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  Helicobacter pylori proteins useful for vaccines and diagnosticsUSPTO Application #: 20070243204Title: Helicobacter pylori proteins useful for vaccines and diagnostics Abstract: This invention provides polypeptides of Helicobacter Pylori cytotoxin associated immunodominant (CAI) antigen. The invention also provides prophylactic and therapeutic vaccines comprising polypeptides of Helicobacter pylori CAI antigen, and methods for preparation. The invention also provides methods of treatment of Helicobacter pylori infection using these vaccines. (end of abstract) Agent: Woodcock Washburn LLP - Philadelphia, PA, US Inventors: Antonello Covacci, Massimo Bugnoli, John Telford, Rino Rappuoli, Giovanni Macchia USPTO Applicaton #: 20070243204 - Class: 424190100 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Antigen, Epitope, Or Other Immunospecific Immunoeffector (e.g., Immunospecific Vaccine, Immunospecific Stimulator Of Cell-mediated Immunity, Immunospecific Tolerogen, Immunospecific Immunosuppressor, Etc.), Amino Acid Sequence Disclosed In Whole Or In Part; Or Conjugate, Complex, Or Fusion Protein Or Fusion Polypeptide Including The Same, Disclosed Amino Acid Sequence Derived From Bacterium (e.g., Mycoplasma, Anaplasma, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070243204. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 09/360,685, filed Jul. 26, 1999 which is a divisional of U.S. application Ser. No. 08/471,491, filed Jun. 6, 1995, now U.S. Pat. No. 6,090,611, which is a divisional of U.S. application Ser. No. 08/256,848, filed Oct. 21, 1994, now abandoned, which is a U.S. national phase application of PCT/EP93/00472, filed Mar. 2, 1993 and PCT/EP93/00158, filed Jan. 25, 1993, which two PCT applications claimed priority benefit of Italian application Serial No. FI 92 A 000052, filed Mar. 2, 1992, the entire contents of each application is incorporated by reference herein. BACKGROUND OF THE INVENTION 1. Field of the Disclosure [0002] The present invention relates generally to certain Helicobacter pylori proteins, to the genes which express these proteins, and to the use of these proteins for diagnostic and vaccine applications. [0003] Helicobacter pylori is a curved, microaerophilic, gram negative bacterium that has been isolated for the first time in 1982 from stomach biopsies of patients with chronic gastritis, Warren et al., Lancet i:1273-75 (1983). Originally named Campylobacter pylori, it has been recognized to be part of a separate genus named Helicobacter, Goodwin et al., Int. 3. Syst. Bacteriol. 39:397-405 (1989). The bacterium colonizes the human gastric mucosa, and infection can persist for decades. During the last few years, the presence of the bacterium has been associated with chronic gastritis type B, a condition that may remain asymptomatic in most infected persons but increases considerably the risk of peptic ulcer and gastric adenocarcinoma. The most recent studies strongly suggest that H. pylori infection may be either a cause or a cofactor of type B gastritis, peptic ulcers, and gastric tumors, see e.g., Blaser, Gastroenterology 93:371-83 (1987); Dooley et al., New Engl. J. Med. 321:1562-66 (1989); Parsonnet et al., New Engl. J. Med. 325:1127-31 (1991). H. pylori is believed to be transmitted by the oral route, Thomas et al., Lancet i:340, 1194 (1992), and the risk of infection increases with age, Graham et al., Gastroenterology 100: 1495-1501 (1991), and is facilitated by crowding, Drumm et al., New Engl. J. Med. 4322:359-63 (1990); Blaser, Clin. Infect. Dis. 15:386-93 (1992). In developed countries, the presence of antibodies against H. pylori antigens increases from less than 20% to over 50% in people 30 and 60 years old respectively, Jones et al., Med. Microbio. 22:57-62 (1986); Morris at al., N.Z. Med. J. 99:657-59 (1986), while in developing countries over 80% of the population are already infected by the age of 20, Graham et al., Digestive Diseases and Sciences 36:1084-88 (1991). [0004] The nature and the role of the virulence factors of H. pylori are still poorly understood. The factors that have been identified so far include the flagella that are probably necessary to move across the mucus layer, see e.g., Leying et al., Mol. Microbiol. 