ELISA or Western blot) with antisera of a patient afflicted with acne.
  • the present invention provides illustrative polynucleotide compositions having sequences set forth in SEQ ID NOs: 1-299, illustrative polypeptide compositions having amino acid sequences set forth in SEQ ID NOs: 300-29,212, antibody compositions capable of binding such polypeptides, and numerous additional embodiments employing these and other compositions, for example in the detection, diagnosis and/or therapy of acne in humans.
  • DNA segment and “polynucleotide” refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms “DNA segment” and “polynucleotide” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • the DNA segments of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man. "Isolated,” as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA segment does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • polynucleotides may be single- stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Propiombacterium acnes protein or a portion thereof) or may comprise a variant or a biological or antigenic functional equivalent of such a sequence.
  • Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native Propiombacterium acnes protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein.
  • variants also encompasses homologous genes of xenogenic origin.
  • two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted using the
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http ://www.ncbi.nlm.nih. govA) .
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0
  • a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • additions or deletions i.e., gaps
  • the percentage is calculated by determimng the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • the present invention encompasses polynucleotide and polypeptide sequences having substantial identity to the sequences disclosed herein, for example those comprising at least 50% or more sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide or polypeptide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below).
  • BLAST analysis using standard parameters, as described below.
  • the present invention provides isolated polynucleotides and polypeptides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein.
  • polynucleotides are provided by this invention, preferably polynucleotides encoding P. acnes polypeptides, that comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths therebetween.
  • intermediate lengths means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1 ,000, and the like.
  • polynucleotides of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • illustrative DNA segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
  • polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology.
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.
  • the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed.
  • suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.
  • the polynucleotides described above e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein.
  • such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
  • nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
  • the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization.
  • nucleic acid segments that comprise a sequence region of at least about a 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility.
  • Longer contiguous identical or complementary sequences e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
  • nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to SEQ ID NOs:l- 299, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment.
  • Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
  • the use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NOs: 1-299, or to any continuous portion of the sequence, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer.
  • the choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence.
  • Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • nucleic acid reproduction technology such as the PCRTM technology of U. S. Patent 4,683,202 (incorporated herein by reference)
  • introducing selected sequences into recombinant vectors for recombinant production and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest.
  • relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.
  • Polynucleotides may be identified, prepared and/or manipulated using any of a variety of well established techniques.
  • a polynucleotide may be identified, as described in more detail below, by screening a microarray of Propiombacterium acnes cDNAs. Such screens may be performed, for example, using a Synteni microarray (Palo Alto, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997).
  • polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as cells containing Propiombacterium ⁇ crces-derived DNA. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized.
  • PCR polymerase chain reaction
  • An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a Propiombacterium acnes cDNA library) using well known techniques.
  • a library cDNA or genomic
  • a library is screened using one or more polynucleotide probes or primers suitable for amplification.
  • a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.
  • a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques.
  • a bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis.
  • cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector.
  • Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
  • the complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones.
  • the resulting overlapping sequences can then assembled into a single contiguous sequence.
  • a full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
  • primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
  • amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 7(5:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
  • sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region.
  • RACE Rapid amplification of cDNA ends
  • RACE rapid amplification of cDNA ends
  • This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 7:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 79:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
  • EST expressed sequence tag
  • Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence.
  • Full length DNA sequences may also be obtained by analysis of genomic fragments.
  • polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
  • polypeptide-encoding nucleotide sequences possessing non- naturally occurring codons it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non- naturally occurring codons.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
  • Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
  • a newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography, for example (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y), or other comparable techniques available in the art.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the nucleotide sequences encoding the polypeptide, or functional equivalents may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y, and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y
  • a variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMN;
  • control elements or "regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.
  • inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.
  • promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G and S. M. Schuster (1989) J. Biol.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • sequences encoding polypeptides may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y; pp. 191-196).
  • An insect system may also be used to express a polypeptide of interest.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding the polypeptide may be cloned into a non- essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
  • a number of viral-based expression systems are generally available.
  • sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
  • cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl.
  • npt which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
  • telomere sequence encoding a polypeptide is inserted within a marker gene sequence
  • recombinant cells containing sequences can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a polypeptide- encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells which contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
  • a variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purifi 3: 263- 281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein.
  • IMIAC immobilized metal ion affinity chromatography
  • polypeptides of the invention may be produced by direct peptide synthesis using solid- phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, biologically functional equivalent polypeptides, and other useful variants, through specific mutagenesis of the underlying polynucleotides that encode them.
  • the technique well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
  • the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the antigenicity of a polypeptide vaccine.
  • the techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
  • site-specific mutagenesis is often used to alter a specific portion of a DNA molecule.
  • a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
  • site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.
  • Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
  • oligonucleotide directed mutagenesis procedure is intended to refer to a process that involves the template-dependent extension of a primer molecule.
  • template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.
  • PCRTM polymerase chain reaction
  • the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated.
  • reverse transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut.
  • the two probe pairs will link to form a single unit.
  • bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs.
  • Qbeta Replicase described in PCT Intl. Pat. Appl. Publ. No.
  • PCT/US87/00880 may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • CPR cyclic probe reaction
  • a probe having a 3' and 5' sequences of non-target DNA and an internal or “middle" sequence of the target protein specific RNA is hybridized to DNA which is present in a sample.
  • the reaction is treated with RNaseH, and the products of the probe are identified as distinctive products by generating a signal that is released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • CPR involves amplifying a signal generated by hybridization of a probe to a target gene specific expressed nucleic acid.
  • modified primers are used in a PCR-like, template and enzyme dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes is added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al, 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer that has sequences specific to the target sequence.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat-denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target-specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target-specific sequences.
  • a polymerase such as T7 or SP6
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification of DNA sequences of the present invention.
  • BIOLOGICAL FUNCTIONAL EQUIVALENTS Modification and changes may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a "functional" molecule that encodes a polypeptide with desirable characteristics. As mentioned above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide.
  • the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within +2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well known approaches, several of which are outlined below for the purpose of illustration.
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of an adenovirus.
  • adenovirus a 36 kb, linear, double- stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5'-tripartite leader
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
  • adenovirus generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kB of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
  • MOI multiplicities of infection
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the currently preferred helper cell line is 293.
  • Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) are employed as follows.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 -10 11 plaque- forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild- type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford- Perricaudet et al, 1990; Rich et al, 1993).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al., 1981).
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, and env sequence integrated in the host cell genome.
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990). 3. ADENO- ASSOCIATED VIRUSES
  • AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replications is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized.
  • AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka and McLaughlin, 1988).
  • the AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs (FIG 2).
  • ITRs There are two major genes in the AAV genome: rep and cap.
  • the rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1-3.
  • Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration.
  • the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery.
  • Three viral promoters have been identified and named p5, pl9, and p40, according to their map position. Transcription from p5 and pl9 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat and Muzyczka, 1984).
  • rAAV rAAV as an expression vector
  • AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.
  • viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al, 1988), lentiviruses, polio viruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al, 1988; Horwich et al, 1990). With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences.
  • Chang et al. (1991) introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al, 1991).
  • CAT chloramphenicol acetyltransferase
  • the expression construct In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may be positioned and expressed at different sites.
  • the nucleic acid encoding the construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • Benvenisty and Reshef (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.
  • DNA is transcribed by polymerases into messenger RNA and translated on the ribosome to yield a folded, functional protein.
  • the native DNA segment coding for a polypeptide described herein, as all such mammalian DNA strands, has two strands: a sense strand and an antisense strand held together by hydrogen bonding.
  • the messenger RNA coding for polypeptide has the same nucleotide sequence as the sense DNA strand except that the DNA thymidine is replaced by uridine.
  • antisense nucleotide sequences will bind to a mRNA and inhibit expression of the protein encoded by that mRNA.
  • the targeting of antisense oligonucleotides to bind mRNA is one mechanism to shut down protein synthesis, and, consequently, represents a powerful and targeted therapeutic approach.
  • the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U. S. Patent 5,739,119 and U. S. Patent 5,759,829, each specifically incorporated herein by reference in its entirety).
  • antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A receptor and human EGF (Jaskulski et al, 1988; Vasanthakumar and Ahmed, 1989; Peris et al, 1998; U. S. Patent 5,801,154; U. S. Patent 5,789,573; U. S. Patent 5,718,709 and U. S. Patent 5,610,288, each specifically incorporated herein by reference in its entirety).
  • Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g.,cancer (U. S. Patent 5,747,470; U.
  • the invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof.
  • the antisense oligonucleotides comprise DNA or derivatives thereof.
  • the oligonucleotides comprise RNA or derivatives thereof.
  • the oligonucleotides are modified DNAs comprising a phosphorofhioated modified backbone.
  • the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof.
  • preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein.
  • antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence (t.e.,in these illustrative examples the rat and human sequences) and determination of secondary structure, T m , binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
  • Highly preferred target regions of the mRNA are those which are at or near the AUG translation initiation codon, and those sequences which were substantially complementary to 5' regions of the mRNA.
  • MPG short peptide vector
  • the MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al, 1997). It was demonstrated in that several molecules of the MPG peptides coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane (Morris et al, 1997).
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endohuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U. S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV.
  • Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.
  • enzymatic nucleic acids act by first binding to a target RNA.
  • Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
  • the enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif.
  • hammerhead motifs are described by Rossi et al. (1992).
  • hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and U. S. Patent 5,631,359 (specifically incorporated herein by reference).
  • hepatitis ⁇ virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); and an example of the Group I intron is described in (U. S. Patent 4,987,071, specifically incorporated herein by reference).
  • ribozyme constructs need not be limited to specific motifs mentioned herein.
  • enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target, such as one of the sequences disclosed herein.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.
  • Small enzymatic nucleic acid motifs may also be used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure.
  • catalytic RNA molecules can be expressed within cells from eukaryotic promoters.
  • any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl. Publ. No.
  • Ribozymes may be added directly, or can be complexed with cationic lipids, lipid complexes, packaged within liposomes, or otherwise delivered to target cells.
