Stabilization of helicobacter urease

Details Stabilization of helicobacter urease Stabilization of helicobacter urease

1999-06-23

2004-03-23

Witz, Jean C. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S184000, C435S188000, C435S228000, C514S002600, C530S350000, C530S402000, C530S408000, C530S409000

Reexamination Certificate

active

06709851

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to genetic methods for stabilizing Helicobacter urease.
Helicobacter pylori
is a gram-negative bacterium and a gastroduodenal pathogen that causes gastritis, gastric and duodenal ulceration, and possibly gastric carcinoma in humans (Graham et al., Am. J. Gastroenterol. 82:283-286, 1987; Homick, N. Eng. J. Med. 316:1598-1600, 1987; Lee et al., Microb. Ecol. Health Dis. 1:1-16, 1988; Cover et al., Annu. Rev. Med. 40:269-285, 1989; Buck, Clin. Microbiol. Rev. 3:1-12, 1990). The bacterium produces large amounts of the enzyme urease, which is a multimeric nickel metallohydrolase that cleaves urea to ammonia and carbon dioxide (Hu et al., Infect. Immun. 58:992-998, 1990).
H. pylori
urease is localized both in the cytosol and on the extracellular surface of the bacterium (Hawtin et al., J. Gen. Microbiol. 136:1995-2000, 1990). Extracellular urease may protect the bacteria in the highly acidic stomach by hydrolyzing urea to ammonia, thereby creating a buffering cloud of ammonia that neutralizes the acid around the bacteria (Ferrero et al., Microbiol. Ecol. Health Dis. 4:121-134, 1991; Mobley et al., In
Helicobacter pylori: Basic Mechanisms to Clinical Cure
, Hunt et al. (Eds.) Kluver Acad. Pub., Dordrecht, 1994). Urease may also contribute to the pathogenicity of
H. pylori
by direct toxicity of ammonia and monochloramine to cells lining the gastric mucosa.
H. pylori
urease is immunogenic to humans and antigenicity is highly conserved among
H. pylori
strains (Ferrero et al., Mol. Microbiol 9:323-333, 1993; Gootz et al., Infect. Immun. 62:793-798, 1994). Antigenic conservation among ureases is the basis of protection of mice against
H. felis
infection when vaccinated with
H. pylori
urease (Ferrero et al., Mol. Microbiol 9:323-333, 1993). Antigenic cross-reactivity has also been demonstrated between
H. pylori
and
H. mustelae
ureases (Gootz et al., Infect. Immun. 62:793-798, 1994).
Urease enzymatic activity is toxic to animals and humans (Thomson et al., Am. J. Med. 35:804-812, 1963; Mobley et al., Microbiol. Rev. 53:85-108, 1985; LeVeen et al., Biomed. Pharmacother. 48(3-4):157-166, 1994). Anti-urease antibodies bind to urease, but generally do not inhibit urease enzymatic activity. For example, there is one report that claims to show inhibition of urease activity by monoclonal antibodies to urease (Nagata et al., Infect. Immun. 60:4826-4831, 1992). We tested monoclonal and polyclonal antibodies to urease and urease subunits, and found no inhibition of urease activity. Thomas et al. (J. Clin. Microbiol. 30:1338-1340) measured urease inhibitory activity in serum samples from children infected with
H. pylori
, and found that among thirteen serum samples showing urease binding activity, only one sample showed any urease inhibitory activity. These observations show that the catalytic and immunogenic domains of urease are different. This is further supported by a recent report that antigenic reactivity of a urease preparation was retained under storage conditions in which enzymatic activity was lost (Perez-Perez, Infect. Immun. 62:299-302, 1994). These results suggest that immunogenicity can be separated from potentially toxic enzymatic activity, which is an important characteristic of a potential vaccine.
Specific human antibody responses to urease are absent or weak in a high proportion, if not the majority, of infected individuals. Antibodies to urease administered to animals together with live Helicobacter protect against infection (Blanchard et al., Infect. Immun. 63:1394-1395, 1995). Together, these observations support the basis for the effectiveness of urease as a vaccine that induces a high-grade, urease-specific immune response protective against
H. pylori.
Nine genes have been identified in the
H. pylori
