US0077.49518B2 (12) United States Patent (10) Patent No.: US 7,749,518 B2 Masignani et al. (45) Date of Patent: Jul. 6, 2010 (54) POLYPEPTIDES FROM NON-TYPEABLE Accession No. G86194(Date: Mar. 2, 2001).* HAEMOPHILUS INFLUENZAE Brusic, et al., “Prediction Of MHC Class II-Binding Peptides Using An Evolutionary Algorithm And Artificial Neural Network.” (75) Inventors: Vega Masignani, Siena (IT): Beatrice Bioinformatics 14(2): 121-130 (1998). Maria Arico, Siena (IT) Carter, “Epitope Mapping Of A Protein Using The Geysen (PEPSCAN) Procedure.” Metheds in Molecular Biology vol. 36. (73) Assignee: Novartis Vaccines and Diagnostics Peptide Analysis Protocols, Dunn & Pennington, Eds. Humana Press, Totowa, NJ, pp. 207-223 (1994). SRL, Siena (IT) Davenport, et al., “An Empirical Method For The Prediction Of (*) Notice: Subject to any disclaimer, the term of this T-Cell Epitopes.” Immunogenetics 42:392–397 (1995). 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Date: Nov. 24, 2005 Hopp, et al., “Retrospective: 12 Years Of Antigenic Determinant Predictions, And More.” Peptide Res 6(4): 183-190 (1993). (65) Prior Publication Data Jameson, et al., “The Antigenic Index: A Novel Algorithm For Pre US 2008/O267966 A1 Oct. 30, 2008 dicting Antigenic Determinants.” Cabios 4(1): 181-186 (1988). Li, et al., “Identification And Characterization Of Genomic Loci (30) Foreign Application Priority Data Unique To The Brazilian Purpuric Fever Clonal Group Of H. influenzae Biogroup Aegyptiud: Functionality Explored Using May 14, 2004 (GB) ................................. O410866.8 Meningococcal Homology.” Mol Microbiol 47(4): 1101-1111 (2003). (51) Int. Cl. Maksyutov, et al., “ADEPT: A Computer Program For Prediction Of A6 IK 39/02 (2006.01) Protein Antigenic Determinants.” Cabios 9(3):291-297 (1993). CI2P 2L/04 (2006.01) Mason, et al., “Nontypable Haemophilus influenzae Gene Expres C7H 2L/04 (2006.01) sion Induced In Vivo In A Chinchilla Model Of Otitis Media.” Inf C07K I4/00 (2006.01) Immun 71(6):3454-3462 (2003). McMichael, "Vaccines For Moraxella Catarrhalis.' Vaccine 19:S101 (52) U.S. Cl. .............. 424/256.1; 424/185.1; 424/190.1; s107 (2001). 424/2001; 435/69.1; 435/69.5; 435/69.7; Meister, et al., “Two Novel T Cell Epitope Prediction Algorithms 530/300, 530/350,536/23.5; 536/23.7 Based On MHC-Binding Motifs; Comparison Of Predicted And (58) Field of Classification Search ....................... None Published Epitopes From Myocobacterium Tuberculosis And HIV See application file for complete search history. Protein Sequences.” Vaccine 13(6):581-591 (1995). (56) References Cited Needleman, et al., “A General Method Applied To The Search For Similarities. In The Amino Acid Sequence Of Two Proteins,” J Mol FOREIGN PATENT DOCUMENTS Biol 48:443-453 (1970). WO WO 91.01146 A1 2, 1991 (Continued) WO WO99,11241 A1 3, 1999 WO WO99/44636 A2 9, 1999 Primary Examiner Robert B Mondesi WO WO99.52549 A1 10, 1999 Assistant Examiner Padma V Baskar WO WOOOO7621 A2 2, 2000 (74) Attorney, Agent, or Firm—Roberta L. Robins; Robins & WO WOOO, 23105 A2 4/2000 Pasternak LLP WO WOOO,56360 A2 9, 2000 WO WOO1,21152 A1 3, 2001 (57) ABSTRACT WO WOO1/21207 A2 3, 2001 WO WOO2,28889 A2 4/2002 WO WO O2/O83723 A 10, 2002 Polypeptides comprising non-typeable Haemophilus influen WO WOO3076575 A2 * 9, 2003 WO WO 2004/078949 A 9, 2004 Zae (NTHii) amino acid sequences. Over 2500 specific NTHi WO WO 2006,138.527 A2 12/2006 proteins are disclosed. The invention also provides related WO WO 2007/084053 A1 7/2007 polypeptides, nucleic acids, antibodies and methods. These can all be used in medicine for treating or preventing disease OTHER PUBLICATIONS and/or infection caused by H. influenzae, such as otitis media. Lederman et al (Molecular Immunology 28: 1171-1181, 1991).* Lietal (Proc. Natl. Acad. Sci., USA 77:3211-3214, 1980).* 6 Claims, No Drawings US 7,749,518 B2 Page 2 OTHER PUBLICATIONS Singhi, et al., “Evaluation Of Polymerase Chain Reaction (PCR) For Diagnosing Haemophilus influenzae B Meningitis.” Ann Trop Raddrizzani, et al., “Epitope Scanner Using Virtual Matrix-Based Microbiol 37(3):190-195 (2003). Algorithms.” Breifings In Bioinformatics 1(2): 179-189 (2000). Welling, et al., “Prediction Of Sequential Antigenic Regions. In Pro teins.” FEBS 188(2): 215-218 (1985). Rice, et al., “EMBOSS: The European Molecular Biology Open Yadav, et al., “Rapid Detection Of Haemophius influenzae By Hel Software Suite.” Trends Genet 16:276-277 (2000). Gene Polymerase Chain Reaction.” Lett Applied Microbiol 37:190 Roberts, et al., “Prediction Of HIV Peptide Epitopes By A Novel 195 (2003). Alforithm.” AIDS Res Hum Retroviruses 12(7):593-610 (1996). * cited by examiner US 7,749,518 B2 1. 