  • the RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers.
  • Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO
  • RNA targets in other species can be utilized when necessary.
  • Hammerhead or hairpin ribozymes may be individually analyzed by computer folding (Jaeger et al, 1989) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 or so bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Ribozymes of the hammerhead or hairpin motif may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized.
  • the method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al. (1987) and in Scaringe et l. (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3 '-end. Average stepwise coupling yields are typically >98%.
  • Hairpin ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).
  • Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'- C-allyl, 2'-flouro, 2'-o-mefhyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.
  • nuclease resistant groups for example, 2'-amino, 2'- C-allyl, 2'-flouro, 2'-o-mefhyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992).
  • Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.
  • Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990; Pieken et al, 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; TJ. S. Patent 5,334,711; and Int. Pat. Appl.
  • Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al, 1993; Zhou et al, 1990). Ribozymes expressed from such promoters can function in mammalian cells (e.g., Kashani-Saber et al, 1992; Ojwang et al, 1992; Chen et al, 1992; Yu et al, 1993; L'Huillier et al, 1992; Lisziewicz et al, 1993).
  • transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, Sindbis virus vectors).
  • plasmid DNA vectors such as adenovirus or adeno-associated vectors
  • viral RNA vectors such as retroviral, semliki forest virus, Sindbis virus vectors.
  • Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. They can also be used to assess levels of the target RNA molecule. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease.
  • ribozymes of this invention include detection of the presence of mRNA associated with an IL-5 related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • PNA peptide nucleic acids
  • PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, 1997).
  • PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA.
  • An excellent review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (1997) and is incorporated herein by reference.
  • PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al, 1991; Hanvey et al, 1992; Hyrup and Nielsen, 1996; Neilsen, 1996).
  • PNAs are neutral molecules
  • PNAs are achiral, which avoids the need to develop a stereoselective synthesis
  • PNA synthesis uses standard Boc (Dueholm et al, 1994) or Fmoc (Thomson et al, 1995) protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used (Christensen et al, 1995).
  • PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA, USA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al, 1995). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
  • PNAs can incorporate any combination of nucleotide bases
  • the presence of adjacent purines can lead to deletions of one or more residues in the product.
  • Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine.
  • PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry.
  • PNA backbone is neutral.
  • PNAs recognize complementary DNA and RNA by Watson-Crick pairing (Egholm et al, 1993), validating the initial modeling by Nielsen et al. (1991).
  • PNAs lack 3' to 5' polarity and can bind in either parallel or antiparallel fashion, with the antiparallel mode being preferred (Egholm et al, 1993).
  • Hybridization of DNA oligonucleotides to DNA and RNA is destabilized by electrostatic repulsion between the negatively charged phosphate backbones of the complementary strands.
  • telomere binding provides clear advantages for molecular recognition and the development of new applications for PNAs.
  • 11-13 nucleotide PNAs inhibit the activity of telomerase, a ribonucleo-protein that extends telomere ends using an essential RNA template, while the analogous DNA oligomers do not (Norton et al, 1996).
  • Neutral PNAs are more hydrophobic than analogous DNA oligomers, and this can lead to difficulty solubilizing them at neutral pH, especially if the PNAs have a high purine content or if they have the potential to form secondary structures. Their solubility can be enhanced by attaching one or more positive charges to the PNA termini (Nielsen et al, 1991).
  • Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by
  • PNAs include use in DNA strand invasion (Nielsen et al, 1991), antisense inhibition (Hanvey et al, 1992), mutational analysis (Orum et al, 1993), enhancers of transcription (Mollegaard et al, 1994), nucleic acid purification (Orum et al, 1995), isolation of transcriptionally active genes (Boffa et al, 1995), blocking of transcription factor binding (Vickers et al, 1995), genome cleavage (Veselkov et al, 1996), biosensors (Wang et al, 1996), in situ hybridization (Trusted et al, 1996), and in a alternative to Southern blotting (Perry-O'Keefe, 1996).
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • This term also does not refer to or exclude post- expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof
  • Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.
  • a polypeptide of the invention will be an isolated polypeptide (or an epitope, variant, or active fragment thereof) derived from a mammalian species.
  • the polypeptide is encoded within a polynucleotide sequence disclosed herein, i.e., in SEQ ID NOs: 1-299, or a sequence which hybridizes under moderately stringent conditions to such a polynucleotide sequence.
  • the polypeptide composition of the invention is selected from among the group consisting of SEQ ID NOs:300-29,212.
  • a polypeptide composition is also understood to comprise one or more polypeptides that are immunologically reactive with antibodies generated against a polypeptide of the invention, particularly a polypeptide encoded by one or more of SEQ ID NOs: 1-299, or a polypeptide having a sequence set forth in any one of SEQ DD NOs: 300-29,212, or to active fragments, or to variants or biological functional equivalents thereof.
  • a polypeptide composition of the present invention is understood to comprise one or more polypeptides that are capable of eliciting antibodies that are immunologically reactive with one or more polypeptides encoded by one or more contiguous nucleic acid sequences contained in SEQ ID NOs: 1-299, or a polypeptide having a sequence set forth in any one of SEQ ID NOs: 300-29,212, or to active fragments, or to strain variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.
  • an active fragment of a polypeptide includes a whole or a portion of a polypeptide which is modified by conventional techniques, e.g., mutagenesis, or by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure function, antigenicity, etc., as a native polypeptide as described herein.
  • the polypeptides of the invention will comprise at least an immunogenic portion of a Propiombacterium acnes protein or a variant thereof, as described herein. Proteins that are Propiombacterium acnes proteins also react detectably within an immunoassay (such as an ELISA) with antisera from a patient infected with Propiombacterium acnes . Polypeptides as described herein may be of any length. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may (but need not) possess further immunogenic or antigenic properties.
  • immunogenic portion is a portion of a protein that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor.
  • immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a Propiombacterium acnes protein or a variant thereof.
  • Certain preferred immunogenic portions include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted.
  • Other preferred immunogenic portions may contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.
  • Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones.
  • antisera and antibodies are "antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins).
  • antisera and antibodies may be prepared as described herein, and using well known techniques.
  • An immunogenic portion of a native Propiombacterium acnes protein is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide.
  • Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.
  • an immunogenic portion of a polypeptide of the present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay).
  • the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70% and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide.
  • preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.
  • a composition may comprise a variant of a native Propiombacterium acnes protein.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from a native Propiombacterium acnes protein in that it will generally have less than five, preferably less than 10 and more preferably less than 25 substitutions, deletions, additions and/or insertions relative to the native polypeptide, such that the immunogenicity of the polypeptide variant is not substantially diminished.
  • the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein.
  • variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein.
  • Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed.
  • Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.
  • the polypeptide fragments and variants provided by the present invention are immunologically reactive with an antibody and/or T- cell that reacts with a full-length P. acnes polypeptide specifically set forth herein.
  • the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90% or more of that exhibited by a full-length P. acnes polypeptide sequence specifically set forth herein.
  • Polypeptide variants encompassed by the present invention include those exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), or 99% or more identity (determined as described above) to the polypeptides disclosed herein.
  • a variant contains conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly- His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
  • a suitable purification matrix such as an affinity matrix or an ion exchange resin.
  • Portions and other variants having less than about 100 amino acids, and generally less than about 50 amino acids may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art.
  • polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. ⁇ 5:2149-2146, 1963.
  • Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known protein.
  • a fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein.
  • Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
  • Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments.
  • Still further fusion partners include affinity tags, which facilitate purification of the protein.
  • Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
  • a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system.
  • DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector.
  • the 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
  • a peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
  • Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues.
  • linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 53:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
  • the linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements.
  • DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
  • Fusion proteins are also provided. Such proteins comprise a polypeptide as described herein together with an unrelated immunogenic protein. Preferably the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 33(5:86-91, 1997).
  • an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926).
  • a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated.
  • the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
  • fusion partners include the non- structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
  • the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion).
  • LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C- terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 70:795-798, 1992).
  • a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
  • polypeptides including fusion proteins and polynucleotides as described herein are isolated.
  • An "isolated" polypeptide or polynucleotide is one that is removed from its original environment.
  • a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
  • the present invention further provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a Propiombacterium acnes protein.
  • agents such as antibodies and antigen-binding fragments thereof, that specifically bind to a Propiombacterium acnes protein.
  • an antibody, or antigen-binding fragment thereof is said to "specifically bind" to a Propiombacterium acnes protein if it reacts at a detectable level (within, for example, an ELISA) with a Propiombacterium acnes protein, and does not react detectably with unrelated proteins under similar conditions.
  • binding refers to a noncovalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex.
  • the binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations.
  • two compounds are said to "bind,” in the context of the present invention, when the binding constant for complex formation exceeds about 10 3 L/mol.
  • the binding constant may be determined using methods well known in the art.
  • Binding agents may be further capable of differentiating between patients with and without Propiombacterium acnes using the representative assays provided herein.
  • antibodies or other binding agents that bind to a Propiombacterium acnes protein will generate a signal indicating its presence in at least about 20% of patients with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the disease.
  • biological samples e.g., blood, sera, sputum and/or urine
  • serum e.g., sera, sputum and/or urine
  • binding agent may be used in combination to improve sensitivity.
  • a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide.
  • a binding agent is an antibody or an antigen-binding fragment thereof.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats).
  • the polypeptides of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. (5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • the polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
  • the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques.
  • immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments.
  • the Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.
  • Monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents.
  • Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
  • Preferred radionuclides include ⁇ , I, I, 131 I, 186 Re, 188 Re, 211 At, and 212 Bi.
  • Preferred drugs include methotrexate, and pyrimidine and purine analogs.
  • Preferred differentiation inducers include phorbol esters and butyric acid.
  • Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
  • a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
  • a linker group which is cleavable during or upon intemalization into a cell.
  • a number of different cleavable linker groups have been described.
  • the mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spitier), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S.
  • Patent No. 4,638,045 to Kohn et al.