urease gene cluster (Cussac et al., J. Bacteriol. 174:2466-2473, 1992; Labigne et al., J. Bacteriol. 173:1920-193, 1992). These include the urease structural genes, encoding UreA and UreB, and the accessory genes, encoding Ure I, E, F, G, and H. These genes have been shown to be essential for urease activity. Hu et al. (Infect. Immun. 60:2657-2666, 1992) expressed genes encoding the UreA and UreB subunits of
H. pylori
urease in
E. coli
and showed that these two genes alone are sufficient to encode a fully assembled apoenzyme, which was structurally and immunologically identical to native urease, but catalytically inactive, due to the absence of nickel ions. Addition of nickel ions alone did not restore catalytic activity. Similar results were reported with other bacterial ureases. In other studies, recombinant
Klebsiella aerogenes
apourease, which differs substantially from
H. pylori
urease in subunit structure and overall amino acid sequence, was shown to be activated in vitro by incubation with carbon dioxide or bicarbonate, together with nickel ions (Park et al., Science 267:1156-1158, 1995). Based on crystallographic analysis, the
K. aerogenes
urease bi-nickel center is thought to include His 272, His 246, His 136, His 134, Asp 360, and Lys 219 of the Ure C subunit. By analogy, the
H. pylori
urease metallocenter may be described as including His 248, His 138, His 136, Asp 362, and Lys 219 of the Ure B subunit.
SUMMARY OF THE INVENTION
We have shown that treatment of Helicobacter urease with compounds that chemically modify urease amino acids can prevent in vitro activation of urease, as well as molecular aggregation of urease. We have also shown that including amino acid substitutions in urease can prevent in vitro activation of urease.
Accordingly, in one aspect, the invention features methods of stabilizing Helicobacter (e.g.,
H. pylori
) urease. In these methods, urease (e.g., recombinant or native urease) is contacted with a compound that modifies an amino acid of the urease. Stabilization of urease according to the invention can prevent activation of the urease, or prevent molecular aggregation of the urease.
Compounds used in these methods can be, for example, sulfhydryl-reactive compounds. For example, iodoacetamide (IAM), iodoacetic acid (IAA), 5-5′-dithio-bis-2-nitrobenzoic acid (DTNB), 2,2′-dithiodipyridine (DTDP), cystine, cystamine, methyl methanethiolsulfonate (MMTS), N-ethyl maleimide (NEM), dinitrofluorobenzene (DNFB), or trinitrobenzene sulfonic acid (TNBSA) can be used. Additional compounds that can be used in the invention are described further below.
Also included in the invention are Helicobacter (e.g.,
H. pylori
) urease polypeptides, for example, recombinant or native urease polypeptides, that have been stabilized by any of the methods described above. The stabilized urease polypeptides can be present in a pharmaceutically acceptable carrier or diluent.
In another aspect, the invention provides Helicobacter (e.g.,
H. pylori
) urease polypeptides having an amino acid mutation (or mutations) that prevents activation of the urease polypeptides. For example, the urease polypeptides can contain an amino acid mutation in urease amino acid histidine 136, histidine 248, or lysine 219 in UreB. The urease polypeptides having such a mutation can be present in a pharmaceutically acceptable carrier or diluent.
Also included in the invention are methods of inducing an immune response to Helicobacter in a patient. In these methods, one of the urease polypeptides described above is administered to a patient that is at risk of developing, but does not have, Helicobacter infection, or to a patient that has Helicobacter infection. The urease polypeptide can be administered to a mucosal surface of the patient, or it can be administered parenterally, e.g., by intravenous, intramuscular, or percutaneous. administration.
As used herein, a polypeptide, such as a Helicobacter urease polypeptide, is said to be “stabilized” if it has been treated or modified so that it maintains its molecular structure, to a degree sufficient to retain immunogenicity. Urease that has been treated with a compound, e.g., iodoacetamide (IAM), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethyl mal

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