2 POLYPEPTIDES FROM NON-TYPEABLE emergence of multiple-antibiotic resistant microorganisms. HAEMOPHILUS INFLUENZAE Progress towards a vaccine is slow due to an incomplete understanding of both the pathogenesis of OM and the RELATED APPLICATIONS immune response to it. The genome sequence of the serotype distrain KW2013 This application is the U.S. National Phase of International has been useful for understanding basic H. influenzae biol Application No. PCT/IB2005/001775, filed May 16, 2005 ogy, but it has not been so useful in countering pathogenic H. and published in English, which claims priority to Great influenzae strains, as serotype d strains are generally not Britain Application No. 0410866.8, filed May 14, 2004. The pathogens. teachings of the above applications are incorporated herein in 10 It is an object of the invention to provide polypeptides for their entirety by reference. use in the development of vaccines for preventing and/or All documents cited herein are incorporated by reference treating infections caused by non-typeable H. influenzae in their entirety. strains. In particular, it is an object to provide polypeptides for use in improved vaccines for preventing and/or treating otitis INCORPORATION BY REFERENCE OF 15 media. The polypeptides may also be useful for diagnostic MATERIAL ON COMPACT DISC purposes, and as targets for antibiotics. This application incorporates by reference the Sequence DISCLOSURE OF THE INVENTION Listing contained on the two compact discs (Copy 1 and Copy 2), filed concurrently herewith, containing the following file: Polypeptides File name: PP022930.0003 SequenceList.txt; created Nov. The invention provides polypeptides comprising the H. 10, 2006; 6,676 KB in size. influenzae amino acid sequences disclosed in the examples. These amino acid sequences are the even SEQ ID NOs TECHNICAL FIELD 25 between 2 and 5080. There are thus 2540 amino acid sequences, and these are referred to as NTHnnnn, where nnnn This invention is in the field of Haemophilus influenzae is a number between 0001 and 2832 (there are 292 NTHnnnn immunology and vaccinology. numbers that have no sequence; see Table I). Further NTHi sequences of the invention are given as SEQID NOS: 5088 BACKGROUND ART 30 onwards. The invention also provides polypeptides comprising Haemophilus influenzae is a small, non-motile, Gram amino acid sequences that have sequence identity to the H. negative coccobacillus. It is a respiratory pathogen that Influenzae amino acid sequences disclosed in the examples. causes a wide spectrum of human infections, including: Depending on the particular sequence, the degree of sequence asymptomatic colonization of the upper respiratory tract (i.e. 35 identity is preferably greater than 50% (e.g. 60%, 70%, 75%, carriage); infections that extend from colonized mucosal Sur 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, faces to cause otitis media (inflammation of the middle ear), 98%, 99% or more). These polypeptides include homologs, bronchitis, conjunctivitis, sinusitis, urinary tract infections orthologs, allelic variants and functional mutants. Typically, and pneumonia; and invasive infections, such as bacteremia, 50% identity or more between two polypeptide sequences is septic arthritis, epiglottitis, pneumonia, empyema, pericardi 40 considered to be an indication of functional equivalence. tis, cellulitis, osteomyelitis and meningitis. H. influenzae was Identity between polypeptides is preferably determined by the first bacterium for which a complete genome sequence the Smith-Waterman homology search algorithm as imple was published 1. mented in the MPSRCH program (Oxford Molecular), using H. influenzae strains are either capsulated (typeable) or an affine gap search with parameters gap open penialty=12 non-capsulated (non-typeable), and there are six major sero 45 and gap extension penalty=1. logical types of capsulated strains (a to f). 95% of H. influ These polypeptide may, compared to the NTHiSequences enzae-caused invasive diseases are caused by H. influenzae of the examples, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, type b (Hib) strains. The most serious manifestation of Hib 9, 10, etc.) conservative amino acid replacements i.e. replace disease is meningitis, but the introduction in the 1980s of ments of one amino acid with another which has a related side vaccines based on conjugated Hib capsular saccharides has 50 chain.