  • serum complement- mediated hydrolysis e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
  • acid-catalyzed hydrolysis e.g., U.S. Patent No. 4,569,789, to Blattler et al.
  • immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group.
  • Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
  • a carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis.
  • a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
  • U.S. Patent No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
  • routes of administration for the antibodies and immunoconjugates may be used. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density, and the rate of clearance of the antibody.
  • T CELLS Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for a Propiombacterium acnes protein.
  • T cells may generally be prepared in vitro or ex vivo, using standard procedures.
  • T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the IsolexTM System, available from Nexell Therapeutics, Inc. (Irvine, CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
  • T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
  • T cells may be stimulated with a Propiombacterium acnes polypeptide, polynucleotide encoding a Propiombacterium acnes polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide.
  • APC antigen presenting cell
  • Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide.
  • a Propiombacterium acnes polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
  • T cells are considered to be specific for a Propiombacterium acnes polypeptide if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide.
  • T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065- 1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
  • T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA).
  • a Propiombacterium acnes polypeptide 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng ml - 25 ⁇ g ml
  • contact with a Propiombacterium acnes polypeptide 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng ml - 25 ⁇ g ml
  • T cells that have been activated in response to a Propiombacterium acnes polypeptide, polynucleotide or polypeptide-expressing APC may be CD4 + and/or CD8 + .
  • Propiombacterium acnes protein- specific T cells may be expanded using standard techniques.
  • the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
  • CD4 + or CD8 + T cells that proliferate in response to a Propiombacterium acnes polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways.
  • the T cells can be re-exposed to a Propiombacterium acnes polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a Propiombacterium acnes polypeptide.
  • one or more T cells that proliferate in the presence of a Propiombacterium acnes protein can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution. PHARMACEUTICAL COMPOSITIONS
  • the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody compositions disclosed herein in pharmaceutically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • the nucleic acid segment, RNA, DNA or PNA compositions that express a polypeptide as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents.
  • agents such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents.
  • the compositions may thus be delivered along with various other agents as required in the particular instance.
  • Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.
  • such compositions may further comprise substituted or derivatized RNA or DNA compositions.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.
  • compositions disclosed herein may be delivered via oral administration to an animal.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al, 1997; Hwang et al, 1998; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar, or both.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • aqueous solution for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifiingal agents, isotonic and abso ⁇ tion delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifiingal agents, isotonic and abso ⁇ tion delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212 (each specifically inco ⁇ orated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et al, 1998) and lysophosphatidyl- glycerol compounds (U. S. Patent 5,725,871, specifically inco ⁇ orated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045 (specifically inco ⁇ orated herein by reference in its entirety).
  • the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells.
  • the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be preferred for the introduction of pharmaceutically- acceptable formulations of the nucleic acids or constructs disclosed herein.
  • liposomes are generally known to those of skill in the art (see for example, Couvreur et al, 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases).
  • liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741,516, specifically inco ⁇ orated herein by reference in its entirety).
  • Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, 1990; Muller et al, 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems.
  • Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath et al, 1986; Balazsovits et al, 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al, 1987), enzymes (Imaizumi et al, 1990a; imaizumi et al, 1990b), viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 1979) into a variety of cultured cell lines and animals.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e., in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
  • Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability.
  • the phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • a characteristic phase-transition temperature results in an increase in permeability to ions, sugars and drugs.
  • proteins can alter the permeability of liposomes.
  • Certain soluble proteins, such as cytochrome c bind, deform and penetrate the bilayer, thereby causing changes in permeability. Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol. The ability to trap solutes varies between different types of liposomes.
  • MLVs are moderately efficient at trapping solutes, but SUNs are extremely inefficient.
  • SUVs offer the advantage of homogeneity and reproducibility in size distribution, however, and a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUNs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs.
  • an important determinant in entrapping compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
  • Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adso ⁇ tion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
  • liposomes The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake.
  • MLVs and LUVs are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the ca
  • SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen.
  • this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs. Targeting is generally not a limitation in terms of the present invention.
  • Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell- type surface.
  • Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types.
  • intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
  • the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987; Quintanar-Guerrero et al, 1998; Douglas et al, 1987).
  • ultrafine particles sized around 0.1 ⁇ m
  • Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
  • Such particles may be are easily made, as described (Couvreur et al, 1980; 1988; zur Muhlen et al, 1998; Zambaux et al. 1998; Pinto- Alphandry et al, 1995 and U. S. Patent 5,145,684, specifically inco ⁇ orated herein by reference in its entirety).
  • compositions are provided for use as vaccines.
  • the vaccines will generally comprise one or more pharmaceutical compositions, such as those discussed above, in combination with an immunostimulant.
  • An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and or cell-mediated) to an exogenous antigen.
  • immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is inco ⁇ orated; see e.g., Fullerton, U.S. Patent No. 4,235,877).
  • Vaccine preparation is generally described in, for example, M.F. Powell and M.J.
  • compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • one or more immunogenic portions of other antigens may be present, either inco ⁇ orated into a fusion polypeptide or as a separate compound, within the composition or vaccine.
  • Illustrative vaccines may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 75:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl Acad. Sci. USA 86:311-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.
  • a vaccine may comprise both a polynucleotide and a polypeptide component.
  • a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein.
  • Such salts may be prepared from pharmaceutically acceptable non- toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
  • organic bases e.g., salts of primary, secondary and tertiary amines and basic amino acids
  • inorganic bases e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • any suitable carrier known to those of ordinary skill in the art may be employed in the vaccine compositions of this invention, the type of carrier will vary depending on the mode of administration.
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactate polyglycolate
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.
  • One may also employ a carrier comprising the particulate-protein complexes described in U.S. Patent No. 5,928,647, which are capable of inducing a class I- restricted cytotoxic T lymphocyte responses in a host.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., g., bacteriostats,
  • Compounds may also be encapsulated within liposomes using well known technology. Any of a variety of immunostimulants may be employed in the vaccines of this invention. For example, one or more adjuvants may be included. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or - 12, may also be used as adjuvants.
  • Cytokines such as GM-CSF or interleukin-2, -7, or - 12, may also be used as adjuvants.
  • the adjuvant composition is preferably designed to induce an immune response predominantly of the Thi type.
  • High levels of Thi -type cytokines e.g., IFN- ⁇ , TNF ⁇ , IL-2 and IL-12
  • high levels of Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • a patient will support an immune response that includes Thi- and Th2-type responses.
  • Thl-type cytokines in which a response is predominantly Thl-type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
  • Preferred adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O- acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Corixa Co ⁇ oration (Seattle, WA; see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thi response.
  • Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462.
  • Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.
  • Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which may be used alone or in combination with other adjuvants.
  • QS21 Amla Biopharmaceuticals Inc., Framingham, MA
  • an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other preferred formulations comprise an oil-in-water emulsion and tocopherol.
  • Advants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which are inco ⁇ orated herein by reference in their entireties.
  • SBAS series of adjuvants e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium
  • Detox Corixa, Hamilton, MT
  • RC-529 Corixa, Hamilton, MT
  • AGPs
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration).
  • sustained release formulations i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 74:1429-1438, 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • Such carriers include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
  • Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Patent No.
  • APCs antigen presenting cells
  • APCs antigen presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti- Propiombacterium acnes effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:501-529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate na ⁇ ve T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF ⁇ , CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4- IBB).
  • cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4- IBB).
  • APCs may generally be transfected with a polynucleotide encoding a Propiombacterium acnes protein (or portion or other variant thereof) such that the Propiombacterium acnes polypeptide, or an immunogenic portion thereof, is expressed on the cell surface.
  • a composition or vaccine comprising such transfected cells may then be used for therapeutic pu ⁇ oses, as described herein.
  • a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456- 460, 1997.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the Propiombacterium acnes polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
  • the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
  • an immunological partner that provides T cell help e.g., a carrier molecule.
  • a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
  • Naccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use.
  • formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • compositions described herein may be used for immunotherapy of acne.
  • pharmaceutical compositions and vaccines are typically administered to a patient.
  • a patient refers to any warm-blooded animal, preferably a human.
  • a patient may or may not be afflicted with acne.
  • the above pharmaceutical compositions and vaccines may be used to prevent the development of acne or to treat a patient afflicted with a acne.
  • Acne may be diagnosed using criteria generally accepted in the art.
  • Administration may be by any suitable method but will most typically be oral or topical.
  • immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against Propiombacterium acnes with the administration of immune response- modifying agents (such as polypeptides and polynucleotides as provided herein).
  • immune response- modifying agents such as polypeptides and polynucleotides as provided herein.
  • immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established Propiombacterium acnes -immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate therapeutic effects and does not necessarily depend on an intact host immune system.
  • agents with established Propiombacterium acnes -immune reactivity such as effector cells or antibodies
  • effector cells include T cells as discussed above, T lymphocytes (such as CD8 + cytotoxic T lymphocytes and CD4 + T-helper lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen- presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein.
  • T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy.
  • the polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164, for example) for passive immunotherapy.
  • Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein.
  • Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art.
  • Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells.
  • cytokines such as IL-2
  • immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy.
  • antigen-presenting cells such as dendritic, macrophage, monocyte, fibroblast and/or B cells
  • antigen-presenting cells may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art.
  • antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system.
  • Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo.
  • a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient.
  • Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary or intraperitoneal administration.
  • compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), orally, sublingually or, preferably, topically.
  • injection e.g., intracutaneous, intramuscular, intravenous or subcutaneous
  • intranasally e.g., by aspiration
  • sublingually e.g., topically.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-acne immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti- Propiombacterium acnes antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's Propiombacterium acnes cells in vitro.
  • the amount of each polypeptide present in a dose ranges from about 25 ⁇ g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome in treated patients as compared to non-treated patients, increases in preexisting immune responses to a Propiombacterium acnes protein generally correlate with an improved clinical outcome.
  • immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
  • Propiombacterium acnes may be detected in a patient based on the presence of one or more Propiombacterium acnes proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum and/or urine) obtained from the patient.
