EP0977482A1 - IDENTIFICATION VON POLYNUKLEOTIDEN, DIE NEUE $i(HELIOBACTER) POLYPEPTIDE IM $i(HELIOBACTER) GENOM KODIEREN - Google Patents

IDENTIFICATION VON POLYNUKLEOTIDEN, DIE NEUE $i(HELIOBACTER) POLYPEPTIDE IM $i(HELIOBACTER) GENOM KODIEREN

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Publication number
EP0977482A1
EP0977482A1 EP98917972A EP98917972A EP0977482A1 EP 0977482 A1 EP0977482 A1 EP 0977482A1 EP 98917972 A EP98917972 A EP 98917972A EP 98917972 A EP98917972 A EP 98917972A EP 0977482 A1 EP0977482 A1 EP 0977482A1
Authority
EP
European Patent Office
Prior art keywords
seq
ghpo
gly
leu
asn
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP98917972A
Other languages
English (en)
French (fr)
Other versions
EP0977482A4 (de
Inventor
Harold Kleanthous
Amal Al-Garawi
Charles Miller
Jean-François TOMB
Raymond Peter Oomen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merieux Oravax
Human Genome Sciences Inc
Original Assignee
Merieux Oravax
Human Genome Sciences Inc
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Publication date
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Publication of EP0977482A1 publication Critical patent/EP0977482A1/de
Publication of EP0977482A4 publication Critical patent/EP0977482A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Gram-negative bacteria
    • C07K16/121—Helicobacter (G); Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to Helicobacter antigens and corresponding polynucleotide molecules that can be used in methods to prevent or treat Helicobacter infection in mammals, such as humans.
  • Helicobacter is a genus of spiral, gram-negative bacteria that colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably H. pylori, H. heilmanii, H. felis, and H. mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been isolated from humans, but at lower frequencies than H. pylori. Helicobacter infects over 50% of adult populations in developed countries and nearly 100% in developing countries and some Pacific rim countries, making it one ofthe most prevalent infections worldwide. Helicobacter is routinely recovered from gastric biopsies of humans with histological evidence of gastritis and peptic ulceration. Indeed, H.
  • pylori is now recognized as an important pathogen of humans, in that the chronic gastritis it causes is a risk factor for the development of peptic ulcer diseases and gastric carcinoma. It is thus highly desirable to develop safe and effective vaccines for preventing and treating Helicobacter infection.
  • Helicobacter antigens have been characterized or isolated. These include urease, which is composed of two structural subunits of approximately 30 and 67 kDa (Hu et al, Infect. Immun. 58:992, 1990; Dunn et al, J. Biol. Chem. 265:9464, 1990; Evans et al, Microbial Pathogenesis 10:15, 1991; Labigne et al, J. Bact, 173: 1920, 1991); the 87 kDa vacuolar cytotoxin (VacA) (Cover et al, J. Biol. Chem. 267:10570, 1992; Phadnis et al, Infect. Immun.
  • urease which is composed of two structural subunits of approximately 30 and 67 kDa (Hu et al, Infect. Immun. 58:992, 1990; Dunn et al, J. Biol. Chem. 265:9464, 1990; Evans et al, Microbial Pathogenesis 10:15,
  • the invention provides polynucleotide molecules that encode Helicobacter polypeptides, designated GHPO 35 (SEQ ID NO:2), GHPO 55 (SEQ ID NO:4), GHPO 78 (SEQ ID NO:6), GHPO 89 (SEQ ID NO: 8), GHPO 129 (SEQ ID NO: 10), GHPO 541 (SEQ ID NO: 12), GHPO 607 (SEQ ID NO:14), GHPO 635 (SEQ ID NO:16), GHPO 701 (SEQ ID NO:18), GHPO
  • GHPO 237 (SEQ ID NO: 130), GHPO 290 (SEQ ID NO: 132), GHPO 293 (SEQ ID NO:134), GHPO 335 (SEQ ID NO:136), GHPO 374 (SEQ ID NO: 138), GHPO 442 (SEQ ID NO:140), GHPO 480 (SEQ ID NO: 142), GHPO 523 (SEQ ID NO: 144), GHPO 610 (SEQ ID NO:146), GHPO 675 (SEQ ID NO: 148), GHPO 690 (SEQ ID NO: 150), GHPO 829 (SEQ ID NO: 152), GHPO
  • GHPO 1084 (SEQ ID NO:236), GHPO 1329 (SEQ ID NO:238), GHPO 1330 (SEQ ID NO:240), GHPO 1346 (SEQ ID NO:242), GHPO 1360 (SEQ ID NO:244), GHPO 1388 (SEQ ID NO:246), GHPO 1411 (SEQ ID NO:248), GHPO 1419 (SEQ ID NO:250), GHPO 1446 (SEQ ID NO:252), GHPO 1469 (SEQ ID NO:254), GHPO 1501 (SEQ ID NO:256), GHPO 1505 (SEQ ID NO:
  • GHPO 1522 (SEQ ID NO:260), GHPO 1525 (SEQ ID NO:262), GHPO 1615 (SEQ ID NO:264), GHPO 1689 (SEQ ID NO:266), GHPO 1733 (SEQ ID NO:268), GHPO 18 (SEQ ID NO:270), GHPO 139 (SEQ ID NO:272), GHPO 142 (SEQ ID NO:274), GHPO 250 (SEQ ID NO:276), GHPO 257 (SEQ ID NO:278), GHPO 325 (SEQ ID NO:280), GHPO 355 (SEQ ID NO:260), GHPO 1525 (SEQ ID NO:262), GHPO 1615 (SEQ ID NO:264), GHPO 1689 (SEQ ID NO:266), GHPO 1733 (SEQ ID NO:268), GHPO 18 (SEQ ID NO:270), GHPO 139 (SEQ ID NO:272), GHPO 142 (SEQ ID NO:27
  • GHPO 1272 (SEQ ID NO:318), GHPO 1345 (SEQ ID NO:320), GHPO 1377 (SEQ ID NO:322), GHPO 1424 (SEQ ID NO:324), GHPO 1430 (SEQ ID NO:326), GHPO 1502 (SEQ ID NO:328), GHPO 1600 (SEQ ID NO:330), GHPO 1714 (SEQ ID NO:332), GHPO 359 (SEQ ID NO:334), GHPO 678 (SEQ ID NO:336), GHPO 708 (SEQ ID NO:338), GHPO 759 (SEQ ID NO:
  • GHPO 847 (SEQ ID NO:342), GHPO 1050 (SEQ ID NO:344), GHPO 1101 (SEQ ID NO:346), GHPO 1120 (SEQ ID NO:348), GHPO 1138 (SEQ ID NO:350), GHPO 1310 (SEQ ID NO:352), GHPO 1320 (SEQ ID NO:354), GHPO 1375 (SEQ ID NO:356), GHPO 1432 (SEQ ID NO:358), GHPO 21 (SEQ ID NO:360), GHPO 282 (SEQ ID NO:362), GHPO 1089
  • GHPO 86 (SEQ ID NO:390), GHPO 99 (SEQ ID NO:392), GHPO 106 (SEQ ID NO:394), GHPO 118 (SEQ ID NO:396), GHPO 122 (SEQ ID NO:398), GHPO 128 (SEQ ID NO:400), GHPO 138 (SEQ ID NO:402), GHPO 153 (SEQ ID NO:404), GHPO 160 (SEQ ID NO:406), GHPO 168 (SEQ ID NO:408), GHPO 179 (SEQ ID NO:410), GHPO 189 (SEQ ID NO:412), GHPO
  • GHPO 382 (SEQ ID NO:450), GHPO 384 (SEQ ID NO:452), GHPO 398 (SEQ ID NO:454), GHPO 409 (SEQ ID NO:456), GHPO 422 (SEQ ID NO:458), GHPO 430 (SEQ ID NO:460), GHPO 446 (SEQ ID NO:462), GHPO 447 (SEQ ID NO:464), GHPO 450 (SEQ ID NO:466), GHPO 451 (SEQ ID NO:468), GHPO 452 (SEQ ID NO:470), GHPO 456 (SEQ ID NO:472), GHPO
  • GHPO 580 (SEQ ID NO:500), GHPO 585 (SEQ ID NO:502), GHPO 599 (SEQ ID NO:504), GHPO 639 (SEQ ID NO:506), GHPO 642 (SEQ ID NO:508), GHPO 647 (SEQ ID NO:510), GHPO 654 (SEQ ID NO:512), GHPO 669 (SEQ ID NO:514), GHPO 710 (SEQ ID NO:516), GHPO 713 (SEQ ID NO:518), GHPO 716 (SEQ ID NO:520), GHPO 718 (SEQ ID NO:522), GHPO
  • GHPO 1301 (SEQ ID NO:630), GHPO 1304 (SEQ ID NO:632), GHPO 1315 (SEQ ID NO:634), GHPO 1319 (SEQ ID NO:636), GHPO 1323 (SEQ ID NO:638), GHPO 1331 (SEQ ID NO:640), GHPO 1332 (SEQ ID NO:642), GHPO 1347 (SEQ ID NO:644), GHPO 1373 (SEQ ID NO:646), GHPO 1376 (SEQ ID NO:648), GHPO 1380 (SEQ ID NO:650), GHPO 1394 (SEQ ID NO:630), GHPO 1304 (SEQ ID NO:632), GHPO 1315 (SEQ ID NO:634), GHPO 1319 (SEQ ID NO:636), GHPO 1323 (SEQ ID NO:638), GHPO 1331 (SEQ ID NO:640), GHPO 1332 (SEQ ID NO:642), GHPO 1347 (S
  • GHPO 846 (SEQ ID NO: 1050), GHPO 875 (SEQ ID NO: 1052), GHPO 892 (SEQ ID NO: 1054), GHPO 902 (SEQ ID NO: 1056), GHPO 904 (SEQ ID NO: 1058), GHPO 906 (SEQ ID NO: 1060), GHPO 908 (SEQ ID NO: 1062), GHPO 921 (SEQ ID NO: 1064), GHPO 923 (SEQ ID NO: 1066), GHPO 926 (SEQ ID NO: 1068), GHPO 933 (SEQ ID NO: 1070), GHPO 939 (SEQ ID NO: 1050), GHPO 875 (SEQ ID NO: 1052), GHPO 892 (SEQ ID NO: 1054), GHPO 902 (SEQ ID NO: 1056), GHPO 904 (SEQ ID NO: 1058), GHPO 906 (SEQ ID NO: 1060), GHPO 908 (SEQ ID NO: 1062), GH
  • GHPO 1103 (SEQ ID NO: 1128), GHPO 1113 (SEQ ID NO:l 130), GHPO 1116 (SEQ ID NO: 1132), GHPO 1123 (SEQ ID NO: 1134), GHPO 1125 (SEQ ID NO:1136), GHPO 1129 (SEQ ID NO:1138), GHPO 1130 (SEQ ID NO:l 140), GHPO 1134 (SEQ ID NO: 1142), GHPO 1161 (SEQ ID NO: 1144), GHPO 1166 (SEQ ID NO: 1146), GHPO 1170 (SEQ ID NO: 1148), GHPO
  • GHPO 1460 (SEQ ID NO:1226), GHPO 1381 (SEQ ID NO:1228), GHPO 1401 (SEQ ID NO: 1230), GHPO 1402 (SEQ ID NO: 1232), GHPO 1403 (SEQ ID NO: 1234), GHPO 1408 (SEQ ID NO: 1236), GHPO 1416 (SEQ ID NO:1238), GHPO 1420 (SEQ ID NO: 1240), GHPO 1428 (SEQ ID NO: 1242), GHPO 1437 (SEQ ID NO: 1244), GHPO 1439 (SEQ ID NO: 1246), GHPO 1460 (SEQ ID NO:1226), GHPO 1381 (SEQ ID NO:1228), GHPO 1401 (SEQ ID NO: 1230), GHPO 1402 (SEQ ID NO: 1232), GHPO 1403 (SEQ ID NO: 1234), GHPO 1408 (SEQ ID NO: 1236), GHPO 1416 (SEQ ID NO:1238), GH
  • GHPO 1504 (SEQ ID NO:1272), GHPO 1510 (SEQ ID NO:1274), GHPO 1518 (SEQ ID NO: 1276), GHPO 1533 (SEQ ID NO: 1278), GHPO 1541 (SEQ ID NO: 1280), GHPO 1544 (SEQ ID NO: 1282), GHPO 1548 (SEQ ID NO:1284), GHPO 1565 (SEQ ID NO:1286), GHPO 1575 (SEQ ID NO:1288), GHPO 1582 (SEQ ID NO:1290), GHPO 1595 (SEQ ID NO:1292), GHPO
  • the invention includes the corresponding polypeptides (i.e., polypeptides encoded by the polynucleotide molecules ofthe invention, or fragments thereof), and monospecific antibodies that specifically bind to these polypeptides.