6:2863-74 (1992); the urease that is necessary to neutralize the acidic environment of the stomach and to allow initial colonization, see e.g., Cussac et al., J. Bacteriol. 174:2466-73 (1992); Perez-Perez et al., J. Infect. Immun. 60:3658-3663 (1992); Austin et al., J. Bacteriol. 174:7470-73 (1992); PCT Publ. No. WO 90/04030; and a high molecular weight cytotoxic protein formed by monomers allegedly having a molecular weight of 87 kDa that causes formation of vacuoles in eukaryotic epithelial cells and is produced by H. pylori strains associated with disease, see e.g., Cover et al., J. Bio. Chem. 267:10570-75 (1992) (referencing a "vacuolating toxin" with a specified 23 amino acid N-terminal sequence); Cover et al., J. Clin. Invest. 90:913-18 (1992); Leunk, Rev. Infect. Dis. 13:5686-89 (1991). Additionally, the following is also known. [0005] H. pylori culture supernatants have been shown by different authors to contain an antigen with a molecular weight of 120, 128, or 130 kDa, Apel at al., Aentralblat fur Bakteriol. Microb. und Hygiene 268:271-76 (1988); Crabtree et al., J. Clin. Pathol 45:733-34 (1992); Cover et al., Infect. Immun. 58:603-10 (1990); Figura et al., H. pylori, gastritis and peptic ulcer (ads. Malfrtheiner et al.), Springer Verlag, Berlin (1990). Whether the difference in size of the antigen described was due to interlaboratory differences in estimating the molecular weight of the same protein, to the size variability of the same antigen, or to actual different proteins was not clear. No nucleotide or amino acid sequence information was given about the protein. This protein is very immunogenic in infected humans because specific antibodies are detected in sera of virtually all patients infected with H. pylori, Gerstenecker et al., Eur. J. Clin. Microbiol. 11:595-601 (1992). [0006] H. pylori heat shock proteins (hsp) have been described, Evans et al., Infect. Immun. 60:2125-27 (1992) (44 amino acid N-terminal sequence and a molecular weight of about 62 kDa); Dunn et al., Infect. Immun. 60:1946-51 (1992) (33 amino acids found in the N-terminal sequence and a molecular weight of about 54 kDa); Austin et al., J. Bacterial. 174:7470-73 (1992) (37 amino acids found in the N-terminal sequence and a molecular weight of about 60 kDa). Austin et al. suggest that these are, in fact, the same protein with identical amino acid sequences at their N-terminus. [0007] For examples of diagnostic tests based on H. pylori lysates or semipurified antigens, see Evans et al., Gastroenterology 96:1004-08 (1989); U.S. Pat. No. 4,882,271; PCT Publ. No. WO 89/08843 (all relating to compositions and assays containing the same having high molecular weight antigens (300-700 kDa) from the outer membrane surface with urease activity); EPO Publ. No. 329 570 (relating to antigenic compositions for detecting H. pylori antibodies having fragments of at least one fragment from the group 63, 57, 45, and 31 kDa). [0008] The percentage of people infected by H. pylori, either in a symptomatic or an asymptomatic form, is very high in both developing and developed countries, and the cost of hospitalization and therapy makes desirable the development of both H. pylori vaccines and further diagnostic tests for this disease. SUMMARY OF THE INVENTION [0009] The present invention describes nucleotide and amino acid sequences for three major H. pylori proteins. Specifically, these are the cytotoxin, the "Cytotoxin Associated Immunodominant" (CAI) antigen, and the heat shock protein. None of the complete amino acid sequences for these proteins has been known, nor have their genes been identified. The present invention pertains to not only these purified proteins and their genes, but also recombinant materials associated therewith, such as vectors and host cells. The present invention provides cytotoxin polypeptides that