  • a biological sample for example, blood, sera, sputum and/or urine
  • proteins may be used as markers to indicate the presence or absence of Propiombacterium acnes.
  • proteins may be useful for the detection of other organisms related to Propiombacterium acnes.
  • the binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample.
  • Polynucleotide primers and probes may be used to detect the level of mRNA encoding a Propiombacterium acnes protein, which is also indicative of the presence or absence of Propiombacterium acnes.
  • the presence or absence of Propiombacterium acnes in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-offvalue.
  • the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample.
  • the bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex.
  • detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin.
  • a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample.
  • the extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent.
  • Suitable polypeptides for use within such assays include full length Propiombacterium acnes proteins and portions thereof to which the binding agent binds, as described above.
  • the solid support may be any material known to those of ordinary skill in the art to which the protein may be attached.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinyl chloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
  • immobilization refers to both noncovalent association, such as adso ⁇ tion, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adso ⁇ tion to a well in a microtiter plate or to a membrane is preferred. In such cases, adso ⁇ tion may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
  • binding agent ranging from about 10 ng to about 10 ⁇ g, and preferably about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of binding agent.
  • Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
  • an appropriate contact time is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual having Propiombacterium acnes.
  • the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
  • Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
  • the second antibody which contains a reporter group, may then be added to the solid support.
  • Preferred reporter groups include those groups recited above.
  • the detection reagent is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate.
  • Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups.
  • Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme).
  • Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
  • the cut-off value for the detection of a Propiombacterium acnes is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without Propiombacterium acnes.
  • a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the test.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7.
  • the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result.
  • the cut-off value on the plot that is the closest to the upper left-hand corner i.e., the value that encloses the largest area
  • a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
  • the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
  • a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for Propiombacterium acnes .
  • the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane.
  • a second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane.
  • the detection of bound second binding agent may then be performed as described above.
  • the strip test format one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
  • Concentration of second binding agent at the area of immobilized antibody indicates the presence of Propiombacterium acnes.
  • concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
  • Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 500 ng.
  • Such tests can typically be performed with a very small amount of biological sample.
  • numerous other assay protocols exist that are suitable for use with the Propiombacterium acnes proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use Propiombacterium acnes polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such Propiombacterium acnes protein-specific antibodies may correlate with the presence of Propiombacterium acnes.
  • Propiombacterium acnes may also, or alternatively, be detected based on the presence of T cells that specifically react with a Propiombacterium acnes protein in a biological sample.
  • a biological sample comprising CD4 + and/or CD8 T cells isolated from a patient is incubated with a Propiombacterium acnes polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected.
  • Suitable biological samples include, but are not limited to, isolated T cells.
  • T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C with polypeptide (e.g., 5 - 25 ⁇ g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of Propiombacterium acnes polypeptide to serve as a control.
  • activation is preferably detected by evaluating proliferation of the T cells.
  • CD8 + T cells activation is preferably detected by evaluating cytolytic activity.
  • Propiombacterium acnes may also, or alternatively, be detected based on the level of mRNA encoding a Propiombacterium acnes protein in a biological sample.
  • at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a Propiombacterium acnes cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the Propiombacterium acnes protein.
  • PCR polymerase chain reaction
  • oligonucleotide probes that specifically hybridize to a polynucleotide encoding a Propionibacterium acnes protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the Propionibacterium acnes protein in a biological sample.
  • oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a Propionibacterium acnes protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length.
  • oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above.
  • Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
  • the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule described herein.
  • Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol, 57:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).
  • compositions described herein may be used as markers for the progression of acne disease by monitoring the presence of Propionibacterium acnes.
  • assays as described above for detection of Propionibacterium acnes may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated.
  • the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed.
  • Certain in vivo diagnostic assays may be performed directly on a lesion.
  • One such assay involves contacting lesion cells with a binding agent.
  • the bound binding agent may then be detected directly or indirectly via a reporter group.
  • binding agents may also be used in histological applications.
  • polynucleotide probes may be used within such applications.
  • multiple Propionibacterium acnes protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of Propionibacterium acnes protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for Propionibacterium acnes proteins provided herein may be combined with assays for other known or implicated acne causing proteins.
  • kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a Propionibacterium acnes protein.
  • Such antibodies or fragments may be provided attached to a support material, as described above.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • kits may be designed to detect the level of mRNA encoding a Propionibacterium acnes protein in a biological sample.
  • kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a Propionibacterium acnes protein.
  • Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a Propionibacterium acnes protein.
  • EXAMPLE 1 IDENTIFICATION OF PROPIONIBACTERIUM A CNES GENOMIC DNA SEQUENCES
  • Propionibacterium acnes (ATCC No. 6919; typed strain) was obtained from ATCC.
  • the lyophilized bacteria was resuspended in 1ml of thioglycollate media and -200 ul transferred to each of several culture tubes containing -15 ml fluid thioglycollate and incubated at 37°C for 7 to 10 days.
  • Each 15 ml dense culture was subsequently used to seed 500 ml culture volumes of thioglycolate media with little void volume (air space) to allow for anaerobic growth.
  • the cultures were grown at 37°C for - 3 weeks or until turbid growth was apparent.
  • P. acnes pellets were harvested by centrifugation and the pellets washed three times with 1 X PBS by multiple resuspension and centrifugation. Final centrifugation was at 10,000 ⁇ m and residual liquid was removed.
  • High molecular weight DNA was prepared as follows: P. acnes pellets were heat inactivated for 20 minutes at 80°C in a water bath followed by enzymatic lysis with a 20mg/ml solution of lysozyme (0.1 Og lysozyme in 5ml TE) in TE (lOmM tris; lmM EDTA) at 37°C for lhr.
  • lOul Prot K (lOmg/ml) and 70ul of 10 % SDS for every 0.5 ml of lysis buffer were added (Step 3 above) and the suspension rocked for - 10 minutes at room temperature. This was followed by incubation at 65°C for 15 minutes and the addition of lOOul of a 5M NaCl solution per 500ul of lysis buffer while still at 65°C. Finally, 80 ul of a 10% CTAB solution (lOg CTAB; 14ml 5M NaCl, 86 ml water) was added for every 500 ul lysis buffer and the incubation continued at 65°C for lOmin.
  • lOg CTAB 14ml 5M NaCl, 86 ml water
  • Genomic DNA was extracted from the lysed pellets by the addition of an equal volume of phenol and a few drops of chloroformtisoamyl alcohol (24:1), followed by mixing. The homogeneous "white” solution was centrifuged and the aqueous phase re- extracted two more times. This was followed by the addition of 0.6 volume of isopropanol to precipitate the genomic DNA.
  • the high molecular weight DNA was randomly sheared by sonication and fragments with an average size of -1,200 bp were purified and made blunt end by a standard filling process. Insert DNA was ligated into a PUC vector and transformed into E. coli host cells to generate a library of independent clones. DNA was isolated from the individual clones were sequenced from the P. acnes genomic library. Overlapping clones representing approximately 8.6 full length genome equivalents were aligned to form 299 linear contigs. Sequences identified in this manner are set forth in SEQ DD NOs: 1-299, and are further identified by contig names and lengths in Table 2. These 299 contigs represent a total assembled length of about 2,656,860 nucleotides covering more than 90%) of the P. acnes genome.
  • ORFs Open reading frames
  • This database is composed of all non-redundant GenBank protein sequences (CDS translations+PDB+SwissProt+PIR). Details of the database can be found at http : / / www.ncbi .nlm.nih. go v / Genbank / index .html .
  • the parameters for the BLASTP search were set to the standard defaults with the exception that a significant "hit” was defined as any match with an "E-value” (expected value) of 10 e-8 or less.
  • the E value represents the odds of a particular stretch of homology observed between two sequences occurring by chance. Therefore, the lower the E value, the higher the statistical probability that the observed homology is real and that the predicted ORF polypeptide represents a authentic translation product.
  • the 28,913 predicted P. acnes ORF amino acid sequences were also used as queries in a TBLASTN sequence homology search of the GenBank non-redundant (nr) nucleic acid database (GenBank nr, release date 11/06/2000).
  • This database comprises all non-redundant nucleic acid sequences (GenBank +EMBL+DDBJ+PDB). Details of the database can be found at: http: / / www.ncbi.nlni.nih.gov/Genbank/index.htrnl.
  • TBLASTN uses a protein query to search a DNA database that is translated in all six reading frames. The parameters for this TBLASTN search were set to the standard defaults with the exception that a significant "hit" was defined as any match with an "E-value" (expected value) of 10 e-8 or less.
  • GeneMark software (Georgia Institute of Technology; Atlanta, GA) is a bioinformatics software program used to identify protein-coding regions from DNA sequence based on Markov models, and is discussed at http://genemark.biology.gatech.edu/GeneMark/. which contains information and references particular to the model.
  • P. acnes were used to identify protein-coding sequences in P. acnes. All predicted P. acnes open reading frames (ORFs) with a size of 50 residues or more, as discussed above, were used for this comparative analysis.
  • ORFs open reading frames
  • the BLAST sequence comparison program was then used to compare these ORFs to amino acid sequences in Genebank protein database (blastp) and amino acid sequences translated from DNA sequences in Genebank DNA sequence (tblastn).
  • GeneMark matrices were used to identify ORFs in the P. acnes genome.
  • the seven organisms used were E.coli, Bacillus subtilis, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma genitalium and Mycobacterium tuberculosis.
  • Each matrix predicts a set of protein coding sequences from the P. acnes genomic sequence. Some protein-coding sequences were predicted from all 7 organisms and some were predicted from a subset of the 7 organisms.
  • the number of times that a particular ORF is predicted by the 7 matrices is listed in Table 4 below as the "Genemark Score".
  • the higher the genemark score the more likely that an ORF represents an authentic P. acnes protein sequence because it is predicted from multiple matrices, indicating that the protein is evolutionarily conserved.
  • Table 4 summarizes the BLAST and GeneMark analysis data ORFs that met the following criteria: i. 50 residues or greater and ii. generated a BLASTP hit with an E value of 10 e-8 or less and/or iii. generated a TBLASTN hit with an E value of 10 e-8 or less and/or iv. was predicted from at least six (6) of the GeneMark matrices.