  • the polypeptides ofthe invention include those having the amino acid sequences shown in the sequence listing (even numbers, up to SEQ ID NO: 1363), as well as mature forms of proteins having sequences shown in the sequence listing in their unprocessed forms, and fragments thereof.
  • the present invention has many applications and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention.
  • the present invention provides (i) methods for producing polypeptides ofthe invention in recombinant host systems and related expression cassettes, vectors, and transformed or transfected cells; (ii) live vaccine vectors, such as pox virus, Salmonella typhimurium, and Vibrio cholerae vectors, that contain polynucleotides ofthe invention (such vaccine vectors being useful in, e.g., methods for preventing or treating Helicobacter infection) in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) therapeutic and/or prophylactic methods involving administration of polynucleotide molecules, either in a naked form or formulated with a delivery vehicle, polypeptides or mixtures of polypeptides, or monospecific antibodies ofthe invention, and related pharmaceutical compositions; (iv) methods for detecting the presence of Helicobacter in biological samples, which can involve the use of polynucleotide molecules, monospecific antibodies, or polypeptide
  • Open reading frames encoding new polypeptides, designated GHPO 35 (SEQ ID NO:2), GHPO 55 (SEQ ID NO:4), GHPO 78 (SEQ ID NO:6), GHPO 89 (SEQ ID NO:8), GHPO 129 (SEQ ID NO: 10), GHPO 541
  • GHPO 28 (SEQ ID NO: 122), GHPO 86 (SEQ ID NO: 124), GHPO 155 (SEQ ID NO: 126), GHPO 157 (SEQ ID NO: 128), GHPO 237 (SEQ ID NO: 130), GHPO 290 (SEQ ID NO: 132), GHPO 293 (SEQ ID NO: 134), GHPO 335 (SEQ ID NO: 136), GHPO 374 (SEQ ID NO: 138), GHPO 442 (SEQ ID NO: 140), GHPO 480 (SEQ ID NO: 142), GHPO 523 (SEQ ID NO: 144), GHPO
  • GHPO 208 (SEQ ID NO:204), GHPO 219 (SEQ ID NO:206), GHPO 445 (SEQ ID NO:208), GHPO 479 (SEQ ID NO:210), GHPO 525 (SEQ ID NO:212), GHPO 535 (SEQ ID NO:214), GHPO 731 (SEQ ID NO:216), GHPO 836 (SEQ ID NO:218), GHPO 879 (SEQ ID NO:220), GHPO 881 (SEQ ID NO:222), GHPO 886 (SEQ ID NO:224), GHPO 893 (SEQ ID NO:226), GHPO
  • GHPO 1446 (SEQ ID NO:252), GHPO 1469 (SEQ ID NO:254), GHPO 1501 (SEQ ID NO:256), GHPO 1505 (SEQ ID NO:258), GHPO 1522 (SEQ ID NO:260), GHPO 1525 (SEQ ID NO:262), GHPO 1615 (SEQ ID NO:264), GHPO 1689 (SEQ ID NO:266), GHPO 1733 (SEQ ID NO:268), GHPO 18 (SEQ ID NO:270), GHPO 139 (SEQ ID NO:272), GHPO 142 (SEQ ID NO:252), GHPO 142 (SEQ ID NO:
  • GHPO 250 (SEQ ID NO:276), GHPO 257 (SEQ ID NO:278), GHPO 325 (SEQ ID NO:280), GHPO 355 (SEQ ID NO:282), GHPO 357 (SEQ ID NO:284), GHPO 454 (SEQ ID NO:286), GHPO 475 (SEQ ID NO:288), GHPO 515 (SEQ ID NO:290), GHPO 527 (SEQ ID NO:292), GHPO 551 (SEQ ID NO:294), GHPO 602 (SEQ ID NO:296), GHPO 626 (SEQ ID NO:298), GHPO
  • GHPO 359 (SEQ ID NO:334), GHPO 678 (SEQ ID NO:336), GHPO 708 (SEQ ID NO:338), GHPO 759 (SEQ ID NO:340), GHPO 847 (SEQ ID NO:342), GHPO 1050 (SEQ ID NO:344), GHPO 1101 (SEQ ID NO:346), GHPO 1120 (SEQ ID NO:348), GHPO 1138 (SEQ ID NO:350), GHPO 1310 (SEQ ID NO:352), GHPO 1320 (SEQ ID NO:354), GHPO 1375 (SEQ ID NO:
  • GHPO 996 (SEQ ID NO:568), GHPO 997 (SEQ ID NO:570), GHPO 1002 (SEQ ID NO:572), GHPO 1026 (SEQ ID NO:574), GHPO 1028 (SEQ ID NO:576), GHPO 1034 (SEQ ID NO:578), GHPO 1038 (SEQ ID NO:580), GHPO 1059 (SEQ ID NO:582), GHPO 1065 (SEQ ID NO:584), GHPO 1072 (SEQ ID NO:586), GHPO 1073 (SEQ ID NO:588), GHPO 1088 (SEQ ID NO:590), GHPO 1091 (SEQ ID NO:592), GHPO 1105 (SEQ ID NO:594), GHPO 1115 (SEQ ID NO: 596), GHPO 1159 (SEQ ID NO:598), GHPO 1177
  • GHPO 1570 (SEQ ID NO:694), GHPO 1588 (SEQ ID NO:696), GHPO 1604 (SEQ ID NO:698), GHPO 1605 (SEQ ID NO:700), GHPO 1619 (SEQ ID NO:702), GHPO 1629 (SEQ ID NO:704), GHPO 1642 (SEQ ID NO:706), GHPO 1654 (SEQ ID NO:708), GHPO 1661 (SEQ ID NO:710), GHPO 1673 (SEQ ID NO:712), GHPO 1687 (SEQ ID NO:714), GHPO 1692 (SEQ ID NO:716), GHPO 1693 (SEQ ID NO:718), GHPO 1699 (SEQ ID NO:720), GHPO 1738 (SEQ ID NO:722), GHPO 1745 (SEQ ID NO:724), GHPO 1746
  • GHPO 30 (SEQ ID NO:752), GHPO 37 (SEQ ID NO:754), GHPO 49 (SEQ ID NO:756), GHPO 51 (SEQ ID NO:758), GHPO 54 (SEQ ID NO:760), GHPO 65 (SEQ ID NO:762), GHPO 66 (SEQ ID NO:764), GHPO 68 (SEQ ID NO:766), GHPO 70 (SEQ ID NO:768), GHPO 77 (SEQ ID NO:770), GHPO 83 (SEQ ID NO:772), GHPO 85 (SEQ ID NO:774), GHPO
  • GHPO 131 (SEQ ID NO:802), GHPO 133 (SEQ ID NO:804), GHPO 140 (SEQ ID NO:806), GHPO 141 (SEQ ID NO:808), GHPO 145 (SEQ ID NO:810), GHPO 147 (SEQ ID NO:812), GHPO 166 (SEQ ID NO:814), GHPO 181 (SEQ ID NO:816), GHPO 187 (SEQ ID NO:818), GHPO 188 (SEQ ID NO: 820), GHPO 192 (SEQ ID NO: 822), GHPO 202 (SEQ ID NO: 824), GHPO
  • GHPO 453 (SEQ ID NO:912), GHPO 455 (SEQ ID NO:914), GHPO 464 (SEQ ID NO:916), GHPO 467 (SEQ ID NO:918), GHPO 468 (SEQ ID NO:920), GHPO 470 (SEQ ID NO:922), GHPO 486 (SEQ ID NO:924), GHPO 487 (SEQ ID NO:926), GHPO 488 (SEQ ID NO:928), GHPO 489 (SEQ ID NO:930), GHPO 498 (SEQ ID NO:932), GHPO 501 (SEQ ID NO:934), GHPO
  • GHPO 763 (SEQ ID NO: 1020), GHPO 771 (SEQ ID NO: 1022), GHPO 774 (SEQ ID NO: 1024), GHPO 776 (SEQ ID NO: 1026), GHPO 783 (SEQ ID NO:1028), GHPO 800 (SEQ ID NO:1030), GHPO 806 (SEQ ID NO:1032), GHPO 807 (SEQ ID NO:1034), GHPO 808 (SEQ ID NO:1036), GHPO 809 (SEQ ID NO: 1038), GHPO 811 (SEQ ID NO: 1040), GHPO 815 (SEQ ID NO:
  • GHPO 1001 (SEQ ID NO: 1090), GHPO 1005 (SEQ ID NO: 1092), GHPO 1033 (SEQ ID NO: 1094), GHPO 1039 (SEQ ID NO: 1096), GHPO 1041 (SEQ ID NO: 1098), GHPO 1043 (SEQ ID NO: 1100), GHPO 1044 (SEQ ID NO: 1102), GHPO 1051 (SEQ ID NO: 1104), GHPO 1058 (SEQ ID NO: 1106), GHPO 1060 (SEQ ID NO:l 108), GHPO 1075 (SEQ ID NO: 1110), GHPO 1077 (SEQ ID NO:1112), GHPO 1082 (SEQ ID NO:1114), GHPO 1083 (SEQ ID NO:1116), GHPO 1086 (SEQ ID NO: 1118), GHPO 1087 (SEQ ID NO:
  • GHPO 1161 (SEQ ID NO: 1144), GHPO 1166 (SEQ ID NO: 1146), GHPO 1170 (SEQ ID NO: 1148), GHPO 1175 (SEQ ID NO: 1150), GHPO 1181 (SEQ ID NO: 1152), GHPO 1186 (SEQ ID NO: 1154), GHPO 1188 (SEQ ID NO: 1156), GHPO 1191 (SEQ ID NO:1158), GHPO 1193 (SEQ ID NO: 1160), GHPO 1196 (SEQ ID NO: 1162), GHPO 1204 (SEQ ID NO: 1164), GHPO
  • GHPO 1575 (SEQ ID NO:1288), GHPO 1582 (SEQ ID NO:1290), GHPO 1595 (SEQ ID NO:1292), GHPO 1597 (SEQ ID NO:1294), GHPO 1599 (SEQ ID NO:1296), GHPO 1601 (SEQ ID NO: 1298), GHPO 1609 (SEQ ID NO:1300), GHPO 1613 (SEQ ID NO: 1302), GHPO 1614 (SEQ ID NO: 1304), GHPO 1626 (SEQ ID NO: 1306), GHPO 1628 (SEQ ID NO: 1308), GHPO
  • GHPO 1695 (SEQ ID NO:1334), GHPO 1697 (SEQ ID NO:1336), GHPO 1701 (SEQ ID NO:1338), GHPO 1719 (SEQ ID NO:1340), GHPO 1723 (SEQ ID NO:1342), GHPO 1732 (SEQ ID NO:1344), GHPO 1739 (SEQ ID NO: 1346), GHPO 1741 (SEQ ID NO:1348), GHPO 1747 (SEQ ID NO:1350), GHPO 1749 (SEQ ID NO:1352), GHPO 1750 (SEQ ID NO: 1354), GHPO 1751 (SEQ ID NO:1356), GHPO 1755 (SEQ ID NO:1358), GHPO 1771 (SEQ ID NO:1360), GHPO 1786 (SEQ ID NO: 1362), and GHPO
  • GHPO 1275, GHPO 1308, GHPO 1600, GHPO 1615, GHPO 536, GHPO 66, GHPO 1363, GHPO 1595, and GHPO 1166 have been shown to be protective antigens that can be used in methods for preventing Helicobacter infection.
  • protective antigen is meant an antigen that is capable of reducing the infection level after challenge, relative to a positive control. Absolute protection from infection, although included in the invention, is not required.
  • Some ofthe new polypeptides are secreted polypeptides that can be produced in their mature forms (i.e., as polypeptides that have been exported through class II or class III secretion pathways) or as precursors that include signal peptides, which can be removed in the course of excretion/secretion by cleavage at the N-terminal end ofthe mature form. (The cleavage site is located at the C-terminal end ofthe signal peptide, adjacent to the mature form.)
