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(12) United States Patent (10) Patent No.: US 8,758,764 B2 Masignani Et Al

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(12) United States Patent (10) Patent No.: US 8,758,764 B2 Masignani Et Al US008758764B2 (12) United States Patent (10) Patent No.: US 8,758,764 B2 Masignani et al. (45) Date of Patent: Jun. 24, 2014 (54) PROTEINS AND NUCLEICACIDS FROM WO WO-O2/O771.83 10, 2002 MENINGITIS/SEPSIS-ASSOCATED WO WO-2004/005535 A2 1/2004 ESCHERICHLA COLI WO WO-2005, O76O10 8, 2005 (75) Inventors: Vega Masignani, Siena (IT); Danilo OTHER PUBLICATIONS Gomes Moriel, Monteriggioni (IT): Houghten et al. (New Approaches to Immunization, Vaccines36, Francesco Berlanda Scorza, Trento Cold Spring Harbor Laboratory, p. 21-25, 1986.* (IT): Nathalie Norais, Siena (IT): Maria McGuinness et al. (Lancet 337: 514-517, Mar. 1991.* Rita Fontana, Siena (IT); Mariagrazia McGuinness et al. (Mol. Microbiol. 7:505-514, Feb. 1993).* Pizza, Siena (IT): Laura Serino, Accession No. Q46837 2002 (or Q2M9M2 or Q46838 or Q6BF5) or Monticiano (IT): Herve Tettelin, Science 277:1453-1474(1997).* Gaithersburg, MD (US) EMBL accession No. AJ617685 (Created on May 11, 2004), "Escherichia coli pathogenicity island V. strain 536.” (73) Assignees: Novartis Vaccines and Diagnostics Srl, UniProt accession No. Q46837 (Integrated on Apr. 16, 2002). “Puta Siena (IT); J. Craig Venter Institute, tive lipprotein acflD homolog.” Rockville, MD (US) Hughes et al. (Aug. 19, 1994). “Sequence analysis of the Vibrio cholerae acfD gene reveals the presence of an overlapping reading (*) Notice: Subject to any disclaimer, the term of this frame, orf7, which encodes a protein that shares sequence similarity patent is extended or adjusted under 35 to the FIiA and FIiC products of Salmonella,", Gene 146(1): 79-82. European Search Report mailed Dec. 23, 2008, for EP patent appli U.S.C. 154(b) by 16 days. cation No. 06748228.1, filed Feb. 17, 2006. European Examination Report mailed Dec. 29, 2008, for EP patent (21) Appl. No.: 11/884,812 application No. 06748228.1, filed Feb. 17, 2006. International Search Report mailed Feb. 15, 2007, for International (22) PCT Filed: Feb. 17, 2006 patent application No. PCT/US2006/005913, field Feb. 17, 2006. Bernadac et al. (1998) “Escherichia coli tol-pal mutants form outer (86). PCT No.: PCT/US2OO6/OO5913 membrane vesicles,” Journal of Bacteriology, 180(18): 4872-4878. S371 (c)(1), Wu (2002) “The relationship between outer membrane vesicles and (2), (4) Date: Mar. 26, 2008 pathogenicity” Foreign Medicine (Microbiology Section) 4:13-15. EBI accession No. Q8FKK3, Mar. 1, 2003 (version 56, dated May 1, 2013). (87) PCT Pub. No.: WO2006/089264 EBI accession No. Q8X6G3, Mar. 1, 2002 (version 71, dated May 1, PCT Pub. Date: Aug. 24, 2006 2013). Dean-Nystrom et al. (2002). "Vaccination of pregnant dams with (65) Prior Publication Data intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection.” Infect Immun. 70(5):2414-8. US 2008/O19347OA1 Aug. 14, 2008 Judge et al. (2004). “Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli O157:H7 Related U.S. Application Data shedding in feces.” Infect Immun. 72(1): 168-75. Perna et al. (2001). "Genome sequence of enterohaemorrhagic (60) Provisional application No. 60/712,720, filed on Aug. Escherichia coli O157:H7.” Nature. 4.09(6819):529-33. 29, 2005, provisional application No. 60/654,632, Welch et al. (2002). “Extensive mosaic structure revealed by the filed on Feb. 18, 2005. complete genome sequence of uropathogenic Escherichia coli.” Proc Natl AcadSci USA. 99(26): 17020-4. (51) Int. Cl. Bonacorsi, S. et al. (Apr. 2000). “Identification of Regions of the A6 IK39/08 (2006.01) Escherichia coli Chromosome Specific for Neonatal Meningitis-as C07K I4/245 (2006.01) sociated Strains.” Infection and Immunity 68(4): 2096-2101. (52) U.S. Cl. Database EMBL-EBI Accession No. AY395687, last updated Jan. USPC ..................... 424/185.1; 424/257.1; 530/300; 27, 2008, located at <http://www.ebi.ac.uk/cgi-bin/ 530/350 dbfetch?db=embl&id=AY395687&style=raw>, last visited on Jun. (58) Field of Classification Search 19, 2008, 18 pages. CPC combination set(s) only. (Continued) See application file for complete search history. (56) References Cited Primary Examiner — Padma V Baskar (74) Attorney, Agent, or Firm — Morrison & Foerster LLP U.S. PATENT DOCUMENTS 2003/O165870 A1 9, 2003 Blattner et al. (57) ABSTRACT 2004/O123343 A1 6/2004 La Rosa et al. Disclosed herein are various open reading frames from a FOREIGN PATENT DOCUMENTS strain of E. coli responsible for neonatal meningitis (MNEC), EP 1342784 A1 9, 2003 and a Subset of these that is of particular interest for preparing WO WO-9822575 5, 1998 compositions for immunising against MNEC infections. WO WO-O1? 