  • ORFs not meeting criteria (i) and at least one of the other criteria (ii) - (iv) were not included in the final list of ORFs described in Table 4.
  • Table 4 contains the following information:
  • ORF SEQ ID SEQ ID assigned to each ORF as defined in Table 3.
  • DNA SEQ ID corresponding DNA SEQ ID as defined in Table 2 from which that ORF was derived (translated).
  • Contig Name Sequence name assigned in original genomic sequence analysis.
  • Frame Reading frame on the DNA contig used to derive the ORF. Forward frames 1, 2 or 3. Reverse frames -1, -2 or -3.
  • ORF length Length of ORF in number of amino acid residues.
  • BLASTP E value E value of best BLASTP hit. If no significant hit was obtained, cell is empty.
  • P. acnes proteins were identified that met the above criteria. This number of P. acnes proteins was identified using a E value threshold of 10 e-8 or smaller, as discussed above, however altering this threshold to 10e-15 gives rise to about 3400 ORFs, altering the threshold to 10e-20 gives rise to about 2900 ORFs and altering the threshold to 10e-25 gives rise to about 2600 ORFs. It is believed that there are roughly 2500-3500 proteins encoded by the P. acnes genome given its size and based on information available for other related microbial organisms. In this regard, certain preferred P.
  • acnes polypeptides of the present invention will included those identified in Table 4 as having a BLASTP and/or TBLASTN E value in the range of 10e-15 to 10e-25, or less. From the results of these combined analyses, one of ordinary skill in the art would view the data of Table 4 as substantially supporting that the proteins identified herein represent authentic translation products of the P. acnes genome.
  • An expression library was constructed as follows: total genomic DNA from the P. acnes strain 6919 was randomly sheared by sonication for 5 seconds at 30% power (Fisher Sonic Dismembrator) and then termini were filled-in with DNA polymerease I (Klenow fragment; Life Technologies, Rockville, MD) at room temperature for 30 minutes, followed by Pfu polymerase (Stratagene, La Jolla, CA) at 72°C for 30 minutes. EcoRI adaptors (Stratagene, La Jolla, CA) were next ligated to the blunted fragments for 48 hours at 4°C and after heat inactivation of the ligase, adaptors were phosphorylated with T4 polynucleotide kinase for 30 minutes at 37°C.
  • DNA fragments were finally size selected with a Sephacryl S-400 HR (Sigma, St. Louis, MO) column before being ligated into an EcoRI-digested Lambda ZAP Express (Stratagene, La Jolla, CA) vector for 48 hrs at 4°C and packaged with Gigapack III Gold packaging extract (Stratagene, La Jolla, CA).
  • the expression library was screened with 1) sera from pooled individuals with history of severe acnes and 2) with a rabbit poly-clonal Ab raised against P. acnes lysate. This resulted in the cloning of several genes.
  • P. acnes (PA) clones are designated as hPA or rPA depending on whether they were cloned using the human (h) or rabbit (r) sera. Eight of the completed clones were obtained with the human sera screen (clones hPA-2,4,14,20,21,23,25 and 29) and nine with the rabbit screening (rPA-2,3,7,15,17,19,28,34b and 40). These clones and their positions on the P. acnes DNA contigs of SEQ DD NOs: 1-299 are shown below in Table 5. Also shown are the SEQ DD NOs: of the polypeptide sequences encoded within the respective regions on the DNA contigs. Thus, certain of these polypeptides are reactive with sera from pooled individuals with history of severe acnes and/or with rabbit poly-clonal antibody raised against P. acnes lysate and therefore represent immunogenic P. acnes proteins.
  • EXAMPLE 5 P. ACNES PROTEINS WITH HOMOLOGIES TO COHEMOLYSIN OF STREPTOCOCCI
  • NCTC737 and two clinical isolates (PA-1 and PA-2) were resolved by SDS-PAGE, silver stained and their protein profiles compared.
  • ⁇ -terminal sequencing of ZI and ZII derived directly from the PNDF blot revealed the amino acid sequences APVAPINAVSATQP ⁇ and SLLDTQKSAKKAFAPA ⁇ respectively.
  • the first -25 to 30 amino acids of the full length protein (FL pre-pro) is rich in hydrophobic residues, suggestive of the presence of a signal peptide.
  • ZI/II are presumably membrane bound/secreted.
  • the N-terminal sequence of ZI is located upstream of ZII suggesting that both forms are derived from the same gene product (possibly resulting from differential processing).
  • mass spec sequencing following tryptic digests of ZI and ZII revealed sequence information similar to those identified by N-terminal sequencing as well as additional internal fragments.
  • the sizes of the ORFs of ZI and ZII are 27.7 (257 residues) and 24-KDa (222 residues) respectively.
  • Mass Spectrometry analysis was conducted as follows. The two bands (ZI and ZII) were excised and digested in-gel with trypsin in order to produce small peptide fragments.
  • the digested proteins were extracted by 50% acetonitrile in 5% acetic acid, and concentrated in a speed vacuum. In order to separate the concentrated peptides on the basis of size, the fragments were run through a capillary LC column that contained C18 resin.
  • K.ATGVLM*EPLVR.V K.ATGVLMEPLVR.V
  • ZI/II The full length protein sequence of ZI/II was subsequently used to search the ORFs of the sequenced P. acnes genome. -The search revealed that ZI/II (SEQ ID NO: 15127) belongs to a family of proteins and shares significant homology with five other related proteins identified in the translated genome of P. acnes (SEQ ID NO: 25540; 6706; 23076; 8751; 27533). Since the entire ORFs of ZI and II were identified in the sequenced genome of the ATCC#6919 culture isolate, failure to detect the corresponding products (in the ATCC#6919) by silver staining of SDS/PAGE gels, cannot be attributed to deletion of the gene but rather from supression of the transcription/translation machinary.
  • LCQ tandem mass spectrometry
  • tandem mass spectra were used to search against P. acnes translated ORFs.
  • the tandem mass spectrometry data were manually reviewed after Sequest search for each experiment.
  • the P. acnes database search results gave the protein sequence, then each identified P. acnes protein was searched against GenBank Protein to find a similarity match (E value, typically E ⁇ 0.001 for a good match) to determine the presumed protein function.
  • E value typically E ⁇ 0.001 for a good match
  • positive identification of a protein was defined by three peptides identified from the same protein. Sometimes a single peptide matched (with a significant match score) was also used as a positive identification for a protein.
  • EXAMPLE 7 P. ACNES PROTEIN IDENTIFICATION
  • EXAMPLE 8 P. ACNES IMMUNOTHERAPEUTIC TARGET IDENTIFICATION
  • enterotoxins as vaccine targets are discussed in Immunology 707:154-160 (2000).
  • Lipoproteins as vaccines are discussed in J. Bacteriol 777:1123-1128 (1995); Infection 24:195-202 (1996); J. Appl. Microbiol. 87-250 (1999); and J. Mol. Biol. 302:1153-1164 (2000).
  • Membrane proteins as vaccines are discussed in Vaccine 77:1952-1961 (2000); J. Biotechnol. 44 ⁇ A5-153 (1996); Vaccine 75:1456-1466 (2000); Vaccine 73:821-829 (1995); New Eng.
  • blastp and tblastn columns of Table 4 were searched using as queries the following keywords: transferase, enterotoxin, lipoprotein, membrane, permease, enterobactin, protease/proteinase, protein A, secret(ed/ion ory), dismutase, adhesin, transporter, haemolysin, penicillin binding protein, sialidase, siderophore, zinc, iron, manganese and lipase. These searches led to the identification of P.
  • Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate) activation.
  • HPTU O-Benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate
  • a Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
  • Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1 :2:2:3).
  • the peptides may be precipitated in cold mefhyl-t-butyl-efher.
  • the peptide pellets may then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC.
  • TFA trifluoroacetic acid
  • a gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may be used to elute the peptides.
  • the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.
  • EXAMPLE 10 Assembly of P. acnes Genomic Sequences using Phrap Analysis.
  • Table 7 A description of these ORF sequences and their sequence identifier numbers are described in Table 7.
  • Column 1 contains the SEQ ID NOs: assigned to the ORFs identified from the initial analysis.
  • Column 2 contains identifiers that define the contig from the initial assembly from which the ORF was derived, the reading frame in which the contig was translated and the coordinates (in amino acids) in that translation that define the start and stop codon.
  • Column 3 contains the sequence identifiers of the newly disclosed ORFs.
  • Column 4 contains identifiers that define the contigs in the Phrap assembly and the ORFs derived from the contigs.
  • Column 5 defines the coordinates (in nucleotides) of the DNA sequence that, translated, represents the new ORF.
  • the information contained in columns 4 and 5 for the newly disclosed ORFs is equivalent to that contained within column 2 for the ORFs from the initial assembly.
  • Secreted P. acnes proteins are likely to be important in the development of protective immunity, both humoral and T cell mediated.
  • primary clinical isolates were generated from two patients; PA1 and PA2, both of whom were suffering from clinically diagnosed acne.
  • the isolates were collected from acne lesions and inoculated into thioglycolate media, where they were amplified in culture.
  • P. acnes stocks were generated, an aliquot of the stocks were spiked into 250 mis of fresh thioglycolate media and cultured for 2-3 weeks. Following culture, the media was centrifuged at 3000 rpm for 30 minutes.
  • the supernatants which contain the secreted proteins, were then filtered through 0.45 ⁇ M cellulose acetate membranes and concentrated to 5-10 mis using Milipore Centricon. The concentrated supernatant was then buffer exchanged three times with 80 mis of phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • acnes ORFs identified using this method include: 6923, 10104, 21845, 6884, 15783, 14514, 13744, 12764, 15938, 8751, 12764, 10682, 23076, 10677, 20846, 14912, 4482, 6109, 6570, 23335, 1550, 21648, 19886, 12317, 9292, 9121, 25103, 15127, 16992, 5027, 11592, 14514, 3659, 383, 848, 2889, 1255, 20476, 16002, 6599, 25913, 4604, 3608, 12013, 16184, 23076, 21368, 21693, 13695, 14638, 20476, 6301, 7993, 28871, 21947, 15693, 18690, 1327, 6660, 24460, 6706, 15127, 24284, 6706, 14163, 11655, 24275, 24275, 1400, 18829,
  • EXAMPLE 12 Integral Membrane Proteins of P. acnes Clinical Isolate PA1
  • This example identifies P. acnes antigens that are expressed on the surface of the bacterium and therefore represent attractive targets against which antibodies are generated.