exhibit substantially no toxicity or substantially reduced toxicity. The present invention also provides CAI and heat shock polypeptides that exhibit no functional contribution to toxicity, or a substantially reduced functional contribution to toxicity. The understanding at the molecular level of the nature and the role of these proteins and the availability of recombinant production has important implications for the development of new diagnostics for H. pylori and for the design of vaccines that may prevent H. pylori infection and treat disease. [0010] As such, these proteins can be used in both vaccine and diagnostic applications. The present invention includes methods for treating and diagnosing those diseases associated with H. pylori. As H. pylori has been associated with type B gastritis, peptic ulcers, and gastric adenocarcinoma, it is hoped that the present invention will assist in early detection and alleviation of these disease states. Currently, diagnosis relies mostly on endoscopy and histological staining of biopsies; existing immunoassays are based on H. pylori lysates or semipurified antigens. Given the heterogeneity found in such assays, correlation with disease state is not yet well established. Thus, the potential for recombinant antigen-based immunoassays, as well as nucleic acid assays for disease detection, is great. At present, there is no commercial vaccine for H. pylori infection or treatment. A recombinant vaccine is thus an object of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIGS. 1A, 1B, and 1C (SEQ ID NO:2) comprise the nucleotide sequence for the cytotoxin (CT) protein. [0012] FIG. 2 (SEQ ID NO:3) is the amino acid sequence for the cytotoxin (CT) protein. [0013] FIG. 3 is a map of the cai gene for the CAI protein and summary of the clones used to identify and sequence this gene. In the middle of FIG. 3, upstream of the D3 box, two short peptide sequences are shown: "NEPIYA" (SEQ ID NO:29) and "EEPIYA" (SEQ ID NO:30). At the bottom of the FIG. 3, the nucleotide (SEQ ID NO: 11) and deduced amino acid sequence (SEQ ID NO: 12) of the cloned segment is shown with peptides D1 (SEQ ID NO: 14), D2 (SEQ ID NO: 16) and D3 (SEQ ID NO: 17) shown boxed. [0014] FIGS. 4A through 4F (SEQ ID NO:4 and SEQ ID NO:5) comprise the nucleotide and amino acid sequences of the CAI antigen. The numbers along the left hand margins of FIGS. 4A, 4C and 4E designate the amino acid positions, and the numbers along the right-hand margins of FIGS. 4B, 4D, and 4F designate the nucleotide positions. Shown boxed in FIG. 4C-D are two repeats of the peptide EFKNGKNKDFSK (SEQ ID NO:9), which are encoded by the nucleic acid sequence of SEQ ID NO: 19. Also shown boxed in FIG. 4C-D are two repeats of the peptide EPIYA (SEQ ID NO: 10), the first of which is encoded by the nucleic acid sequence of SEQ ID NO:20, the second of which is encoded by the nucleic acid sequence of SEQ ID NO:21. Also shown boxed in FIG. 4C-D is the peptide FPLKRHDKVDDLSKV (SEQ ID NO:28), which is encoded by the nucleic acid sequence of SEQ ID NO:22. [0015] FIGS. 5A, 5B and 5C (SEQ ID NO:7 and SEQ ID NO:6) comprise the nucleotide and amino acid sequences of the heat shock protein (hsp). DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS A. General Methodology [0016] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION (1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION AND TRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE (R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C. Blackwell eds 1986). [0017] Standard abbreviations for nucleotides and amino acids are used in this specification. All publications, patents, and patent applications cited herein are incorporated by reference. Continue reading... Full patent description for Helicobacter pylori proteins useful for vaccines and diagnostics Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Helicobacter pylori proteins useful for vaccines and diagnostics patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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