  • GHPO proteins listed above. Examples of such polynucleotides are those encoding GHPO 35 (SEQ ID NO:l), GHPO 55 (SEQ ID NO:3), GHPO 78 (SEQ ID NO:5), GHPO 89 (SEQ ID NO:7), GHPO 129 (SEQ ID NO:9), GHPO 541 (SEQ ID NO: 11), GHPO 607 (SEQ ID NO: 13), GHPO 635 (SEQ ID NO: 15), GHPO 701 (SEQ ID NO: 17), GHPO 712 (SEQ ID NO:19), GHPO 761 (SEQ ID NO:21), GHPO 838 (SEQ ID NO:23), GHPO 1034 (SEQ ID NO:25), GHPO 1085 (SEQ ID NO:27), GHPO 1213 (SEQ ID NO:29), GHPO
  • GHPO 1490 (SEQ ID NO: 107), GHPO 1559 (SEQ ID NO: 109), GHPO 1651 (SEQ ID NO: 111), GHPO 1726 (SEQ ID NO: 113), GHPO 1780 (SEQ ID NO:l 15), GHPO 895 (SEQ ID NO:l 17), GHPO 1447 (SEQ ID NO:l 19), GHPO 28 (SEQ ID NO:121), GHPO 86 (SEQ ID NO:123), GHPO 155 (SEQ ID NO: 125), GHPO 157 (SEQ ID NO: 127), GHPO 237 (SEQ ID NO: 129),
  • GHPO 290 (SEQ ID NO: 131), GHPO 293 (SEQ ID NO: 133), GHPO 335 (SEQ ID NO: 135), GHPO 374 (SEQ ID NO: 137), GHPO 442 (SEQ ID NO: 139), GHPO 480 (SEQ ID NO: 141), GHPO 523 (SEQ ID NO: 143), GHPO 610 (SEQ ID NO: 145), GHPO 675 (SEQ ID NO: 147), GHPO 690 (SEQ ID NO: 149), GHPO 829 (SEQ ID NO: 151), GHPO 850 (SEQ ID NO: 153), GHPO 876 (SEQ ID NO: 155), GHPO 984 (SEQ ID NO: 157), GHPO 989 (SEQ ID NO: 159), GHPO 1111 (SEQ ID NO:161), GHPO 1145 (SEQ ID NO:163),
  • GHPO 1256 (SEQ ID NO: 165), GHPO 1264 (SEQ ID NO: 167), GHPO 1316 (SEQ ID NO: 169), GHPO 1368 (SEQ ID NO: 171), GHPO 1442 (SEQ ID NO: 173), GHPO 1506 (SEQ ID NO: 175), GHPO 1543 (SEQ ID NO: 177), GHPO 1574 (SEQ ID NO: 179), GHPO 1627 (SEQ ID NO: 181), GHPO 1657 (SEQ ID NO: 183), GHPO 1664 (SEQ ID NO: 185), GHPO 1694 (SEQ ID NO: 165), GHPO 1264 (SEQ ID NO: 167), GHPO 1316 (SEQ ID NO: 169), GHPO 1368 (SEQ ID NO: 171), GHPO 1442 (SEQ ID NO: 173), GHPO 1506 (SEQ ID NO: 175), GHPO 1543 (SEQ ID NO: 177), GH
  • GHPO 1345 (SEQ ID NO:319), GHPO 1377 (SEQ ID NO:321), GHPO 1424 (SEQ ID NO:323), GHPO 1430 (SEQ ID NO:325), GHPO 1502 (SEQ ID NO:327), GHPO 1600 (SEQ ID NO:329), GHPO 1714 (SEQ ID NO:331), GHPO 359 (SEQ ID NO:333), GHPO 678 (SEQ ID NO:335), GHPO 708 (SEQ ID NO:337), GHPO 759 (SEQ ID NO:339), GHPO 847 (SEQ ID NO:319), GHPO 1377 (SEQ ID NO:321), GHPO 1424 (SEQ ID NO:323), GHPO 1430 (SEQ ID NO:325), GHPO 1502 (SEQ ID NO:327), GHPO 1600 (SEQ ID NO:329), GHPO 1714 (SEQ ID NO:331), GHPO 359
  • GHPO 1050 (SEQ ID NO:343), GHPO 1101 (SEQ ID NO:345), GHPO 1120 (SEQ ID NO:347), GHPO 1138 (SEQ ID NO:349), GHPO 1310 (SEQ ID NO:351), GHPO 1320 (SEQ ID NO:353), GHPO 1375 (SEQ ID NO:355), GHPO 1432 (SEQ ID NO:357), GHPO 21 (SEQ ID NO:359), GHPO 282 (SEQ ID NO:361), GHPO 1089 (SEQ ID NO:363), GHPO 1141 (SEQ ID NO:341), GHPO 1050 (SEQ ID NO:343), GHPO 1101 (SEQ ID NO:345), GHPO 1120 (SEQ ID NO:347), GHPO 1138 (SEQ ID NO:349), GHPO 1310 (SEQ ID NO:351), GHPO 1320 (SEQ ID NO:353), GHPO
  • GHPO 284 (SEQ ID NO:427), GHPO 296 (SEQ ID NO:429), GHPO 300 (SEQ ID NO:431), GHPO 305 (SEQ ID NO:433), GHPO 319 (SEQ ID NO:435), GHPO 330 (SEQ ID NO:437), GHPO 340 (SEQ ID NO:439), GHPO 342 (SEQ ID NO:441), GHPO 344 (SEQ ID NO:443), GHPO 358 (SEQ ID NO:445), GHPO 373 (SEQ ID NO:447), GHPO 382 (SEQ ID NO:449), GHPO
  • GHPO 478 SEQ ID NO:477), GHPO 491 (SEQ ID NO:479), GHPO 511 (SEQ ID NO:481), GHPO 519 (SEQ ID NO:483), GHPO 526 (SEQ ID NO:485), GHPO 534 (SEQ ID NO:487), GHPO 536 (SEQ ID NO:489), GHPO 542 (SEQ ID NO:491), GHPO 544 (SEQ ID NO:493), GHPO 576 (SEQ ID NO:495), GHPO 578 (SEQ ID NO:497), GHPO 580 (SEQ ID NO:499), GHPO
  • GHPO 1225 (SEQ ID NO:609), GHPO 1228 (SEQ ID NO:611), GHPO 1229 (SEQ ID NO:613), GHPO 1231 (SEQ ID NO:615), GHPO 1236 (SEQ ID NO:617), GHPO 1242 (SEQ ID NO:619), GHPO 1248 (SEQ ID NO:621), GHPO 1270 (SEQ ID NO:623), GHPO 1271 (SEQ ID NO:625), GHPO 1298 (SEQ ID NO:627), GHPO 1301 (SEQ ID NO:629), GHPO 1304 (SEQ ID NO:609), GHPO 1228 (SEQ ID NO:611), GHPO 1229 (SEQ ID NO:613), GHPO 1231 (SEQ ID NO:615), GHPO 1236 (SEQ ID NO:617), GHPO 1242 (SEQ ID NO:619), GHPO 1248 (SEQ ID NO:621), GH
  • GHPO 1315 (SEQ ID NO:633), GHPO 1319 (SEQ ID NO:635), GHPO 1323 (SEQ ID NO:637), GHPO 1331 (SEQ ID NO:639), GHPO 1332 (SEQ ID NO:641), GHPO 1347 (SEQ ID NO:643), GHPO 1373 (SEQ ID NO:645), GHPO 1376 (SEQ ID NO:647), GHPO 1380 (SEQ ID NO:649), GHPO 1394 (SEQ ID NO:651), GHPO 1407 (SEQ ID NO:653), GHPO 1415
  • GHPO 1560 SEQ ID NO:689
  • GHPO 1564 SEQ ID NO:691
  • GHPO 1570 SEQ ID NO:693
  • GHPO 1588 SEQ ID NO:695
  • GHPO 1604 SEQ ID NO:697
  • GHPO 1605 SEQ ID NO:699
  • GHPO 1619 SEQ ID NO:701
  • GHPO 1629 SEQ ID NO:703
  • GHPO 1642 SEQ ID NO:705
  • GHPO 1654 SEQ ID NO:707
  • GHPO 1661 SEQ ID NO:709
  • GHPO 7 (SEQ ID NO:735), GHPO 8 (SEQ ID NO:737), GHPO 9 (SEQ ID NO:739), GHPO 10 (SEQ ID NO:741), GHPO 12 (SEQ ID NO:743), GHPO 25 (SEQ ID NO:745), GHPO 27 (SEQ ID NO:747), GHPO 29 (SEQ ID NO:749), GHPO 30 (SEQ ID NO:751), GHPO 37 (SEQ ID NO:753), GHPO 49 (SEQ ID NO:755), GHPO 51 (SEQ ID NO:757), GHPO 54 (SEQ ID NO:
  • GHPO 65 (SEQ ID NO:761), GHPO 66 (SEQ ID NO:763), GHPO 68 (SEQ ID NO:765), GHPO 70 (SEQ ID NO:767), GHPO 77 (SEQ ID NO:769), GHPO 83 (SEQ ID NO:771), GHPO 85 (SEQ ID NO:773), GHPO 87 (SEQ ID NO:775), GHPO 91 (SEQ ID NO:777), GHPO 92 (SEQ ID NO:779), GHPO 96 (SEQ ID NO:781), GHPO 97 (SEQ ID NO:783), GHPO
  • GHPO 192 (SEQ ID NO:821), GHPO 202 (SEQ ID NO:823), GHPO 204 (SEQ ID NO:825), GHPO 205 (SEQ ID NO:827), GHPO 212 (SEQ ID NO:829), GHPO 218 (SEQ ID NO:831), GHPO 226 (SEQ ID NO:833), GHPO 231 (SEQ ID NO:835), GHPO 236 (SEQ ID NO:837), GHPO 239 (SEQ ID NO:839), GHPO 245 (SEQ ID NO:841), GHPO 246 (SEQ ID NO:843), GHPO
  • GHPO 326 SEQ ID NO:871
  • GHPO 331 SEQ ID NO:873
  • GHPO 343 SEQ ID NO: 875
  • GHPO 345 SEQ ID NO: 877
  • GHPO 346 SEQ ID NO:879
  • GHPO 352 SEQ ID NO:881
  • GHPO 355 SEQ ID NO:883
  • GHPO 363 SEQ ID NO:885
  • GHPO 369 SEQ ID NO:887
  • GHPO 376 SEQ ID NO:889
  • GHPO 378 SEQ ID NO:891
  • GHPO 388 SEQ ID NO:893
  • GHPO 470 (SEQ ID NO:921), GHPO 486 (SEQ ID NO:923), GHPO 487 (SEQ ID NO:925), GHPO 488 (SEQ ID NO:927), GHPO 489 (SEQ ID NO:929), GHPO 498 (SEQ ID NO:931), GHPO 501 (SEQ ID NO:933), GHPO 504 (SEQ ID NO:935), GHPO 512 (SEQ ID NO:937), GHPO 517 (SEQ ID NO:939), GHPO 520 (SEQ ID NO:941), GHPO 528 (SEQ ID NO:943), GHPO 530 (SEQ ID NO:945), GHPO 532 (SEQ ID NO:947), GHPO 548 (SEQ ID NO:949), GHPO 561 (SEQ ID NO:951), GHPO 564 (SEQ ID NO:953), GHPO
  • GHPO 612 (SEQ ID NO:981), GHPO 615 (SEQ ID NO:983), GHPO 632 (SEQ ID NO:985), GHPO 633 (SEQ ID NO:987), GHPO 637 (SEQ ID NO:989), GHPO 651 (SEQ ID NO:991), GHPO 663 (SEQ ID NO:993), GHPO 686 (SEQ ID NO:995), GHPO 693 (SEQ ID NO:997), GHPO 698 (SEQ ID NO:999), GHPO 703 (SEQ ID NO: 1001), GHPO 704 (SEQ ID NO: 1003),
  • GHPO 705 (SEQ ID NO: 1005), GHPO 707 (SEQ ID NO: 1007), GHPO 721 (SEQ ID NO: 1009), GHPO 727 (SEQ ID NO: 1011), GHPO 728 (SEQ ID NO: 1013), GHPO 733 (SEQ ID NO: 1015), GHPO 758 (SEQ ID NO: 1017), GHPO 763 (SEQ ID NO: 1019), GHPO 771 (SEQ ID NO: 1021), GHPO 774 (SEQ ID NO: 1023), GHPO 776 (SEQ ID NO: 1025), GHPO 783 (SEQ ID NO: 1005), GHPO 707 (SEQ ID NO: 1007), GHPO 721 (SEQ ID NO: 1009), GHPO 727 (SEQ ID NO: 1011), GHPO 728 (SEQ ID NO: 1013), GHPO 733 (SEQ ID NO: 1015), GHPO 758 (SEQ ID NO: 1017), GH
  • GHPO 991 SEQ ID NO:1085)
  • GHPO 998 SEQ ID NO:1087
  • GHPO 1001 SEQ ID NO: 1089
  • GHPO 1005 SEQ ID NO: 1091
  • GHPO 1033 SEQ ID NO: 1093
  • GHPO 1039 SEQ ID NO: 1095
  • GHPO 1041 SEQ ID NO: 1097
  • GHPO 1043 SEQ ID NO: 1099
  • GHPO 1044 SEQ ID NO: 1101
  • GHPO 1051 SEQ ID NO: 1103
  • GHPO 1058 SEQ ID NO: 1105)
  • GHPO 1060 (SEQ ID NO:1107), GHPO 1075 (SEQ ID NO:1109), GHPO 1077 (SEQ ID NO:l l l l), GHPO 1082 (SEQ ID NO:1113), GHPO 1083 (SEQ ID NO:1115), GHPO 1086 (SEQ ID NO:1117), GHPO 1087 (SEQ ID NO: 1119), GHPO 1090 (SEQ ID NO: 1121), GHPO 1097 (SEQ ID NO: 1123), GHPO 1098 (SEQ ID NO: 1125), GHPO 1103 (SEQ ID NO: 1127), GHPO
  • GHPO 1401 (SEQ ID NO:1229), GHPO 1402 (SEQ ID NO: 1231), GHPO 1403 (SEQ ID NO: 1233), GHPO 1408 (SEQ ID NO: 1235), GHPO 1416 (SEQ ID NO: 1237), GHPO 1420 (SEQ ID NO: 1239), GHPO 1428 (SEQ ID NO: 1241), GHPO 1437 (SEQ ID NO: 1243), GHPO 1439 (SEQ ID NO: 1245), GHPO 1460 (SEQ ID NO: 1247), GHPO 1463 (SEQ ID NO: 1249),
  • GHPO 1472 (SEQ ID NO: 1251), GHPO 1474 (SEQ ID NO: 1253), GHPO 1484 (SEQ ID NO: 1255), GHPO 1489 (SEQ ID NO: 1257), GHPO 1494 (SEQ ID NO: 1259), GHPO 1495 (SEQ ID NO: 1261), GHPO 1498 (SEQ ID NO: 1263), GHPO 1499 (SEQ ID NO: 1265), GHPO 1500 (SEQ ID NO: 1267), GHPO 1503 (SEQ ID NO:1269), GHPO 1504 (SEQ ID NO:1271), GHPO
  • GHPO 1749 (SEQ ID NO:1351), GHPO 1750 (SEQ ID NO: 1353), GHPO 1751 (SEQ ID NO:1355), GHPO 1755 (SEQ ID NO:1357), GHPO 1771 (SEQ ID NO:1359), GHPO 1786 (SEQ ID NO:1361), and GHPO 1789 (SEQ ID NO:1363).