  • 35 mis of P. acnes isolate PA1 (the generation of which was described above) was harvested by centrifugation at 3700 rpm for 30 minutes. The pellet was then washed three times in PBS and resuspended in 5mls of a 1% TX-114 (a non-ionic detergent) solution. The pellet was resuspended and rocked at 4°C for 1 hour. The homogenate was then centrifuged at 14,000 rpm to pellet the debris.
  • the supernatant was then collected in a fresh tube, incubated at 37°C for 15 minutes, followed by centrifugation at room temperature for 15 minutes at 14,000 rpm. This resulted in the separation of the suspension into an upper aqueous phase and a lower TX-114 phase.
  • Transmembrane and surface lipid anchored proteins are generally extracted by the detergent and get partitioned to the lower detergent rich phase, while the hydrophilic proteins partition into the upper detergent poor phase.
  • the two phases were re-extracted twice prior to a final concentration step.
  • the two phases were then analyzed using SDS-PAGE and Western Blot analysis. Two sera were used in the analysis of the components of the aqueous and TX-114 phases. One was a mouse anti- whole PA1 formulated in AS2 and the second was a rabbit anti-PAl CFP.
  • the 35kDa band from the aqueous phase was identified as ORF 16536, a lysophospholipase.
  • the 24kDa band from the aqueous phase was identified as ORF 23076, a CAMP factor.
  • the 60kDa band from the aqueous phase was identified as ORF 6868, a GROEL heat shock protein.
  • the 25kDa band from the TX-114 phase was identified as ORF 15217, the 24kDa band was identified as ORF 6706, and the 23kDa band was identified as ORF 15217, all of which encode CAMP factors.
  • the 75kDa from the TX-114 phase identified two ORFs; ORF 25620, a peptide binding protein related to ABC transporters and ORF 25274, a serine/threonine protein kinase protein.
  • ORFs 6706, 15217, 25620, and 25274 are thus believed to be membrane bound proteins, making them attractive targets for the generation of antibodies, either actively or passively, against P. acnes.
  • the rationale behind raising a serum against the whole bacterium (unlysed bacteria) was to target proteins that are present or exposed on the surface of the bacterium and thus available as targets for antibody production.
  • the fact that the mouse sera generated against the whole bacterium detected proteins in the aqueous phase is a good indication that these proteins are also surface exposed and therefore valuable candidates for antibody-based therapeutics.
  • EXAMPLE 13 In silico Identification of Antibody- Accessible P. acnes Polypeptides.
  • This example describes the identification of antibody accessible P. acnes polypeptides. Identification of such proteins provides valuable information on the generation of a vaccine capable of inducing an antibody response specifically reactive against these peptides. These proteins were identified by comparing the sequences of different P. acnes ORFs described above with the sequences of known surface exposed/secreted, immunogenic determinants and/or virulence factors harbored by other pathogenic bacteria. P. acnes ORFs that displayed significant identity to known proteins of these types from other pathogenic bacteria are described below in Table 8. Described in Table 8, Column 1, is the original SEQ ID NO of the ORF.
  • Column 3 contains the E value of the closest known homologue to the P. acnes ORF of interest with its corresponding E value.
  • Columns 4, 5, and 6, contain information on the nature, location and function of the P. acnes ORF of interest. This information was also deduced from the primary sequence of the P. acnes ORF.
  • Column 7 contains information on whether the protein is predicted to contain any transmembrane domains. This data was generated using psort analysis. Psort is a computer program, which predicts the subcellular localization sites of proteins based on their amino acid sequence.
  • Column 8 provides further information as to why the P. acnes ORFs of interest constitute attractive targets for an antibody-based immuno therapeutics.
  • EXAMPLE 14 The identification of HLA-A2 peptides derived from P. acnes.
  • HLA-A2 binding peptides of several predicted P. acnes proteins were identified by analyzing the full-length open- reading frames (ORF) of these proteins and running them through "Episeek," a program used to predict MHC binding peptides. The program used is based on the algorithm published by Parker, K.C. et al, J. Immunol. 152(1 ⁇ :163-175 (1994) (incorporated by reference herein in its entirety). The peptide epitopes identified by this approach are described below in Table 9.
  • mice Males; ⁇ 10-12 weeks old were treated with a 3x concentrated 3 week old culture (stationary phase) of the clinical P. acnes isolate PA1, while 2 counterparts were treated with virgin culture medium (thioglycolate) alone. These mice spontaneously develop comedon like structures, and therefore have had some limited utility for testing the anti-hyperkeratinization effect of certain compounds. However, these lesions appear very infrequently and are not P. acnes induced. Thus, the backs of hairless mice were rubbed with gauze soaked in warm water in order to remove the excess of sebum secretion and render the follicle openings accessible to the bacteria.
  • mice were then blotted and painted with the bacterial suspension until the mice started shivering (indicating that the effects of the anesthesia were wearing off).
  • the mice started shivering (indicating that the effects of the anesthesia were wearing off).
  • one of the two mice that had received live P. acnes culture on its skin developed numerous visible comedon-like structures, while the two control animals did not. The lesions healed spontaneously within about one week.
  • a random shear genomic expression library was constructed in the Lambda ZAP Express (Stratagene) vector with genomic DNA from P. acnes ATCC#6919, essentially as described in Example 4.
  • the library was screened with a pool of 5 sera from individuals with history of disease (acne) to identify immunoreactive clones recognized by the IgA subclass.
  • 80k plaque forming units (pfu) of the unamplified genomic PA6919 expression library was screened with the pooled adsorbed human sera (from donors D65, D328, D339, D349, D398) diluted 1/25.
  • Plaque lifts were incubated O/N at 4°C in sera, washed then subsequently incubated in AP conjugated affinipure F(ab')2 Fragment Goat anti-human serum IgA, alpha chain specific (Jackson Immunoresearch) diluted 1/1000. Positive plaques were visualized using BCIP/NBT (Gibco/BRL). Pure clones were isolated following secondary and tertiary screening. This resulted in the purification of 29 independent clones identified as hPalgA (for human P. acnes IgA clones). The clones were sequenced in both directions (5 prime and 3 prime) and searched against the P. acnes genomic sequences identified in the above examples.
  • a random shear genomic expression library was constructed in the Lambda ZAP Express (Stratagene) vector with genomic DNA from P. acnes ATCC isolate 6919.
  • the library was screened with a rabbit polyclonal sera generated against P. acnes lysate. 80k pfu of the unamplified genomic PA6919 expression library was screened. Positive plaques were visualized using BCIP/NBT (Gibco/BRL). Pure clones were isolated following secondary and tertiary screening. This resulted in the purification of independent clones identified as rPA (for rabbit P. acnes clones). The clones were sequenced in both directions (5 prime and 3 prime) and searched against the P. acnes genomic sequence described in the above examples.
  • proteins from lysed, cultured P. acnes bacteria or from the secreted culture supernatant were run out by SDS-PAGE. Gels were electroblotted onto PVDF membrane and stained with Coomassie Brilliant Blue. The resulting bands were cut with a clean razor blade and sequenced by Edman degradation. Sequences were then searched against a database of P. acnes ATCC#6919 protein sequences described above and full length amino acid sequences were determined. Using this approach, P.
  • acnes proteins were identified corresponding to a CAMP-like factor (SEQ ID NOs: 15127), a GroES-like protein (SEQ ID NO: 6923), Paf21b60 (SEQ ID NO: 25103), Paf21b25 (SEQ ID NO: 24679), a secreted ymga protein (SEQ ID NO: 2373), a secreted bacillopepfidase protein (SEQ ID NO: 12764) and a secreted GroES protein (SEQ ID NO: 6923).
  • Two random shear genomic expression libraries were constructed in the Lambda ZAP Express (Stratagene) vector with genomic DNA from P. acnes clinical isolates PA-1 and PA-2, essentially as described in Example 4. These libraries were screened with either a pool of 5 sera from individuals with no evidence of inflammatory acne (BBF clones) or with a pool of sera from individuals symptomatic for inflammatory acne (INF clones). Following these screens, sequencing of the PA1 and PA2 reactive inserts resulted in the identification of 5' and 3' sequences. Searching these insert sequences against the database of genomic DNA from P. acnes ATCC#6919 (e.g., Example 1) allowed for the identification of the corresponding gene(s) and the encoded immunoreactive protein(s).
  • Table 12 contains the following information: clone names, insert sizes in base pairs, the homologous ATCC#6919 contigs and nucleotide positions corresponding to the cloned insert sequences, percent identity of the cloned PA1 and PA2 insert sequences with the corresponding ATCC#6919 sequences, the SEQ ID NOs: of the ATCC#6919 open reading frames that are encoded by the cloned insert sequences, and the predicted cellular location of the encoded proteins using psort analysis.
  • the table also conatins information on the number of times a clone was hit during a serological screen with either a BBF sera pool from asymptomatic donors, a sera pool from symptomatic donors (e.g. INFxn, CJ INFxn), or sera from rabbit anti- whole P. acnes (CJ rabbit).
  • a BBF sera pool from asymptomatic donors e.g. INFxn, CJ INFxn
  • CJ rabbit rabbit anti- whole P. acnes
  • SEQ ID NOs: 4687 a secreted or membrane protein
  • 21693 a membrane protein
  • HtaA hemin transport- associated protein A
  • these represent attractive prophylactic and/or immunotherapeutic targets (e.g., Schmitt, M.P. and Drazek, E. 2001. Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species. J. Bacteriol. 183:1476-1481).