66572 A2 9, 2001 WO WO-0216439 2, 2002 WO WO-O2/O5932O 8, 2002 16 Claims, 4 Drawing Sheets US 8,758,764 B2 Page 2 (56) References Cited Janke, B. etal. (May 15, 2001). “A SubtractiveHybridisation Analy sis of Genomic Differences between the Uropathogenic E. coli Strain OTHER PUBLICATIONS 536 and the E. coli K-12 Strain MG 1655.” FEMS Microbiology Letters 199(1):61-66. Gonzalez, M. et al. (May 1, 2001). "Adaptation of Signature-tagged Mutagenesis to Escherichia coli K1 and the Infant-rat Model of Rode, C. et al. (Jan. 1999). “Type-specific Contributions to Chromo Invasive Disease.” FEMS Microbiology Letters 198(2):125-128. some Size Differences in Escherichia coli,' Infection and Immunity Henry, T. et al. (Jul. 2004). “Improved Methods for Producing Outer 67(1):230-236. Membrane Vesicles in Gram-negative Bacteria.” Research in Zhang, L. et al. (Apr. 2000). “Molecular Epidemiologic Approaches Microbiology 155(6):437-446. to Urinary Tract Infection Gene Discovery in Uropathogenic International Search Report mailed on Feb. 15, 2007, for PCT Patent Escherichi coli,' Infection and Immunity 68(4):2009-2015. Application No. PCT/US2006/005913 filed on Feb. 17, 2006, 6 pageS. * cited by examiner U.S. Patent Jun. 24, 2014 Sheet 1 of 4 US 8,758,764 B2 FIGURE 1 1,E+09 1,E+08 1,E+07 1,E+06 1,E+05 1,E+04 1,E+03 1,E+02 V QY HE3034 HE3034 HE3034 FISIOL. FISIOL. FISIOL. U.S. Patent Jun. 24, 2014 Sheet 2 of 4 US 8,758,764 B2 FIGURE 2 O 1 2 3 4 timepoint -(- HE3034 O DH5 a O a FISOL. timepoint o C. a OMVIHE o O p OMV DH5 O. O. d. FSO. U.S. Patent Jun. 24, 2014 Sheet 3 of 4 US 8,758,764 B2 FIGURE 3 Challenge 10 7 CFU O 1 2 3 4. timepoint a Cd a roN mO ctrl. 400000 35OOOO 300000 250000 2OOOOO 150000 100000 50000 U.S. Patent Jun. 24, 2014 Sheet 4 of 4 US 8,758,764 B2 FIGURE 4 timepoint a {X a beA Our Ctrl. US 8,758,764 B2 1. 2 PROTEINS AND NUCLECACDS FROM There is thus a need for improved ExPEC vaccines, includ MENINGITIS/SEPSIS-ASSOCATED ing a need to move away from crude cell lysates and towards ESCHERICHLA COLI better-defined molecules, and a need to identify further anti gens that are Suitable for inclusion in vaccines, particularly RELATED APPLICATIONS antigens that are prevalent among clinical ExpEC strains without also being found in commensal strains. Within the This application claims the benefit of U.S. Provisional ExPEC group, there is a particular need to identify antigens Application No. 60/654,632, filed Feb. 18, 2005 and U.S. suitable for immunising against MNEC strains. Provisional Application No. 60/712,720, filed Aug. 29, 2005. One way of addressing these needs was reported in refer The teachings of both these applications are incorporated 10 ence 5, where the inventors looked for genes present in herein in their entirety by reference. genomes of MLEE types B2 and D but absent from MLEE types A and B1. Further comparative approaches, based on TECHNICAL FIELD subtractive hybridisation, have been reported in references 6 & 7. Virulence genes in ExPEC strains have also been This invention is in the field of Escherichia coli biology, 15 identified in reference 8. Reference 9 discloses an analysis of and in particular relates to immunogens for use in immunis four pathogenicity islands in UPEC E. coli strain 536. ing against extraintestinal pathogenic E. coli (EXPEC) Reference 10 used the genome sequence of UPEC (O6:K2: strains. H1) strain CFTO73 (11.12 to identify sequences not present in non-pathogenic E. coli Strains. Reference 13 discloses a BACKGROUND ART comparison of the genome sequence of E. coli human pyelo nephritis isolate 536 (O6:K15:H31), anUPEC, with sequence Few microorganisms are as Versatile as E. coli. As well as data for strains CFTO73 (UPEC), EDL933 (enterohemor being an important member of the normal intestinal microf rhagic) and MG1655 (non-pathogenic laboratory strain). lora of mammals, it has been widely exploited as a host in Genome sequences of pathogenic strains are available in the recombinant DNA technology. In addition, however, E. coli 25 databases under accession numbers AE005174, BAO00007 can also be a deadly pathogen. and NC-004431. A sequence from a non-pathogenic strain is E. coli strains have traditionally been classified as either available under accession number U00096. commensal or pathogenic, and pathogenic strains are then It is an object of the invention to provide further antigens Sub-classified as intestinal or extraintestinal strains. Classifi for use in immunisation against pathogenic E. coli Strains, cation may also be based on the Kantigens. The best-studied 30 particularly against ExPEC Strains, and more particularly K antigen is K1, which is considered to be the major against MNEC strains. determinant of virulence among those strains of E. coli that cause neonatal meningitis. The K1 antigen is a homopolymer SUMMARY OF THE INVENTION of C-2.8-linked sialic acid, like the capsular saccharide of serogroup B meningococcus.
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