  • An isolated polynucleotide ofthe invention encodes (i) a polypeptide having an amino acid sequence that is homologous to a Helicobacter amino acid sequence of a polypeptide, the Helicobacter amino acid sequence being selected from the group consisting ofthe amino acid sequences shown in the sequence listing (even numbers, up to SEQ ID NO: 1364), or (ii) a derivative of the polypeptide.
  • polynucleotides included in the invention can also encode polypeptides that lack signal sequences, as well as other polypeptide or peptide fragments ofthe full-length polypeptides.
  • isolated polynucleotide is defined as a polynucleotide that is removed from the environment in which it naturally occurs.
  • a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part ofthe bacterial genome, as a result of, e.g., a cloning event (amplification), is "isolated.”
  • an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous, at the 5 ' or 3' ends, in the naturally occurring genome.
  • isolated polynucleotides can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.
  • a polynucleotide ofthe invention can consist of RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of RNA or DNA.
  • the polynucleotide can be double-stranded or single-stranded and, if single-stranded, can be the coding (sense) strand or the non-coding (anti- sense) strand.
  • sequences that encode polypeptides ofthe invention can be (a) the coding sequence as shown in any ofthe nucleotide sequences ofthe sequence listing (odd numbers, up to SEQ ID NO: 1363); (b) a ribonucleotide sequence derived by transcription of (a); or (c) a different coding sequence that, as a result ofthe redundancy or degeneracy ofthe genetic code, encodes the same polypeptides as the polynucleotide molecules having the sequences illustrated in any ofthe nucleotide sequences ofthe sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • the polypeptide can be one that is naturally secreted or excreted by, e.g., H. felis, H. mustelae, H. heilmanii, or H. pylori.
  • polypeptide or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
  • homologous amino acid sequence is meant an amino acid sequence that differs from an amino acid sequence shown in the sequence listing (even numbers, up to SEQ ID NO: 1364), or an amino acid sequence encoded by a nucleotide sequence shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363), by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the specific antigenicity ofthe polypeptide.
  • such a sequence is at least 75%o, more preferably at least 80%, and most preferably at least 90% identical to an amino acid sequence shown in the sequence listing (even numbers, up to SEQ ID NO: 1364).
  • Homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in the sequence listing (even numbers, up to SEQ ID NO: 1364).
  • amino acid sequence that is substantially identical is meant a sequence that is at least 90%), preferably at least 95%, more preferably at least 97%, and most preferably at least 99% identical to an amino acid sequence of reference and that differs from the sequence of reference, if at all, by a majority of conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions among amino acids ofthe same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. Homology can be measured using sequence analysis software (e.g.,
  • homologous polynucleotide sequences are defined in a similar way.
  • a homologous sequence is one that is at least 45%, more preferably at least 60%>, and most preferably at least 85% identical to a coding sequence of any ofthe nucleotide sequences set forth in the sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • Polypeptides having a sequence homologous to any one ofthe sequences shown in the sequence listing include naturally-occurring allelic variants, as well as mutants or any other non- naturally occurring variants that are analogous in terms of antigenicity, to a polypeptide having a sequence as shown in the sequence listing (even numbers, up to SEQ ID NO: 1364).
  • an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function ofthe polypeptide.
  • biological function is meant a function ofthe polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival ofthe cells.
  • the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • the biological function is distinct from the antigenic function.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature. For example, a bacterial species, e.g., H.
  • pylori is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not identical in each ofthe strains. Such an allelic variation can be equally reflected at the polynucleotide level.
  • allelic variants of polypeptide antigens comes from, e.g., studies ofthe Helicobacter urease antigen.
  • the amino acid sequence of Helicobacter urease varies widely from species to species, yet cross-species protection occurs, indicating that the urease molecule, when used as an immunogen, is highly tolerant of amino acid variations. Even among different strains ofthe single species H. pylori, there are amino acid sequence variations.
  • H. pylori urease protects mice from H. felis infection (Michetti et al, Gasfroenterology 107:1002, 1994).
  • UreA and UreB which contain distinct amino acid sequences, are both protective antigens against Helicobacter infection (Michetti et al, supra).
  • H. pylori strain CPM630 H. pylori strain CPM630; Lee et al, J. Infect. Dis.l72:161, 1995); recombinant UreA + UreB apoenzyme expressed from pORN214 (UreA and UreB sequences differ from H. pylori strain CPM630 by one and two amino acid changes, respectively; Lee et al, supra, 1995); a UreA-glutathione-S- transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al, Acta Gastro-Enterologica Belgica 56:54, 1993); UreA + UreB holoenzyme purified from H. pylori strain ⁇ CTC11637 (Marchetti et al,
  • UreA-MBP fusion protein (UreA from H. pylori strain 85P; Ferrero et al, Infection and Immunity 62:4981, 1994); a UreB-MBP fusion protein (UreB from H. pylori strain 85P; Ferrero et al, supra); a UreA- MBP fusion protein (UreA from H felis strain ATCC 49179; Ferrero et al, supra); a UreB-MBP fusion protein (UreB from H. felis strain ATCC 49179;
  • Polynucleotides, e.g., D ⁇ A molecules, encoding allelic variants can easily be obtained by polymerase chain reaction (PCR) amplification of genomic bacterial D ⁇ A extracted by conventional methods.
  • PCR polymerase chain reaction
  • Suitable primers can be designed based on the nucleotide sequence information provided in the sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • a primer consists of 10 to 40, preferably 15 to 25 nucleotides.
  • primers containing C and G nucleotides in proportions sufficient to ensure efficient hybridization, e.g., an amount of C and G nucleotides of at least 40%, preferably 50%, ofthe total nucleotide amount.
  • primers that can be used to isolate the polynucleotides ofthe invention from different Helicobacter strains can readily design primers that can be used to isolate the polynucleotides ofthe invention from different Helicobacter strains.
  • Experimental conditions for carrying out PCR can readily be determined by one skilled in the art and an illustration of carrying out PCR is provided in Example 2.
  • restriction endonuclease recognition sites that contain, typically, 4 to 6 nucleotides (for example, the sequences 5'-
  • GGATCC-3' (BamHI) or 5'-CTCGAG-3' (Xhol)
  • Restriction sites can be selected by those skilled in the art so that the amplified DNA can be conveniently cloned into an appropriately digested vector, such as a plasmid.
  • Useful homologs that do not occur naturally can be designed using known methods for identifying regions of an antigen that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, sequences ofthe antigen from different species can be compared to identify conserved sequences.
  • Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.
  • Polypeptide fragments ofthe invention can be derived from a polypeptide having a sequence homologous to any ofthe sequences ofthe sequence listing (even numbers, up to SEQ ID NO: 1364), to the extent that the fragments retain the substantial antigenicity ofthe parent polypeptide (specific antigenicity).
  • Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part ofthe parent polypeptide, while retaining specific antigenicity.
  • polypeptide derivatives should be about at least 12 amino acids in length to maintain antigenicity.
  • they can be at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.
  • polypeptide derivatives e.g., polypeptide fragments
  • polypeptide fragments can be designed using computer-assisted analysis of amino acid sequences in order to identify sites in protein antigens having potential as surface-exposed, antigenic regions (Hughes et al, Infect. Immun. 60(9):3497, 1992). For example, the
  • Laser Gene Program from DNA Star can be used to obtain hydrophilicity, antigenic index, and intensity index plots for the polypeptides ofthe invention.
  • This program can also be used to obtain information about homologies ofthe polypeptides with known protein motifs.
  • One skilled in the art can readily use the information provided in such plots to select peptide fragments for use as vaccine antigens. For example, fragments spanning regions ofthe plots in which the antigenic index is relatively high can be selected. One can also select fragments spanning regions in which both the antigenic index and the intensity plots are relatively high. Fragments containing conserved sequences, particularly hydrophilic conserved sequences, can also be selected.
  • Polypeptide fragments and polypeptides having large internal deletions can be used for revealing epitopes that are otherwise masked in the parent polypeptide and that may be of importance for inducing a protective T cell- dependent immune response. Deletions can also remove immunodominant regions of high variability among strains.
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction enzyme treatment ofthe cloned DNA molecules, or by the method of Kunkel et al. (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at Stratagene).
  • a polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative ofthe invention fused, e.g., at the N- or C-terminal end, to any other polypeptide (hereinafter referred to as a peptide tail).
  • a product can be easily obtained by translation of a genetic fusion, i.e., a hybrid gene.
  • Vectors for expressing fusion polypeptides are commercially available, and include the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives ofthe invention.
  • fusion polypeptides included in invention includes a polypeptide or polypeptide derivative ofthe invention fused to a polypeptide having adjuvant activity, such as, e.g., subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such as, e.g., subunit B of either cholera toxin or E. coli heat-labile toxin.
  • the polypeptide ofthe invention can be fused to the N-terminal end or, preferably, to the C-terminal end ofthe polypeptide having adjuvant activity.
  • a polypeptide fragment ofthe invention can be fused within the amino acid sequence ofthe polypeptide having adjuvant activity. Spacer sequences can also be included, if desired.
  • the polynucleotides ofthe invention encode Helicobacter polypeptides in precursor or mature form. They can also encode hybrid precursors containing heterologous signal peptides, which can mature into polypeptides ofthe invention.
  • heterologous signal peptide is meant a signal peptide that is not found in the naturally-occurring precursor of a polypeptide ofthe invention.
  • a polynucleotide ofthe invention hybridizes, preferably under stringent conditions, to a polynucleotide having a sequence as shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • Hybridization procedures are, e.g., described by Ausubel et al. (supra); Silhavy et al. (Experiments with Gene
  • Tm melting temperature
  • hybridization temperature (Th) is approximately 20 to 40°C, 20 to 25 °C, or, preferably, 30 to 40°C below the calculated Tm.
  • optimal temperature and salt conditions can be readily determined empirically in preliminary experiments using conventional procedures. For example, stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, (i) within 4-16 hours at 42 °C, in 6 x SSC containing 50% formamide or (ii) within 4-16 hours at 65 °C in an aqueous 6 x SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)).
  • Tm 4 x (G+C) + 2 (A+T).
  • a polynucleotide molecule ofthe invention containing RNA, DNA, or modifications or combinations thereof, can have various applications.
  • a polynucleotide molecule can be used (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Helicobacter infection, (iii) as a vaccine agent, in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Helicobacter strains that can over-express a polynucleotide ofthe invention or express it in a non-toxic, mutated form.
  • vaccine vectors such as poxviruses
  • elements e.g., a promoter
  • a recombinant expression system can be selected from procaryotic and eucaryotic hosts.
  • Eucaryotic hosts include, for example, yeast cells (e.g., Saccharomyces cerevisiae or Pichia Pastoris), mammalian cells (e.g., COS1,
  • NIH3T3, or JEG3 cells NIH3T3, or JEG3 cells
  • arthropods cells e.g., Spodoptera frugiperda (SF9) cells
  • plant cells e.g., a procaryotic host such as E. coli is used.
  • Bacterial and eucaryotic cells are available from a number of different sources that are known to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland).
  • an expression cassette includes a constitutive or inducible promoter that is functional in the selected host system; a ribosome binding site; a start codon (ATG); if necessary, a region encoding a signal peptide, e.g., a lipidation signal peptide; a polynucleotide molecule ofthe invention; a stop codon; and, optionally, a 3' terminal region (translation and/or transcription terminator).
  • the signal peptide-encoding region is adjacent to the polynucleotide ofthe invention and is placed in the proper reading frame.
  • the signal peptide-encoding region can be homologous or heterologous to the polynucleotide molecule encoding the mature polypeptide and it can be specific to the secretion apparatus ofthe host used for expression.
  • the open reading frame constituted by the polynucleotide molecule ofthe invention, alone or together with the signal peptide, is placed under the control ofthe promoter so that transcription and translation occur in the host system.
  • Promoters and signal peptide-encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (U.S. Patent No. 5,028,530; Cagnon et al, Protein Engineering 4(7) : 843 , 1991 ); the promoter of the bacteriophage T7 RNA polymerase gene, which is functional in a number of E. coli strains expressing T7 polymerase (U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al, J. Bact. 169:5692, 1987).
  • the expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.
  • Expression vectors e.g., plasmids or viral vectors
  • plasmids or viral vectors can be chosen from, for example, those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987) and can purchased from various commercial sources. Methods for transforming or transfecting host cells with expression vectors are well known in the art and will depend on the host system selected, as described in Ausubel et al (supra).
  • a recombinant polypeptide ofthe invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane.
  • the polypeptide can then be recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation ofthe cell culture.
  • the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method known to a person skilled in the art, such as by genetic fusion to a small affinity-binding domain.
  • Antibody-based affinity purification methods are also available for purifying a polypeptide ofthe invention extracted from a Helicobacter strain. Antibodies useful for immunoaffinity purification ofthe polypeptides ofthe invention can be obtained using methods described below.
  • Polynucleotides ofthe invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid.
  • Therapeutic or prophylactic efficacy of a polynucleotide ofthe invention can be evaluated as is described below.
  • a vaccine vector such as a poxvirus, containing a polynucleotide molecule ofthe invention placed under the control of elements required for expression;
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector ofthe invention (iv) a method for inducing an immune response against Helicobacter in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Helicobacter infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector ofthe invention to elicit an immune response, e.g., a protective or therapeutic immune response to Helicobacter; and (v) a method for preventing and/or treating a Helicobacter
  • the third aspect ofthe invention encompasses the use of a vaccine vector ofthe invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • a vaccine vector ofthe invention can express one or several polypeptides or derivatives ofthe invention, as well as at least one additional Helicobacter antigen such as a urease apoenzyme or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a vaccine vector can include an additional polynucleotide molecules encoding, e.g., urease subunit A, B, or both, or a cytokine, placed under the control of elements required for expression in a mammalian cell.
  • composition ofthe invention can include several vaccine vectors, each of which being capable of expressing a polypeptide or derivative ofthe invention.
  • a composition can also contain a vaccine vector capable of expressing an additional Helicobacter antigen such as urease apoenzyme, a subunit, fragment, homolog, mutant, or derivative thereof, or a cytokine such as IL-2 or IL-12.
  • a vaccine vector ofthe invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the administration can be achieved in a single dose or repeated at intervals.
  • the appropriate dosage depends on various parameters that are understood by those skilled in the art, such as the nature ofthe vaccine vector itself, the route of administration, and the condition ofthe mammal to be vaccinated (e.g., the weight, age, and general health ofthe mammal).
  • Live vaccine vectors that can be used in the invention include viral vectors, such as adenoviruses and poxviruses, as well as bacterial vectors, e.g.,
  • adenovirus vector as well as a method for constructing an adenovirus vector capable of expressing a polynucleotide molecule ofthe invention, is described in U.S. Patent No. 4,920,209.
  • Poxvirus vectors that can be used in the invention include, e.g., vaccinia and canary pox viruses, which are described in U.S. Patent No. 4,722,848 and U.S. Patent No.
  • Poxvirus vectors capable of expressing a polynucleotide ofthe invention can be obtained by homologous recombination, as described in Kieny et al. (Nature 312:163, 1984) so that the polynucleotide ofthe invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
  • the dose of viral vector vaccine for therapeutic or prophylactic use, can be from about lxlO 4 to about lxlO 11 , advantageously from about lxl 0 7 to about lxl 0 10 , or, preferably, from about lxl 0 7 to about lxl 0 9 plaque-forming units per kilogram.
  • viral vectors are administered parenterally, for example, in 3 doses that are 4 weeks apart. Those skilled in the art will recognize that it is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector ofthe invention and thereby minimizing the immune response to the viral vector itself.
  • Non-toxicogenic Vibrio cholerae mutant strains that can be used in live oral vaccines are described by Mekalanos et al. (Nature 306:551, 1983) and in U.S. Patent No. 4,882,278 (strain in which a substantial amount ofthe coding sequence of each ofthe two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSl DNA sequences).
  • An effective vaccine dose of a V. cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a polynucleotide molecule ofthe invention can contain, e.g., about lxlO 5 to about lxlO 9 , preferably about lxlO 6 to about lxl 0 8 viable bacteria in an appropriate volume for the selected route of administration.
  • Preferred routes of administration include all mucosal routes, but, most preferably, these vectors are administered intranasally or orally.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361.
  • Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally.
  • a polynucleotide ofthe invention can be inserted into the bacterial genome or it can remain in a free state, for example, carried on a plasmid.
  • An adjuvant can also be added to a composition containing a bacterial vector vaccine.
  • a number of adjuvants that can be used are known to those skilled in the art.
  • preferred adjuvants can be selected from the list provided below.
  • a composition of matter containing a polynucleotide ofthe invention, together with a diluent or carrier containing a therapeutically or prophylactically effective amount of a polynucleotide ofthe invention
  • a method for inducing an immune response against a polynucleotide ofthe invention containing a therapeutically or prophylactically effective amount of a polynucleotide ofthe invention
  • Helicobacter in a mammal, by administering to the mammal an immunogenically effective amount of a polynucleotide ofthe invention to elicit an immune response, e.g., a protective immune response to Helicobacter; and (iv) a method for preventing and or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, by administering a prophylactic or therapeutic amount of a polynucleotide ofthe invention to an individual in need of such treatment.
  • a Helicobacter e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii
  • the fourth aspect ofthe invention encompasses the use of a polynucleotide ofthe invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • the fourth aspect ofthe invention preferably includes the use of a polynucleotide molecule placed under conditions for expression in a mammalian cell, e.g., in a plasmid that is unable to replicate in mammalian cells and to substantially integrate into a mammalian genome.
  • Polynucleotides (for example, DNA or RNA molecules) ofthe invention can also be administered as such to a mammal as a vaccine.
  • a DNA molecule ofthe invention When a DNA molecule ofthe invention is used, it can be in the form of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al, Molec.
  • the desmin promoter (Li et al, Gene 78:243, 1989; Li et al, J. Biol. Chem. 266:6562, 1991; Li et al, J. Biol. Chem. 268: 10403, 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, e.g., in WO 94/21797 and by Hartikka et al (Human Gene Therapy 7:1205, 1996).
  • the polynucleotide ofthe invention can encode a precursor or a mature form of a polypeptide ofthe invention.
  • the precursor sequence can be homologous or heterologous.
  • a eucaryotic leader sequence can be used, such as the leader sequence ofthe tissue-type plasminogen factor (tPA).
  • a composition ofthe invention can contain one or several polynucleotides ofthe invention. It can also contain at least one additional polynucleotide encoding another Helicobacter antigen, such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof.
  • DNA molecules ofthe invention and/or additional DNA molecules to be included in the same composition are carried in the same plasmid.
  • Standard methods can be used in the preparation of therapeutic polynucleotides ofthe invention.
  • a polynucleotide can be used in a naked form, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g., calcium phosphate, or any other fransfection-facilitating agent.
  • the polynucleotide can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier.
  • a physiologically acceptable solution such as sterile saline or sterile buffered saline
  • the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.
  • a polynucleotide can be associated with agents that assist in cellular uptake.
  • It can be, e.g., (i) complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.
  • a chemical agent that modifies cellular permeability such as bupivacaine (see, e.g., WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.
  • Anionic and neutral liposomes are well-known in the art (see, e.g.,
  • Liposomes A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
  • Cationic lipids can also be used for gene delivery.
  • Such lipids include, for example, LipofectinTM, which is also known as DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2- bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine), and cholesterol derivatives.
  • DOTMA N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP 1,2- bis(oleyloxy)-3-(trimethylammonio)propane
  • DDAB dimethyldioctadecylammonium bromide
  • DOGS dioctadecylamid
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092).
  • DOPE dioleyl phosphatidylethanolamine
  • Other transfection- facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, e.g., WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397.
  • spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane see, for example, WO 93/18759
  • membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
  • Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and by Tang et al (Nature 356:152,
  • the microparticle-coated polynucleotides can be injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
  • the amount of DNA to be used in a vaccine recipient depends, e.g., on the strength ofthe promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition ofthe mammal intended for administration (e.g., the weight, age, and general health ofthe mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 ⁇ g to about
  • 1 mg preferably, from about 10 ⁇ g to about 800 ⁇ g, and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the route of administration can be any conventional route used in the vaccine field.
  • a polynucleotide ofthe invention can be administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, or urinary tract surface, or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • the choice of administration route will depend on, e.g., the formulation that is selected.
  • a polynucleotide formulated in association with bupivacaine is advantageously administered into muscle.
  • the formulation can be advantageously injected via intravenous, intranasal (for example, by aerosolization), intramuscular, intradermal, and subcutaneous routes.
  • a polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or subcutaneous routes.
  • such a composition can also contain an adjuvant.
  • a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable.
  • nucleotide probe or primer having a sequence found in, or derived by degeneracy ofthe genetic code from, a sequence shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to polynucleotide molecules having sequences homologous to any of those shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363), or to a complementary or anti-sense sequence of any of those shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363).
  • probes are significantly shorter than the full-length sequences shown in the sequence listing. For example, they can contain from about 5 to about 100, preferably from about 10 to about 80 nucleotides.
  • probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of a sequence as shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363), or a sequence complementary to any of such sequences.
  • Probes can contain modified bases, such as inosine, methyl-5- deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6- purine.
  • Sugar or phosphate residues can also be modified or substituted.
  • a deoxyribose residue can be replaced by a polyamide (Nielsen et al, Science 254: 1497, 1991) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate esters.
  • the 2'-hydroxyl group on ribonucleotides can be modified by addition of, e.g., alkyl groups.
  • Probes ofthe invention can be used in diagnostic tests, or as capture or detection probes. Such capture probes can be immobilized on solid supports, directly or indirectly, by covalent means or by passive adsorption.
  • a detection probe can be labeled by a detectable label, for example a label selected from radioactive isotopes; enzymes, such as peroxidase and alkaline phosphatase; enzymes that are able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate; compounds that are chromogenic, fluorogenic, or luminescent; nucleotide base analogs; and biotin.
  • Probes ofthe invention can be used in any conventional hybridization method, such as in dot blot methods (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol. Biol.
  • Primers used in the invention usually contain about 10 to 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), an elongation process, or a reverse transcription method. In a diagnostic method involving PCR, the primers can be labeled.
  • the invention also encompasses (i) a reagent containing a probe of the invention for detecting and/or identifying the presence of Helicobacter in a biological material; (ii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) the sample is exposed to a probe ofthe invention, for example, a capture probe, a detection probe, or both, under stringent hybridization conditions, so that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is contacted with at least one, or, preferably two, primers ofthe invention, and amplified by the polymerase chain reaction, and (d) an amplified DNA
  • a sixth aspect ofthe invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide ofthe invention.
  • a "substantially purified polypeptide” is defined as a polypeptide that is separated from the environment in which it naturally occurs and or a polypeptide that is free of most ofthe other polypeptides that are present in the environment in which it was synthesized.
  • the polypeptides ofthe invention can be purified from a natural source, such as a Helicobacter strain, or can be produced using recombinant methods.
  • Homologous polypeptides or polypeptide derivatives encoded by polynucleotides ofthe invention can be screened for specific antigenicity by testing cross-reactivity with an antiserum raised against a polypeptide having an amino acid sequence as shown in the sequence listing (even numbers, up to SEQ ID NO: 1364). Briefly, a monospecific hyperimmune antiserum can be raised against a purified reference polypeptide as such or as a fusion polypeptide, for example, an expression product of MBP, GST, or His-tag systems, or a synthetic peptide predicted to be antigenic. The homologous polypeptide or derivative that is screened for specific antigenicity can be produced as such or as a fusion polypeptide.
  • the material After being transferred to a filter, such as a nitrocellulose membrane, the material is incubated with the monospecific hyperimmune antiserum, which is diluted in a range of dilutions from about 1 : 50 to about 1 :5000, preferably from about
  • the product to be screened can be used as the coating antigen.
  • a purified preparation is preferred, but a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation of about 10 ⁇ g protein/ml is distributed into wells of a 96-well ELISA plate. The plate is incubated for about 2 hours at 37°C, then overnight at 4°C. The plate is washed with phosphate buffer saline (PBS) contaimng 0.05% Tween 20 (PBS/Tween buffer) and the wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA), to prevent non-specific antibody binding.
  • PBS phosphate buffer saline
  • BSA bovine serum albumin
  • the plate After 1 hour of incubation at 37 °C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA, and 100 ⁇ l dilutions are added to each well. The plate is incubated for 90 minutes at 37°C, washed, and evaluated using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when the specific antibodies used were raised in rabbits. Incubation is carried out for about 90 minutes at
  • a purified product is preferred, although a whole cell extract can be used.
  • a solution ofthe product at a concentration of about 100 ⁇ g/ml is serially diluted two-fold with 50 mM Tris-HCl (pH 7.5).
  • a filter such as a 0.45 ⁇ m nitrocellulose membrane, set in a 96-well dot blot apparatus (Biorad).
  • the buffer is removed by applying vacuum to the system.
  • Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried.
  • the membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum diluted from about 1:50 to about 1:5000, preferably about 1:500.
  • the reaction is detected using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when rabbit antibodies are used. Incubation is carried out for about 90 minutes at 37 °C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is then measured visually by the appearance of a colored spot, e.g., by colorimetry.
  • a positive reaction is associated with detection of a colored spot for reactions carried out with a dilution of at least about 1 :50, preferably, of at least about 1 :500.
  • Therapeutic or prophylactic efficacy of a polypeptide or polypeptide derivative ofthe invention can be evaluated as described below.
  • a composition of matter containing a polypeptide ofthe invention together with a diluent or carrier containing a therapeutically or prophylactically effective amount of a polypeptide ofthe invention
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide ofthe invention containing a therapeutically or prophylactically effective amount of a polypeptide ofthe invention
  • a method for inducing an immune response against Helicobacter in a mammal by administering to the mammal an immunogenically effective amount of a polypeptide ofthe invention to elicit an immune response, e.g., a protective immune response to Helicobacter
  • a method for preventing and/or treating a Helicobacter e.g., H. pylori, H. felis, H.
  • this aspect ofthe invention includes the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • the immunogenic compositions ofthe invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
  • a parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the choice ofthe administration route depends upon a number of parameters, such as the adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or oral route will be preferred, and if a lipid formulation or an aluminum compound is used,
  • the subcutaneous or intramuscular route is most preferred.
  • the choice of administration route can also depend upon the nature ofthe vaccine agent.
  • a polypeptide ofthe invention fused to CTB or to LTB will be best administered to a mucosal surface.
  • a composition ofthe invention can contain one or several polypeptides or derivatives ofthe invention. It can also contain at least one additional Helicobacter antigen, such as the urease apoenzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a polypeptide or polypeptide derivative can be formulated into or with liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response.
  • liposomes such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs)
  • VLPs virus-like particles
  • Adjuvants other than liposomes can also be used in the invention and are well known in the art (see, for example, the list provided below).
  • Administration can be achieved in a single dose or repeated as necessary at intervals that can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly or monthly intervals.
  • a vaccine antigen ofthe invention can be administered mucosally in an amount ranging from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • the dose usually should not exceed about 1 mg, and is, preferably, about 100 ⁇ g.
  • the polynucleotides and polypeptides ofthe invention can be used sequentially as part of a multi-step immunization process.
  • a mammal can be initially primed with a vaccine vector ofthe invention, such as a pox virus, e.g., via a parenteral route, and then boosted twice with a polypeptide encoded by the vaccine vector, e.g., via the mucosal route.
  • liposomes associated with a polypeptide or polypeptide derivative ofthe invention can be used for priming, with boosting being carried out mucosally using a soluble polypeptide or polypeptide derivative ofthe invention, in combination with a mucosal adjuvant (e.g., LT).
  • a mucosal adjuvant e.g., LT
  • Polypeptides and polypeptide derivatives ofthe invention can also be used as diagnostic reagents for detecting the presence of an ⁇ -Helicobacter antibodies, e.g., in blood samples.
  • Such polypeptides can be about 5 to about 80, preferably, about 10 to about 50 amino acids in length and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative is produced and can be purified using known methods.
  • the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product can be used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise monospecific antibodies against the polypeptide or polypeptide derivative.
  • the eighth aspect ofthe invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • monospecific antibody an antibody that is capable of reacting with a unique, naturally-occurring Helicobacter polypeptide.
  • An antibody ofthe invention can be polyclonal or monoclonal.
  • Monospecific antibodies can be recombinant, e.g., chimeric (e.g., consisting of a variable region of murine origin and a human constant region), humanized (e.g., a human immunoglobulin constant region and a variable region of animal, e.g., murine, origin), and/or single chain.
  • Both polyclonal and monospecific antibodies can also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments.
  • the antibodies ofthe invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes.
  • the antibodies ofthe invention which can be raised to a polypeptide or polypeptide derivative ofthe invention, can be produced and identified using standard immunological assays, e.g., Western blot assays, dot blot assays, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology, John Wiley
  • the antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigens in a sample, such as a biological sample.
  • the antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative ofthe invention. As is discussed further below, the antibodies can also be used in prophylactic and therapeutic passive immunization methods.
  • a ninth aspect ofthe invention provides (i) a reagent for detecting the presence of Helicobacter in a biological sample that contains an antibody, polypeptide, or polypeptide derivative ofthe invention; and (ii) a diagnostic method for detecting the presence of Helicobacter in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative ofthe invention, so that an immune complex is formed, and detecting the complex as an indication ofthe presence of Helicobacter in the sample or the organism from which the sample was derived.
  • the immune complex is formed between a component ofthe sample and the antibody, polypeptide, or polypeptide derivative, and that any unbound material can be removed prior to detecting the complex.
  • a polypeptide reagent can be used for detecting the presence of anti-Helicobacter antibodies in a sample, e.g., a blood sample, while an antibody ofthe invention can be used for screening a sample, such as a gastric extract or biopsy sample, for the presence of Helicobacter polypeptides.
  • the reagent e.g., the antibody, polypeptide, or polypeptide derivative ofthe invention
  • the reagent can be in a free state or can be immobilized on a solid support, such as, for example, on the interior surface of a tube or on the surface, or within pores, of a bead. Immobilization can be achieved using direct or indirect means.
  • Direct means include passive adsorption (i.e., non-covalent binding) or covalent binding between the support and the reagent.
  • indirect means is meant that an anti-reagent compound that interacts with the reagent is first attached to the solid support.
  • an anti-reagent compound that interacts with the reagent can serve as an anti-reagent, provided that it binds to an epitope that is not involved in recognition of antibodies in biological samples.
  • Indirect means can also employ a ligand-receptor system, for example, a molecule, such as a vitamin, can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase.
  • a process for purifying, from a biological sample, a polypeptide or polypeptide derivative ofthe invention which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody ofthe invention.
  • the antibody can be polyclonal or monospecific, and preferably is ofthe IgG type.
  • Purified IgGs can be prepared from an antiserum using standard methods (see, e.g., Coligan et al, supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described, for example, by Harlow et al.
  • a biological sample such as an H. pylori extract, preferably in a buffer solution
  • a chromatography material which is, preferably, equilibrated with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative ofthe invention (i.e., the antigen) is allowed to adsorb onto the material.
  • the chromatography material such as a gel or a resin coupled to an antibody ofthe invention, can be in batch form or in a column.
  • the unbound components are washed off and the antigen is eluted with an appropriate elution buffer, such as a glycine buffer, a buffer containing a chaotropic agent, e.g., guanidine HC1, or a buffer having high salt concentration (e.g., 3 M MgCl 2 ).
  • an appropriate elution buffer such as a glycine buffer, a buffer containing a chaotropic agent, e.g., guanidine HC1, or a buffer having high salt concentration (e.g., 3 M MgCl 2 ).
  • Eluted fractions are recovered and the presence ofthe antigen is detected, e.g., by measuring the absorbance at 280 nm.
  • An antibody ofthe invention can be screened for therapeutic efficacy as follows.
  • a composition of matter containing a monospecific antibody ofthe invention together with a diluent or carrier;
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody ofthe invention and
  • a method for treating or preventing Helicobacter e.g., H. pylori, H felis, H. mustelae, or H. heilmanii
  • the eleventh aspect ofthe invention includes the use of a monospecific antibody ofthe invention in the preparation of a medicament for treating or preventing Helicobacter infection.
  • the monospecific antibody can be polyclonal or monoclonal, and is, preferably, predominantly ofthe IgA isotype.
  • the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, optionally, in the presence of a bicarbonate buffer.
  • systemic administration not requiring a bicarbonate buffer, can be carried out.
  • a monospecific antibody ofthe invention can be administered as a single active agent or as a mixture with at least one additional monospecific antibody specific for a different Helicobacter polypeptide.
  • the amount of antibody and the particular regimen used can be readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibody over one week, or three doses per day of about 100 to 1,000 mg of antibody over two or three days, can be effective regimens for most purposes.
  • Therapeutic or prophylactic efficacy can be evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H. felis mouse model and the procedures described by Lee et al. (Eur. J. Gasfroenterology & Hepatology 7:303, 1995) or Lee et al (J. Infect. Dis.
  • H felis strain of the model can be replaced with another Helicobacter strain.
  • the efficacy of polynucleotide molecules and polypeptides from H pylori is, preferably, evaluated in a mouse model using an H. pylori strain. Protection can be determined by comparing the degree of Helicobacter infection in the gastric tissue assessed by, for example, urease activity, bacterial counts, or gastritis, to that of a control group. Protection is shown when infection is reduced by comparison to the control group.
  • Such an evaluation can be made for polynucleotides, vaccine vectors, polypeptides, and polypeptide derivatives, as well as for antibodies ofthe invention.
  • an antibody ofthe invention can be administered to the gastric mucosa of mice previously challenged with an H pylori strain, as described, e.g., by Lee et al (supra). Then, after an appropriate period of time, the bacterial load ofthe mucosa can be estimated by assessing urease activity, as compared to a control. Reduced urease activity indicates that the antibody is therapeutically effective.
  • Adjuvants that can be used in any ofthe vaccine compositions described above are described as follows.
  • Adjuvants for parenteral administration include, for example, aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen can be precipitated with, or adsorbed onto, the aluminum compound using standard methods. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), can also be used in parenteral administration.
  • Adjuvants that can be used for mucosal administration include, for example, bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A, the pertussis toxin (PT), and combinations, subunits, toxoids, or mutants thereof.
  • CT cholera toxin
  • LT E. coli heat-labile toxin
  • PT pertussis toxin
  • a purified preparation of native cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity.
  • CTB native cholera toxin subunit B
  • Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity.
  • a mutant having reduced toxicity is used. Suitable
  • Additional LT mutants that can be used in the methods and compositions ofthe invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110- Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, and polylactide glycolide (PLGA) microspheres, can also be used in mucosal administration.
  • MPLA bacterial monophosphoryl lipid A
  • PLGA polylactide glycolide
  • Adjuvants useful for both mucosal and parenteral administrations can also be used.
  • Any pharmaceutical composition ofthe invention, containing a polynucleotide, polypeptide, polypeptide derivative, or antibody ofthe invention can be manufactured using standard methods. It can be formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution, such as phosphate buffer saline, optionally, including a bicarbonate salt, such as sodium bicarbonate, e.g., 0.1 to 0.5 M. Bicarbonate can advantageously be added to compositions intended for oral or intragastric administration.
  • a diluent or carrier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
  • the invention also includes methods in which gastroduodenal infections, such as Helicobacter infection, are treated by oral administration of a Helicobacter polypeptide ofthe invention and a mucosal adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof.
  • antibiotics including, e.g., macrolides, tetracyclines, ⁇ -lactams, aminoglycosides, quinolones, penicillins, and derivatives thereof
  • antibiotics include, e.g., amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin
  • antisecretory agents including, e.g., H 2 - receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine, and roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and anticholinergic agents (e.g., pirenzepin
  • compositions for carrying out these methods i.e., compositions containing a Helicobacter antigen (or antigens) ofthe invention, an adjuvant, and one or more ofthe above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • Amounts ofthe above-listed compounds used in the methods and compositions ofthe invention can readily be determined by one skilled in the art. In addition, one skilled in the art can readily design treatment/immunization schedules.
  • the non- vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.
  • Methods and pharmaceutical compositions ofthe invention can be used to treat or to prevent Helicobacter infections and, accordingly, gastroduodenal diseases associated with these infections, including acute, chronic, and afrophic gastritis, and peptic ulcer diseases, e.g., gastric and duodenal ulcers.
  • Example 1 describes identification of genes, such as genes that encode the polypeptides ofthe invention, in the Helicobacter genome, as well as identification of signal sequences, and primer design for amplification of genes lacking signal sequences.
  • Example 2 describes cloning of DNA molecules encoding polypeptides ofthe invention into a vector that provides a histidine tag, and production and purification ofthe resulting his-tagged fusion proteins.
  • Example 3 describes methods for cloning DNA encoding the polypeptides of the invention so that they can be produced without his-tags
  • Example 4 describes methods for purifying recombinantly produced polypeptides ofthe invention.
  • EXAMPLE 1 Identification of genes in the H. pylori genome, identification of signal sequences, and primer design for amplification of genes lacking signal sequences l.A. Creating H. pylori genomic databases
  • the H. pylori genome was provided as a text file containing a single contiguous string of nucleotides that had been determined to be 1.76 Megabases in length.
  • the complete genome was split into 17 separate files using the program SPLIT (Creativity in Action), giving rise to 16 contigs, each containing 100,000 nucleotides, and a 17 th contig containing the remaining 76,000 nucleotides.
  • a header was added to each ofthe 17 files using the format: >hpg0.txt (representing contig 1), .hpgl.txt (representing contig 2), etc.
  • the resulting 17 files, named hpgO through hpg 16 were then copied together to form one file that represented the plus strand ofthe complete H.
  • a negative strand database ofthe H. pylori genome was created similarly by first creating a reverse complement ofthe positive strand using the program SeqPup (D.G. Gilbert, Indiana University Biology Department) and then performing the same procedure as described above for the plus strand. This database was given the designation "N.”
  • ORFs open reading frames
  • FASTA Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988.
  • FASTA was used for searching either a DNA sequence against either of the gene databases (" ⁇ " and/or "N"), or a peptide sequence against the ORF library ("O").
  • TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database (" ⁇ " and/or "N” libraries). Potential frameshifts also being resolved, FASTX was used for searching the translated reading frames of a DNA sequence against either a DNA database, or a peptide sequence against the protein database.
  • the FASTA searches against the constructed DNA databases identified exact nucleotide coordinates on one or more ofthe isolated contigs, and therefore the location ofthe target DNA. Once the exact location ofthe target sequence was known, the contig identified to carry the gene was exported into the software package MapDraw (DNAStar, Inc.) and the gene was isolated. Gene sequences with flanking DNA was then excised and copied into the EditSeq. Software package (DNAStar, Inc.) for further analysis. l.D. Identification of signal sequences
  • the deduced protein encoded by a target gene sequence is analyzed using the PROTEAN software package (DNAStar, Inc.). This analysis predicts those areas ofthe protein that are hydrophobic by using the Kyte-Doolittle algorithm, and identifies any potential polar residues preceding the hydrophobic core region, which is typical for many signal sequences. For confirmation, the target protein is then searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many signal sequences and hydrophobic regions in general, is the identification of predicted prokaryotic lipid attachment sites. Where confirmation between the two approaches is apparent at the N-terminus of any protein, putative cleavage sites are sought.
  • this includes the presence of either an Alanine (A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic region.
  • A Alanine
  • S Serine
  • G Glycine
  • C Cysteine
  • the gene sequence that specifies the signal sequence is omitted.
  • the 5'-end ofthe gene- specific portion ofthe N-terminal primer is designed to start at the first codon beyond the cleavage site. In the case of lipoproteins, the 5'-end ofthe N- terminal primer begins at the second codon, immediately after the modifiable residue at position +1 post-cleavage.
  • EXAMPLE 2 Preparation of isolated DNA encoding the polypeptides of the invention, and production of these polypeptides as histidine-tagged fusion proteins
  • H. pylori strain ORV2001 stored in LB medium containing 50% glycerol at -70 °C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% C0 2 , 5-7% 0 2 , 85-87% N 2 ). Cells are harvested, washed with phosphate buffer saline (PBS) (pH 7.2), and
  • DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech).
  • PCR amplification DNA molecules encoding the polypeptides ofthe invention are amplified from genomic DNA, as can be prepared as is described above, by the Polymerase Chain Reaction (PCR) using primers that can readily be designed by one skilled in the art. Specific examples of primers that can be used in the invention are shown in Table 1. As specific examples, to amplify genes encoding GHPO 147, GHPO 615, GHPO 961, GHPO 1282, GHPO 296, and
  • GHPO 840 the following primers can be used:
  • GHPO 147 5'-CTGAATTCGAATGAAAAGAATTTTAGTCTCT-3' (SEQ ID NO: 1365), and 5'-CCGCTCGAGTTAAAACTCATAATTCAAAT-3' (SEQ ID NO: 1366).
  • GHPO 615 5 * -CGCGGATCCGAAGACATGTGCAACCGATG-3' (SEQ ID NO: 1367), and
  • GHPO 1282 5'-GCGGATCCTTTTCTTCAATGTTTG-3" (SEQ ID NO:1371), and
  • GHPO 296 5'-CCGAATTCGGTTATAAAGCCCCT-3' (SEQ ID NO: 1373), and
  • GHPO 840 5'-CGCGGATCCGAGGAAATAGCATGTTAATAACC-3' (SEQ ID NO: 1375), and
  • the N-terminal and C-terminal primers for each clone can each include a 5' clamp and a restriction enzyme recognition sequence for cloning purposes (for example, Bam ⁇ l (GGATCC) and Xhol (CTCGAG) recognition sequences).
  • GGATCC Bam ⁇ l
  • CTCGAG Xhol
  • Amplification of gene-specific DNA is carried out using Vent DNA Polymerase (New England Biolabs) or Taq DNA polymerase (Appligene), according to the manufacturer's instructions.
  • the reaction mixture which is brought to a final volume of 100 ⁇ l with distilled water, is as follows: dNTPs mix 200 ⁇ M lOx ThermoPol buffer 10 ⁇ l primers 300 nM each
  • Appropriate amplification reaction conditions can readily be determined by one skilled in the art. For example, the following conditions can be used for amplification of DNA encoding GHPO 615 using the primers set forth above: initial denaturation at 94°C for 5 minutes, 25 cycles of denaturation at 97°C for 30 seconds, hybridization at 55°C for 1 minute, and elongation at 72°C for 2 minutes, using Vent DNA polymerase.
  • the following conditions can be used: initial denaturation at 94°C for 5 minutes, 25 cycles of denaturation at 94°C for 30 seconds, hybridization at 45°C for 30 seconds, and elongation at 72°C for 30 seconds, followed by a final elongation at 72°C for 7 minutes, using Vent DNA polymerase.
  • the following conditions can be used for amplification of DNA encoding GHPO 840 using the primers set forth above: 25 cycles of denaturation at 97°C for 30 seconds, hybridization at 55°C for 1 minute, and elongation at 72°C for 2 minutes using Vent DNA polymerase.
  • Table 1 sets forth conditions for using the primers listed therein.
  • a single PCR product is thus amplified and then is digested at 37 °C for 2 hours with BamHI and Xhol together in a 20 ⁇ l reaction volume.
  • the digested product is ligated to similarly cleaved pET28a (Novagen) that is dephosphorylated prior to the ligation by treatment with Calf Intestinal Alkaline Phosphatase (CIP).
  • CIP Calf Intestinal Alkaline Phosphatase
  • the ligation reaction (20 ⁇ l) is carried out at 14 °C overnight and then is used to transform 100 ⁇ l fresh E. coli XL 1 -blue competent cells (Novagen). The cells are incubated on ice for 2 hours, heat-shocked at 42 °C for 30 seconds, and returned to ice for 90 seconds. The samples are then added to
  • PCR is performed with the gene-specific primers under the conditions set forth above and transformant DNA is confirmed to contain the desired insert. If PCR-positive, one ofthe five plasmid DNA samples (500 ng) extracted from the E. coli XL 1 -blue cells is used to transform competent BL21 ( ⁇ DE3) E. coli competent cells (Novagen; as described previously). Transformants (10) are picked, plated onto selective kanamycin (50 ⁇ g/ml)- containing LB agar plates, and stored as a research stock in LB containing
  • One ml of frozen glycerol stock prepared as described in 2.C. is used to inoculate 50 ml of LB medium containing 25 ⁇ g/ml kanamycin in a 250 ml
  • the flask is incubated at 37°C for 2 hours or until the absorbance at 600 nm (OD 600 ) reaches 0.4-1.0.
  • the culture is stopped from growing by placing the flask at 4°C overnight. The following day, 10 ml ofthe overnight culture is used to inoculate 240 ml LB medium containing kanamycin (25 ⁇ g/ml), with the initial OD 600 being about 0.02-0.04.
  • Four flasks are inoculated for each ORF.
  • the cells are grown to an OD 600 of 1.0 (about 2 hours at 37°C), a 1 ml sample is harvested by centrifugation, and the sample is analyzed by SDS-PAGE to detect any leaky expression.
  • the remaining culture is induced with 1 mM IPTG and the induced cultures are grown for an additional 2 hours at 37°C.
  • the final OD 600 reading is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Two hundred and fifty ml of buffer are used for each 1 L of culture and the cells are recovered by centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and the pellets are stored at -45°C.
  • Pellets obtained using the methods described in 2.D. are thawed and resuspended in 95 ml of 50 mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final concentrations of 100 ⁇ M and 100 ⁇ g/ml, respectively. The mixture is homogenized with magnetic stirring at 5°C for 30 minutes.
  • Benzonase (Merck) is added to a final concentration of 1 U/ml, in the presence of 10 mM MgCl2, to ensure total digestion ofthe DNA.
  • the suspension is sonicated (Branson Sonifier 450) for 3 cycles of 2 minutes each at maximum output.
  • the homogenate is centrifuged at 19,000 x g for 15 minutes and both the supernatant and the pellet are analyzed by SDS-PAGE to detect the cellular location ofthe target protein in the soluble or insoluble fractions, as is described further below.
  • the elution profile is monitored by measuring the absorbance ofthe fractions at 280 nm. Fractions corresponding to the protein peak are pooled, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 ⁇ m membrane, and stored at -45°C.
  • the target protein is expressed in the insoluble fraction (pellets obtained using the methods described in 2.E.)
  • purification is conducted under denaturing conditions. NaCl, imidazole, and urea are added to the resuspended pellet to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole, and 6 M urea (buffer C). After complete solubilization, the mixture is filtered through a 0.45 ⁇ m membrane and loaded onto an IMAC column.
  • the purification procedures on the IMAC column are the same as are described in 2.E.I., except that 6 M urea is included in all ofthe buffers used and 10 column volumes of buffer C are used to wash the column after protein loading, instead of 50 column volumes.
  • the protein fractions eluted from the IMAC column with buffer D (buffer C containing 500 mM imidazole) are pooled.
  • Arginine is added to the solution to a final concentration of 0.5 M, and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) to progressively decrease the concentration of urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45 °C.
  • a first alternative involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a pellet obtained as is described in 2.E. is homogenized in a solution of 5 mM imidazole, 500 mM sodium chloride, and
  • the pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0).
  • the urea-solubilized protein is diluted with an equal volume of 2 M arginine, 50 mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to remove the urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45°C.
  • a second alternative involves the use of a strong denaturant, such as guanidine hydrochloride. Briefly, a pellet obtained as is described in 2.E.
  • ⁇ -mercaptoethanol is added to the eluted protein to a final concentration of 1 mM, and then the eluted protein is passed through a Sephadex G-25 column equilibrated in 0.1 M acetic acid. Protein eluted from the column is slowly added to 4 volumes of 50 mM phosphate buffer (pH 7.0), and the protein remains in solution.
  • mice Groups of 10 OF 1 mice (IFFA Credo) are immunized rectally with 25 ⁇ g ofthe purified recombinant protein, admixed with 1 ⁇ g of cholera toxin (Berna) in physiological buffer. Mice are immunized on days 0, 7, 14, and 21. Fourteen days after the last immunization, the mice are challenged with H. pylori strain ORV2001, grown in liquid media (the cells are grown on agar plates, as described in 2.A., and, after harvest, are resuspended in Brucella broth; the flasks are then incubated overnight at 37 °C). Fourteen days after challenge, the mice are sacrificed and their stomachs are removed. The amount of H. pylori is determined by measuring the urease activity in the stomach and by culture.
  • 2.G. Production of monospecific polyclonal antibodies 2.G.I. Hyperimmune rabbit antiserum New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 ⁇ g of a purified fusion polypeptide, as obtained using the methods described in 2.E.I. or 2.E.2., in the presence of Freund's complete adjuvant and in a total volume of approximately 2 ml. Twenty one and 42 days after the initial injection, booster doses, which are identical to the priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Fifteen days after the last injection, animal serum is recovered, decomplemented, and filtered through a 0.45 ⁇ m membrane.
  • mice are injected subcutaneously with 10-50 ⁇ g of a purified fusion polypeptide as obtained using the methods described in 2.E.1. or 2.E.2., in the presence of Freund's complete adjuvant and in a volume of approximately 200 ⁇ l. Seven and 14 days after the initial injection, booster doses, which are identical to the priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Twenty one and 28 days after the initial infection, mice receive 50 ⁇ g ofthe antigen alone infraperitoneally.
  • mice are also injected infraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al, Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 5th Ed. Washington DC, American Public Health Association, 1979). Ascites fluid is collected 10-13 days after the last injection.
  • the N-terminal primers are designed to include the ribosome binding site ofthe target gene, the ATG start site, and any signal sequence and cleavage site.
  • the N-terminal primers can include a 5' clamp and a restriction endonuclease recognition site, such as that for BamHI (GGATCC), which facilitates subsequent cloning.
  • the C-terminal primers can include a restriction endonuclease recognition site, such as that for Xhol (CTCGAG), which can be used in subsequent cloning, and a TAA stop codon.
  • Amplification of genes encoding the polypeptides ofthe invention can be carried out using Thermalase DNA Polymerase under the conditions described above in Example 2.
  • Vent DNA polymerase New England Biolabs
  • Pwo DNA polymerase Boehringer Mannheim
  • Taq Taq
  • DNA polymerase (Appligene) can be used, according to instructions provided by the manufacturers.
  • a single PCR product for each clone is amplified and cloned into appropriately cleaved pET 24 (e.g., BamHl-Xh ⁇ cleaved pET 24), resulting in the construction of a franscriptional fusion that permits expression of the proteins without His-tags.
  • the expressed products can be purified as denatured proteins that are refolded by dialysis into 1 M arginine.
  • Cloning into pET 24 allows transcription ofthe genes from the T7 promoter, which is supplied by the vector, but relies upon binding ofthe RNA- specific DNA polymerase to the intrinsic ribosome binding sites ofthe genes, and thereby expression ofthe complete ORF.
  • the amplification, digestion, and cloning protocols that can be used in this method are as described above for constructing translational fusions.
  • EXAMPLE 4 Purification of the polypeptides of the invention by immunoaffinity
  • An immune serum as prepared as is described in section 2.G., is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH 8.0). The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the column.
  • IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are collected as 5 ml fractions to each of which is added 0.25 ml 1 M Tris-HCl (pH 8.0). The optical density ofthe eluate is measured at 280 nm and fractions containing the IgG antibodies are pooled, dialyzed against 50 mM Tris-HCl
  • CNBr-activated Sepharose 4B gel (1 g of dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430- 01) is suspended in 1 mM HCl buffer and washed with a buchner by adding small quantities of 1 mM HCl buffer. The total volume of buffer is 200 ml per gram of gel. Purified IgG antibodies are dialyzed for 4 hours at 20 ⁇ 5°C against
  • IgG antibodies are mixed with the gel overnight at 5 ⁇ 3°C.
  • the gel is packed into a chromatography column and is washed with 2 column volumes of
  • the gel is then transferred to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature, and washed twice with 2 column volumes of PBS. The gel is then stored in 1/10,000 PBS/merthiolate. The amount of IgG antibodies coupled to the gel is determined by measuring the optical density (OD) at 280 nm ofthe IgG solution and the direct eluate, plus washings.
  • OD optical density
  • the adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is recovered in 3 ml fractions, to each of which is added 150 ⁇ l of 1 M sodium phosphate buffer (pH 8.0). Absorption is measured at 280 nm for each fraction; those fractions containing the antigen are pooled and stored at -20 °C.
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • GAG CCT AAA AAA AGT CAT ATT TAT TTT GGG GCT ATG GTG GGT TTA GCT 152 Glu Pro Lys Lys Ser His lie Tyr Phe Gly Ala Met Val Gly Leu Ala 20 25 30
  • AGC ACG ATC GAT CGC CAC CAC CGC ATA GAG CTT GGG GCT AAA ATC CCT 536 Ser Thr lie Asp Arg His His Arg lie Glu Leu Gly Ala Lys lie Pro 150 155 160
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic RNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AAA AAG ATT GAT ATA GCT AGG GGG ATT TAT CCT ACA GAG ACT TTT GTA 255 Lys Lys He Asp He Ala Arg Gly He Tyr Pro Thr Glu Thr Phe Val 50 55 60
  • GGC AAG GTG ATT GAT TCT ATA GCG TGC GGG AAC GCT AGG GCC AAT AAA 543 Gly Lys Val He Asp Ser He Ala Cys Gly Asn Ala Arg Ala Asn Lys 145 150 155
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE (A) NAME/KEY: Coding Sequence
  • GGC ATG GTG GGA TCT ATT TTC TAT GAT GGC ACG AAG AAG TTT GAA GAC 344 Gly Met Val Gly Ser He Phe Tyr Asp Gly Thr Lys Lys Phe Glu Asp 85 90 95
  • AAA CCT CAT CGT TTC CTC ATA GAA GGC TTT TAT TAC CTT TCG
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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EP98917972A 1997-04-01 1998-04-01 Identification von polynukleotiden, die neue heliobacter polypeptide im heliobacter genom kodieren Withdrawn EP0977482A4 (de)

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US902615 1997-07-29
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Publication number Priority date Publication date Assignee Title
DE19847628C2 (de) * 1998-10-15 2000-10-19 Chiron Behring Gmbh & Co Helicobacter pylori-Impfstoff
US6238894B1 (en) 1998-11-04 2001-05-29 Diane Taylor α1,2 fucosyltransferase
AU5399800A (en) * 1999-05-31 2000-12-18 Creatogen Aktiengesellschaft Essential gene and gene products for identifying, developing and optimising immunological and pharmacological active ingredients for the treatment of microbial infections
WO2001008486A1 (en) * 1999-08-03 2001-02-08 Smithkline Beecham Corporation CoaA POLYPEPTIDES AND POLYNUCLEOTIDES AND METHODS THEREOF
AUPQ347199A0 (en) 1999-10-15 1999-11-11 Csl Limited Novel polypeptide fragments
US20030180330A1 (en) * 2000-04-27 2003-09-25 Meyer Thomas F Method for identifying helicobacter antigens
SE0001988D0 (sv) * 2000-05-29 2000-05-29 A & Science Invest Ab Novel polypeptides and use thereof
WO2002000888A1 (en) * 2000-06-28 2002-01-03 National Research Council Of Canada Helicobacter pylori heptosyl transferase polypeptides
US20040033240A1 (en) * 2000-07-05 2004-02-19 Bruno Guy Immunological combinations for prophylaxis and therapy of helicobacter pylori infection
ATE468391T1 (de) * 2001-08-24 2010-06-15 Kyowa Hakko Bio Co Ltd Alpha-1,2-fucosyl-transferase und diese codierende dna
KR20030024129A (ko) * 2001-09-17 2003-03-26 국윤호 헬리코박터 파일로리의 탐지 및 동정 방법
AU2003218551A1 (en) * 2002-03-26 2003-10-08 National Research Council Of Canada Helicobacter flagellar, motility polypeptides
JP3823268B2 (ja) * 2002-05-30 2006-09-20 独立行政法人科学技術振興機構 ヘリコバクター・ピロリの細胞死誘導剤
WO2005023851A1 (en) * 2003-09-05 2005-03-17 Karolinska Innovations Ab Plasminogen/plasmin binding polypeptides and nucleic acids therefore
KR20060129229A (ko) * 2003-11-21 2006-12-15 에이스 바이오사이언시즈 에이/에스 표면에 위치한 빈창자캄필로박터 폴리펩티드
US10828358B2 (en) 2015-12-14 2020-11-10 Technische Universität München Helicobacter pylori vaccines
KR101985335B1 (ko) * 2017-11-01 2019-06-03 연세대학교 원주산학협력단 피시알-리버스 블랏 교잡 어세이 기반 헬리코박터 파이로리균 및 그 균의 항생제 내성에 관련된 유전자의 검출을 확인할 수 있는 진단법 및 그 응용
CN113754727B (zh) * 2020-09-30 2022-07-12 广州派真生物技术有限公司 腺相关病毒突变体及其应用
PE20251773A1 (es) * 2022-11-29 2025-07-14 Jeil Pharmaceutical Co Ltd Composicion para eliminar helicobacter pylori que comprende zastaprazan o una sal farmaceuticamente aceptable del mismo
CN118063569B (zh) * 2024-04-24 2024-08-30 上海金翌生物科技有限公司 一种幽门螺杆菌分泌蛋白及其在检测幽门螺杆菌中的应用

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GB8928625D0 (en) * 1989-12-19 1990-02-21 3I Res Expl Ltd H.pylori dna probes

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