  • these proteins were hit multiple times in the serological expression cloning screen. Approximately 32% of all BBF clones identified encoded one of these two proteins, suggesting that the proteins are highly immunogenic.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions et des méthodes destinées au traitement et au diagnostic de l'acné vulgaire et d'autres affections associées. Ces compositions peuvent comprendre une ou plusieurs protéines de Propionibacterium acnes, des parties immunogènes correspondantes, ou des polynucléotides codant pour ces parties. Dans un autre mode de réalisation, une composition thérapeutique peut comprendre un anticorps se liant à une protéine de Propionibacterium acnes, une cellule présentatrice d'antigène exprimant une protéine de Propionibacterium acnes, ou un lymphocyte T spécifique pour les cellules exprimant cette protéine. Lesdites compositions peuvent être utilisées, par exemple, dans la prévention et/ou le traitement de l'acné.
PCT/US2002/032727 2001-10-15 2002-10-11 Compositions et methodes pour le traitement et le diagnostic de l'acne vulgaire WO2003033515A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US97882501A 2001-10-15 2001-10-15
US09/978,825 2001-10-15

Publications (1)

Publication Number Publication Date
WO2003033515A1 true WO2003033515A1 (fr) 2003-04-24

Family

ID=25526424

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/032727 WO2003033515A1 (fr) 2001-10-15 2002-10-11 Compositions et methodes pour le traitement et le diagnostic de l'acne vulgaire

Country Status (1)

Country Link
WO (1) WO2003033515A1 (fr)

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047313A1 (fr) * 2003-11-14 2005-05-26 Peptide Door Co., Ltd. Peptide ou proteine apte a fixer le lipide a et le lps, adn codant ledit peptide ou ladite proteine et utilisation dudit peptide ou de ladite proteine, adsorbant de lps ou agent de neutralisation de poison de lps
WO2005058944A2 (fr) * 2003-12-12 2005-06-30 The Government Of The United States, As Represented By The Secretary Of The Department Of Health & Human Services Peptides immunogenes de xage-1
WO2006046072A3 (fr) * 2004-10-28 2007-01-25 Ares Trading Sa Proteines renfermant vwfa et/ou le domaine ant_ig
EP1725662A4 (fr) * 2004-03-15 2008-03-05 Tissugen Pty Ltd Etiologie infectieuse de maladie prostatique et procede d'identification d'agents etiologiques
WO2010138769A1 (fr) * 2009-05-28 2010-12-02 The United States Government As Represented By The Department Of Veterans Affairs Séquences d'acides aminés qui améliorent la solubilité de conjugués peptidiques
US7973135B2 (en) 2006-07-12 2011-07-05 Oncotx, Inc. Compositions and methods for targeting cancer-specific transcription complexes
US8088890B2 (en) * 2008-09-26 2012-01-03 Fibrex Medical Research & Development Gmbh Peptides and peptidomimetic compounds, the manufacturing thereof as well as their use for preparing a therapeutically and/or preventively active pharmaceutical composition
US20130123132A1 (en) * 2011-11-15 2013-05-16 Ingen Biosciences Method for Diagnosing Propionibacterium Bacterial Infections
WO2013142378A1 (fr) 2012-03-17 2013-09-26 The Regents Of The University Of California Diagnostic rapide et traitements personnalisés de l'acné
US20130289238A1 (en) * 2012-04-26 2013-10-31 Bristol-Myers Squibb Company Par4 agonist peptides
US20140271670A1 (en) * 2013-03-15 2014-09-18 University Medical Pharmaceuticals Compositions for treating skin disorders
US20160022831A1 (en) * 2004-09-07 2016-01-28 Sanford-Burnham Medical Research Institute Peptides that Selectively Home to Heart Vasculature and Related Conjugates and Methods
WO2016138245A3 (fr) * 2015-02-27 2016-10-20 The Regents Of The University Of Michigan Réactifs peptidiques de la claudine 1 et méthodes associées
US9546198B2 (en) 2007-10-12 2017-01-17 Cancer Research Technology Limited Cyclic peptides as ADAM protease inhibitors
US9889165B2 (en) 2016-04-21 2018-02-13 Naked Biome, Inc. Compositions and methods for treatment of skin disorders
CN109456390A (zh) * 2018-12-27 2019-03-12 西华师范大学 一种人工合成多肽h-473及其应用
US10584344B2 (en) 2014-06-17 2020-03-10 Crown Laboratories, Inc. Genetically modified bacteria and methods for genetic modification of bacteria
US10745447B2 (en) 2015-09-28 2020-08-18 The University Of North Carolina At Chapel Hill Methods and compositions for antibody-evading virus vectors
WO2021165543A1 (fr) 2020-02-21 2021-08-26 Origimm Biotechnology Gmbh Traitement immunitaire prophylactique et thérapeutique de propionibacterium acnes
US11504404B2 (en) 2016-02-24 2022-11-22 Crown Laboratories, Inc. Skin probiotic formulation
EP3904368A4 (fr) * 2018-12-26 2023-02-22 Skinmed Co., Ltd. Peptides inhibiteurs du récepteur de l'acétylcholine et leurs utilisations
WO2023225234A1 (fr) * 2022-05-18 2023-11-23 Matrubials Inc. Peptides dérivés de lait de mammifère à activité antimicrobienne
US11905523B2 (en) 2019-10-17 2024-02-20 Ginkgo Bioworks, Inc. Adeno-associated viral vectors for treatment of Niemann-Pick Disease type-C
US11976096B2 (en) 2018-04-03 2024-05-07 Ginkgo Bioworks, Inc. Antibody-evading virus vectors
US11981914B2 (en) 2019-03-21 2024-05-14 Ginkgo Bioworks, Inc. Recombinant adeno-associated virus vectors
US12060390B2 (en) 2018-04-03 2024-08-13 Ginkgo Bioworks, Inc. Antibody-evading virus vectors
WO2024188165A1 (fr) * 2023-03-10 2024-09-19 成都威斯克生物医药有限公司 Vaccin à base de protéine recombinante anti-acné, procédé de préparation et utilisation
US12104163B2 (en) 2020-08-19 2024-10-01 Sarepta Therapeutics, Inc. Adeno-associated virus vectors for treatment of Rett syndrome
US12102710B2 (en) 2020-06-23 2024-10-01 Crown Laboratories, Inc. Probiotic skin formulations
US12116384B2 (en) 2018-04-03 2024-10-15 Ginkgo Bioworks, Inc. Virus vectors for targeting ophthalmic tissues

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5574007A (en) * 1990-08-03 1996-11-12 Asahi Kasei Kogyo Kabushiki Kaisha Polypeptide capable of interacting with thrombin
US5858750A (en) * 1997-11-25 1999-01-12 Incyte Pharmaceuticals, Inc. Human retinol dehydrogenase type II homolog
US5994081A (en) * 1998-04-27 1999-11-30 Incyte Pharamaceuticals, Inc. Human keratins
US6057108A (en) * 1998-06-23 2000-05-02 Incyte Pharmaceuticals, Inc. Human ARF-related proteins
US6489152B1 (en) * 2000-02-29 2002-12-03 Millennium Pharmaceuticals, Inc. 32670, novel human phosphatidylserine synthase-like molecules and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5574007A (en) * 1990-08-03 1996-11-12 Asahi Kasei Kogyo Kabushiki Kaisha Polypeptide capable of interacting with thrombin
US5858750A (en) * 1997-11-25 1999-01-12 Incyte Pharmaceuticals, Inc. Human retinol dehydrogenase type II homolog
US5994081A (en) * 1998-04-27 1999-11-30 Incyte Pharamaceuticals, Inc. Human keratins
US6057108A (en) * 1998-06-23 2000-05-02 Incyte Pharmaceuticals, Inc. Human ARF-related proteins
US6489152B1 (en) * 2000-02-29 2002-12-03 Millennium Pharmaceuticals, Inc. 32670, novel human phosphatidylserine synthase-like molecules and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BENTLEY ET AL.: "Complete genome sequence of the model actinomycete streptomyces coelicolor A3(2)", NATURE, vol. 417, May 2002 (2002-05-01), pages 141 - 147, XP002233530 *
FERRETTI ET AL.: "Complete genome sequence of an M1 strain of streptococcus pyogenes", PROC. NATL. ACAD. SCI. USA, vol. 98, no. 8, April 2001 (2001-04-01), pages 4658 - 4663, XP002168716 *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047313A1 (fr) * 2003-11-14 2005-05-26 Peptide Door Co., Ltd. Peptide ou proteine apte a fixer le lipide a et le lps, adn codant ledit peptide ou ladite proteine et utilisation dudit peptide ou de ladite proteine, adsorbant de lps ou agent de neutralisation de poison de lps
WO2005058944A2 (fr) * 2003-12-12 2005-06-30 The Government Of The United States, As Represented By The Secretary Of The Department Of Health & Human Services Peptides immunogenes de xage-1
WO2005058944A3 (fr) * 2003-12-12 2006-01-05 Us Gov Health & Human Serv Peptides immunogenes de xage-1
EP1725662A4 (fr) * 2004-03-15 2008-03-05 Tissugen Pty Ltd Etiologie infectieuse de maladie prostatique et procede d'identification d'agents etiologiques
US20160022831A1 (en) * 2004-09-07 2016-01-28 Sanford-Burnham Medical Research Institute Peptides that Selectively Home to Heart Vasculature and Related Conjugates and Methods
US8222371B2 (en) 2004-10-28 2012-07-17 Ares Trading S.A. vWFA and/or ANT—IG domain containing proteins
WO2006046072A3 (fr) * 2004-10-28 2007-01-25 Ares Trading Sa Proteines renfermant vwfa et/ou le domaine ant_ig
EA013251B1 (ru) * 2004-10-28 2010-04-30 Арес Трейдинг С.А. БЕЛКИ, СОДЕРЖАЩИЕ ДОМЕНЫ vWFA И/ИЛИ ANT_IG
US7973135B2 (en) 2006-07-12 2011-07-05 Oncotx, Inc. Compositions and methods for targeting cancer-specific transcription complexes
