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(12) Patent Application Publication (10) Pub. No.: US 2016/0244489 A1 MASIGNAN Et Al

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US 20160244489A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0244489 A1 MASIGNAN et al. (43) Pub. Date: Aug. 25, 2016 (54) PROTEINS AND NUCLEICACIDS FROM application No. 1 1/884,812, filed on Mar. 26, 2008, MENINGITIS/SEPSIS-ASSOCATED now Pat. No. 8,758,764, filed as application No. PCT/ ESCHERICHA COL US2006/005913 on Feb. 17, 2006. (71) Applicants: GlaxoSmithKline Biologicals SA, (60) Provisional application No. 60/712,720, filed on Aug. Rixensart (BE); J. Craig Venter 29, 2005, provisional application No. 60/654,632, Institute, Inc., Rockville, MD (US) filed on Feb. 18, 2005. (72) Inventors: Vega MASIGNANI, Siena (IT): Danilo Gomes MORIEL, Monteriggioni (IT): Publication Classification Francesco BERLANDA SCORZA, Trento (IT): Nathalie NORAIS, Siena (51) Int. Cl. (IT): Maria Rita FONTANA, Siena C07K I4/245 (2006.01) (IT); Mariagrazia PIZZA, Siena (IT): A6139/08 (2006.01) Laura SERINO, Monticiano (IT): C07K 6/2 (2006.01) Herve TETTELIN, Gaithersburg, MD (52) U.S. Cl. (US) CPC ........... C07K 14/245 (2013.01); C07K 16/1232 (73) Assignees: GlaxoSmithKline Biologicals SA, (2013.01); A61 K39/0258 (2013.01); A61 K Rixensart (BE); J. Craig Venter 2039/523 (2013.01) Institute, Inc., Rockville, MD (US) (21) Appl. No.: 15/150,204 (57) ABSTRACT (22) Filed: May 9, 2016 Disclosed herein are various open reading frames from a Related U.S. Application Data strain of E. coli responsible for neonatal meningitis (MNEC), (62) Division of application No. 14/293,967, filed on Jun. 2, and a Subset of these that is of particular interest for preparing 2014, now Pat. No. 9,334.313, which is a division of compositions for immunising against MNEC infections. Patent Application Publication Aug. 25, 2016 Sheet 1 of 4 US 2016/0244489 A1 Ft. A 1,E-02 --&-ES-0---3-3-------------- HE3034 iHE3034 E334 FSO. FS. FSO Patent Application Publication Aug. 25, 2016 Sheet 2 of 4 US 2016/0244489 A1 timepoint mk-mo iii E3034 soooooooos -5 a O is FSO. tirect c. C. c. 3W - ria a WW 5 c a FSO. Patent Application Publication Aug. 25, 2016 Sheet 3 of 4 US 2016/0244489 A1 F. 3 Chaiienge ( 7 CF Ss tirepoint a K a re. took hooooo tir. 3i 40 350 3. 25 5. 0. s Patent Application Publication Aug. 25, 2016 Sheet 4 of 4 US 2016/0244489 A1 tirepoint ce & 3 eA wo-Con Cir. F. 48 US 2016/0244489 A1 Aug. 25, 2016 PROTEINS AND NUCLECACDS FROM 0009 Most previous ExPEC vaccines have been based on MENINGITIS/SEPSIS-ASSOCATED cell lysates or on cellular structures. SOLCOUROVACTM ESCHERICHA COL includes ten different heat-killed bacteria including six ExPEC strains, and a successful phase II clinical trial was CROSS REFERENCE TO RELATED reported in reference 2. URO-VAXOMTM is an oral tablet APPLICATIONS vaccine containing lyophilised bacterial lysates of 18 selected 0001. This application is a divisional of U.S. patent appli E. coli strains 3. Baxter Vaccines developed a UTI vaccine cation Ser. No. 14/293,967, filed Jun. 2, 2014, now U.S. Pat. based on pili from 6 to 10 different strains, but this product No. 9,334.313, which is a divisional of U.S. patent applica has been abandoned. MedImmune developed a product called tion Ser. No. 1 1/884.812, claiming an international filing date MEDI516 based on the Fimhadhesin complex 4, but phase of Feb. 17, 2006, now U.S. Pat. No. 8,758,764, which is a U.S. II clinical trials shows inadequate efficacy. Moreover, there National Phase of PCT/US2006/005913, filed Feb. 17, 2006, was a risk with this vaccine that it would also affect non which claims the benefit of U.S. Provisional Application No. pathogenic FimEI'? strains in the normal intestinal flora, and 60/654,632, filed Feb. 18, 2005 and U.S. Provisional Appli it was expected that it would be effective against UPEC cation No. 60/712,720, filed Aug. 29, 2005, all of which are strains only, because of its bladder-specific adherence mecha hereby incorporated by reference in the present disclosure in nism, leaving other ExPEC strains uncontrolled. their entirety. 0010. There is thus a need for improved ExPEC vaccines, including a need to move away from crude cell lysates and TECHNICAL FIELD towards better-defined molecules, and a need to identify fur ther antigens that are Suitable for inclusion in vaccines, par 0002. This invention is in the field of Escherichia coli ticularly antigens that are prevalent among clinical ExpEC biology, and in particular relates to immunogens for use in strains without also being found in commensal Strains. Within immunising against extraintestinal pathogenic E. coli (Ex the ExPEC group, there is a particular need to identify anti PEC) strains. gens Suitable for immunising against MNEC strains. 0011. One way of addressing these needs was reported in SUBMISSION OF SEQUENCE LISTING ON reference 5, where the inventors looked for genes present in ASCII TEXT FILE genomes of MLEE types B2 and D but absent from MLEE 0003. The content of the following submission on ASCII types A and B1. Further comparative approaches, based on text file is incorporated herein by reference in its entirety: a subtractive hybridisation, have been reported in references 6 computer readable form (CRF) of the Sequence Listing (file & 7. Virulence genes in ExPEC strains have also been iden name: 529552001711 SeqList.txt, date recorded: May 4, tified in reference 8. Reference 9 discloses an analysis of four 2016, size: 27.806 KB). pathogenicity islands in UPEC E. coli strain 536. 0012 Reference 10 used the genome sequence of UPEC BACKGROUND ART (O6:K2:H1) strain CFTO73 (11,12] to identify sequences not present in non-pathogenic E. coli strains. Reference 13 dis 0004 Few microorganisms are as versatile as E. coli. As closes a comparison of the genome sequence of E. coli human well as being an important member of the normal intestinal pyelonephritis isolate 536 (O6:K15:H31), an UPEC, with microflora of mammals, it has been widely exploited as a host sequence data for strains CFTO73 (UPEC), EDL933 (entero in recombinant DNA technology. In addition, however, E. hemorrhagic) and MG1655 (non-pathogenic laboratory coli can also be a deadly pathogen. strain). Genome sequences of pathogenic strains are available 0005 E. coli strains have traditionally been classified as either commensal or pathogenic, and pathogenic strains are in the databases under accession numbers AE005174, then Sub-classified as intestinal or extraintestinal strains. BAO00007 and NC-004431. A sequence from a non-patho Classification may also be based on the K antigens. The genic strain is available under accession number U00096. best-studied Kantigen is K, which is considered to be the 0013. It is an object of the invention to provide further major determinant of virulence among those strains of E. coli antigens for use in immunisation against pathogenic E. coli that cause neonatal meningitis. The K1 antigen is a strains, particularly against ExPEC Strains, and more particu homopolymer of C-2.8-linked sialic acid, like the capsular larly against MNEC strains. saccharide of serogroup B meningococcus. SUMMARY OF THE INVENTION 0006 More recent taxonomic techniques such as multilo cus enzyme electrophoresis (MLEE) classify E. coli into five 0014. The inventors have identified various open reading phylogenetic groups (A, B1, B2, D&E), and these groupings frames from a strain of E. coli responsible for neonatal men do not match the traditional ones. For instance, MLEE group ingitis (MNEC), and have identified a subset of these that is of B1 includes both commensal and pathogenic strains, and particularinterest for preparing compositions for immunising group D includes both intestinal and extraintestinal strains. against MNEC infections. 0007. The extraintestinal pathogenic strains (or “ExPEC 0015. In one aspect, the present invention relates to a strains 1) of E. coli fall into MLEE groups B2 and D, and polypeptide comprising: (a) an amino acid sequence selected include both uropathogenic (UPEC) strains and meningitis/ from the group consisting of SEQID NOS 706, 2, 4, 6, 8, 10. sepsis-associated (MNEC) strains. UPEC strains cause uri 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36,38, 40, 42, 44, nary tract infections (UTIs), and are a common cause of 46,48, 50, 52,54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, cystitis. They also cause pyelonephritis (and its complica 80, 82,84, 86, 88,90, 92,94, 96, 98, 100, 102, 104,106, 108, tions such as sepsis) and catheter-associated infections. 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 0008 MNEC strains cause neonatal meningitis (0.1 cases 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, per 1000 live births) with case fatality rates ranging from 25 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, to 40%, and are also responsible for around"/6 of sepsis cases. 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, US 2016/0244489 A1 Aug. 25, 2016 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 302, 304,306, 308, 310, 312,314, 316, 318,320, 322, 324, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 326, 328,330, 332, 334, 336,338, 340, 342, 344, 346, 348, 1720, 1722, 1724, 1726,
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