US10472393B2 (en) 2007-10-12 2019-11-12 Cancer Research Technology Limited Method for inhibiting ADAM proteases with cyclic peptides
US9546198B2 (en) 2007-10-12 2017-01-17 Cancer Research Technology Limited Cyclic peptides as ADAM protease inhibitors
EP2197898B1 (fr) * 2007-10-12 2018-05-30 Cancer Research Technology Limited Inhibition de la protéase
US8088890B2 (en) * 2008-09-26 2012-01-03 Fibrex Medical Research & Development Gmbh Peptides and peptidomimetic compounds, the manufacturing thereof as well as their use for preparing a therapeutically and/or preventively active pharmaceutical composition
WO2010138769A1 (fr) * 2009-05-28 2010-12-02 The United States Government As Represented By The Department Of Veterans Affairs Séquences d'acides aminés qui améliorent la solubilité de conjugués peptidiques
US9155801B2 (en) 2009-05-28 2015-10-13 The United States Government As Represented By The Department Of Veterans Affairs Amino acid sequences which enhance peptide conjugate solubility
US20130123132A1 (en) * 2011-11-15 2013-05-16 Ingen Biosciences Method for Diagnosing Propionibacterium Bacterial Infections
EP2594580A1 (fr) * 2011-11-15 2013-05-22 InGen Biosciences Procédé pour le diagnostic des infections bactériennes de propionibacterium
US9528997B2 (en) * 2011-11-15 2016-12-27 Diaxonhit Method for diagnosing Propionibacterium bacterial infections
EP3002292A1 (fr) * 2011-11-15 2016-04-06 Diaxonhit Procédé pour le diagnostic des infections bactériennes de propionibacterium
JP2021013395A (ja) * 2012-03-17 2021-02-12 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ざ瘡の高速診断および個人化された治療
JP2015512255A (ja) * 2012-03-17 2015-04-27 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ざ瘡の高速診断および個人化された治療
CN104364394A (zh) * 2012-03-17 2015-02-18 加州大学评议会 痤疮的快速诊断和个体化治疗
US10364473B2 (en) 2012-03-17 2019-07-30 The Regents Of The University Of California Fast diagnosis and personalized treatments for acne
EP2825676A1 (fr) * 2012-03-17 2015-01-21 The Regents of The University of California Diagnostic rapide et traitements personnalisés de l'acné
JP2019050833A (ja) * 2012-03-17 2019-04-04 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ざ瘡の高速診断および個人化された治療
EP3692999A1 (fr) * 2012-03-17 2020-08-12 The Regents of the University of California Diagnostic et traitements personnalisés rapides de l'acné
US11692229B2 (en) 2012-03-17 2023-07-04 The Regents Of The University Of California Fast diagnosis and personalized treatments for acne
WO2013142378A1 (fr) 2012-03-17 2013-09-26 The Regents Of The University Of California Diagnostic rapide et traitements personnalisés de l'acné
EP2825676B1 (fr) * 2012-03-17 2017-12-20 The Regents of The University of California Diagnostic rapide et traitements personnalisés de l'acné
US9605024B2 (en) 2012-04-26 2017-03-28 Bristol-Myers Squibb Company PAR4 agonist peptides
US20130289238A1 (en) * 2012-04-26 2013-10-31 Bristol-Myers Squibb Company Par4 agonist peptides
US9303065B2 (en) 2012-04-26 2016-04-05 Bristol-Myers Squibb Company PAR4 agonist peptides
JP2015516983A (ja) * 2012-04-26 2015-06-18 ブリストル−マイヤーズ スクイブ カンパニーＢｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ Ｃｏｍｐａｎｙ Ｐａｒ４アゴニストペプチド
US8927688B2 (en) * 2012-04-26 2015-01-06 Bristol-Myers Squibb Company PAR4 agonist peptides
US20140271670A1 (en) * 2013-03-15 2014-09-18 University Medical Pharmaceuticals Compositions for treating skin disorders
US12049633B2 (en) 2014-06-17 2024-07-30 Crown Laboratories, Inc. Genetically modified bacteria and methods for genetic modification of bacteria
US10584344B2 (en) 2014-06-17 2020-03-10 Crown Laboratories, Inc. Genetically modified bacteria and methods for genetic modification of bacteria
WO2016138245A3 (fr) * 2015-02-27 2016-10-20 The Regents Of The University Of Michigan Réactifs peptidiques de la claudine 1 et méthodes associées
US10746738B2 (en) 2015-02-27 2020-08-18 The Regents Of The University Of Michigan Claudin-1 peptide reagents and methods
US11208438B2 (en) 2015-09-28 2021-12-28 The University Of North Carolina At Chapel Hill Methods and compositions for antibody-evading virus vectors
US10745447B2 (en) 2015-09-28 2020-08-18 The University Of North Carolina At Chapel Hill Methods and compositions for antibody-evading virus vectors
US11504404B2 (en) 2016-02-24 2022-11-22 Crown Laboratories, Inc. Skin probiotic formulation
US11207357B2 (en) 2016-04-21 2021-12-28 Symbiome, Inc. Compositions and methods for treatment of skin disorders
US9889165B2 (en) 2016-04-21 2018-02-13 Naked Biome, Inc. Compositions and methods for treatment of skin disorders
US10293007B2 (en) 2016-04-21 2019-05-21 Naked Biome, Inc. Compositions and methods for treatment of skin disorders
US11976096B2 (en) 2018-04-03 2024-05-07 Ginkgo Bioworks, Inc. Antibody-evading virus vectors
US12116384B2 (en) 2018-04-03 2024-10-15 Ginkgo Bioworks, Inc. Virus vectors for targeting ophthalmic tissues
US12091435B2 (en) 2018-04-03 2024-09-17 Ginkgo Bioworks, Inc. Antibody-evading virus vectors
US12060390B2 (en) 2018-04-03 2024-08-13 Ginkgo Bioworks, Inc. Antibody-evading virus vectors
EP3904368A4 (fr) * 2018-12-26 2023-02-22 Skinmed Co., Ltd. Peptides inhibiteurs du récepteur de l'acétylcholine et leurs utilisations
CN109456390A (zh) * 2018-12-27 2019-03-12 西华师范大学 一种人工合成多肽h-473及其应用
CN109456390B (zh) * 2018-12-27 2021-11-16 西华师范大学 一种人工合成多肽h-473及其应用
US11981914B2 (en) 2019-03-21 2024-05-14 Ginkgo Bioworks, Inc. Recombinant adeno-associated virus vectors
US11905523B2 (en) 2019-10-17 2024-02-20 Ginkgo Bioworks, Inc. Adeno-associated viral vectors for treatment of Niemann-Pick Disease type-C
WO2021165543A1 (fr) 2020-02-21 2021-08-26 Origimm Biotechnology Gmbh Traitement immunitaire prophylactique et thérapeutique de propionibacterium acnes
US12102710B2 (en) 2020-06-23 2024-10-01 Crown Laboratories, Inc. Probiotic skin formulations
US12104163B2 (en) 2020-08-19 2024-10-01 Sarepta Therapeutics, Inc. Adeno-associated virus vectors for treatment of Rett syndrome
WO2023225234A1 (fr) * 2022-05-18 2023-11-23 Matrubials Inc. Peptides dérivés de lait de mammifère à activité antimicrobienne
WO2024188165A1 (fr) * 2023-03-10 2024-09-19 成都威斯克生物医药有限公司 Vaccin à base de protéine recombinante anti-acné, procédé de préparation et utilisation

Similar Documents

Publication Publication Date Title
WO2003033515A1 (fr) Compositions et methodes pour le traitement et le diagnostic de l'acne vulgaire
CA2407352A1 (fr) Compositions et procedes pour la therapie et le diagnostic de l'acne vulgaire
US6630574B1 (en) Compositions and methods for the therapy and diagnosis of lung cancer
JP5053497B2 (ja) 乳癌の治療および診断のための組成物および方法
WO2001040269A2 (fr) Compositions et methodes destinees au traitement et au diagnostic du cancer du sein
EP1297147B1 (fr) Compositions pour le diagnostic et le traitement de l'infection du virus de l'herpes
US6746846B1 (en) Methods for diagnosing lung cancer
US6844325B2 (en) Compositions for the treatment and diagnosis of breast cancer and methods for their use
AU2001273128A1 (en) Compositions and methods for the diagnosis and treatment of herpes simplex virus infection
US20020022591A1 (en) Compositions and methods for the therapy and diagnosis of ovarian cancer
WO2000078960A2 (fr) Compositions et methodes destinees au traitement et au diagnostic du cancer du sein
US6667154B1 (en) Compositions and methods for the therapy and diagnosis of lung cancer
WO2001070976A2 (fr) Compositions et procedes de therapie et de diagnostic du cancer des ovaires et de l'endometre
WO2001073031A2 (fr) Compositions et methodes destinees au traitement et au diagnostic du cancer de l'ovaire
WO2002002623A2 (fr) Compositions et methodes de diagnostic et de traitement du cancer des poumons
US6686447B1 (en) Compositions and methods for the therapy and diagnosis of lung cancer
US6914132B1 (en) Compositions and methods for the therapy and diagnosis of lung cancer
WO2003020108A2 (fr) Compositions et procedes destines a diagnostiquer et a traiter une infection par le virus de l'herpes simplex
WO2002012332A2 (fr) Compositions et procedes pour la therapie et le diagnostic du cancer du colon
US20020111308A1 (en) Compositions and methods for the therapy and diagnosis of pancreatic cancer
WO2001090154A2 (fr) Compositions et procedes therapeutiques et diagnostiques du cancer ovarien
US20030008299A1 (en) Compositions and methods for the therapy and diagnosis of ovarian cancer
US20020137911A1 (en) Compositions and methods for the therapy and diagnosis of pancreatic cancer
WO2002012329A2 (fr) Compositions et procedes pour la therapie et le diagnostic du cancer de la tete et du cou
KR20030008369A (ko) 유방암의 치료 및 진단용 조성물 및 방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ PL PT RO RU SE SG SI SK SL TJ TM TR TT TZ UA US UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP