Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
2000-12-22
2004-08-24
Smith, Lynette R. F. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C424S198100, C530S313000, C530S326000, C530S350000
Reexamination Certificate
active
06780969
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to peptide compositions that are useful as immunogens for the prevention of urinary tract infection. The peptide immunogen of the present invention comprises a FimH Adhesin Functional Site-Derived (FAFSD) target peptide and a helper T cell epitope (Th) having multiple class II MHC binding motifs. Optionally, the immunogenic peptide further comprises an invasin domain, which acts as a general immune stimulator. The helper T cell epitope and the invasin domain enable the host to generate an immune response specific against the FAFSD target peptide to prevent the adherence of
Escherichia coli
and other enterobacteria to the bladder mucosa and confer protection against urinary tract infection.
BACKGROUND OF THE INVENTION
Urinary tract infection (UTI) is one of the most common disorders in women and children, resulting in 7-8 million physician and hospital visits per year at a cost of over $1 billion. It is estimated that by age 30, roughly 50 percent of women have had at least one incidence of UTI with 2-10 percent having recurrent UTI. Females are generally more prone to UTI because of their anatomy. Recent studies have shown that, on average, women who are 18-40 years old suffer 1-2 infections over a two year period. Older women are at risk with the incidence being as high as 30%. In most cases, UTI is not life threatening. Standard antibiotics usually offer quick relief, but when left untreated, the chronic recurrence of urinary tract infection can cause kidney damage and even death. A vaccine would reduce this toll but there has been little success in the development of a practicable vaccine for UTI (Service,
Science
1997, 276:533).
Earlier attempts to produce a UTI vaccine using whole fimbriae were not successful in protecting against a broad range of disease-causing bacteria. Intact whole fimbriae do not elicit a strong antibody response to FimH adhesin. (Hanson and Brinton,
Nature
1988, 332:265; Johnson, 1991; U.S. Pat. No. 4,454,117, Langermann et al, 1997). Vaccines comprising peptides of the major fimbrial protein FimA have been reported (Schmidt et al,
J Exp Med
, 1985; 161:705; U.S. Pat. No. 4,740,585). However, antibodies raised against FimA are not anti-adhesive and do not block attachment. Furthermore, vaccines based on the major components of the fimbriae contain variable sites and are expected to provide a narrow typespecific protection (Abraham et al, 1988).
Certain strains of
Escherichia coli
(
E. coli
) are the main cause of UTI. While many factors contribute to the initiation and progression of UTI, it is widely accepted that attachment of bacteria to tissue in the urinary tract is a first step in the initiation of active infection. A number of studies have pointed to a role for “fimbriae” or “pilus” organelles, the long filamentous proteinaceous appendages on the surface of
E. coli
, as the primary means by which the bacteria fasten onto urogenital tissue to establish an infection. Studies have shown that an overwhelming majority of the uropathogenic
E. coli
isolates express mannose-binding type 1 fimbriae (Johnson,
Clin Microbiol Rev
, 1991; 4:80).
Specifically, a cluster of eight to nine closely associated genes located in the bacterial chromosome are responsible for the biogenesis, assembly and function of type 1 fimbriae (Klemm & Christiansen,
Mol Gen Genet
, 1987; 208:439-445). Each type 1 fimbrial filament is 1-2 &mgr;m long with a diameter of 7 nm. It is a heteropolymer comprising a major subunit FimA and three minor subunits FimF, FimG, and FimH. The FimA subunits constitute >95% of the total fimbrial proteins and are arranged in a tight right-handed helix forming a central axial hole (Klemm & Christiansen, 1987; Johnson, 1991).
More specifically, type 1 fimbriae has, as a minor component, the mannose-binding FimH adhesin which is serologically conserved throughout the Enterobacteriaceae genera (Abraham et al,
Nature
, 1988; 336:682). Immune electron microscopy has revealed FimH to be placed strategically at the distal fimbrial tips and along the fimbriae at various intervals (Abraham et al,
J Bacteriol
, 1987; 169:5530). The FimH molecules that are localized at the fimbrial tips appear to be complexed with FimG in a flexible fibrillum structure (Jones et al.,
Proc Natl Acad Sci USA
, 1995; 92:2081). The presence of FimH is important for initiating bacterial infections in the urinary tract (Langermann et al,
Science
, 1997; 276:607). The mannose-binding domain of FimH is localized at the amino terminus region of FimH (Jones et al, 1995). The mannose-binding site is believed to promote attachment of the bacteria to D-mannose-containing receptors on the host mucosa cells. In fact, antibodies specific to residues of the amino terminus region of FimH inhibited attachment by type 1 fimbriated
E. coli
to human buccal cells and to the mouse bladder epithelium (Abraham et al, 1987; Thankavel et al,
J Clin Invest
, 1997; 100:1123). This indicates involvement of the mannose-binding domain of FimH in the adherence of fimbriae as a potential virulence determinant.
In light of the role played by FimH in promoting the adherence of
E. coli
, a more detailed structure-function study was conducted to map the functionally important domains within the FimH molecule. FimH is folded into two domains belonging to the all-beta class connected by a short linker. The full sequence of the FimH molecule is shown in FIG.
1
. The NH
2
-terminal mannose-binding lectin domain comprises residues 1 to156, and the COOH-terminal fimbriae domain, which anchors the adhesin to the fimbriae, comprises residues 160 to 279. The lectin domain of FimH is an 11-stranded elongated &bgr; barrel with a jellyroll-like topology. A pocket capable of accommodating a monomannose unit is located at the tip of the domain, distal from the connection to the pilin domain (Choudhury et al,
Science
, 1999; 285:1061).
The identification the tip adhesins of FimH, as a key virulence factor provided a specific target for vaccine development. In contrast to the variability of the major fimbrial protein, FimH is conserved throughout the genera of the Enterobacteriaceae. This has implications for the development of broadly protective vaccines against UTI (Abraham et al, 1988).
Recent vaccine development has been focused against the presumed FimH virulence determinant and have been more successful. Antibodies were raised in mice to two forms of FimH protein: 1) a complex containing the periplasmic chaperone FimC bound to full-length FimH protein, and 2) a naturally occurring mannose-binding FimH truncate corresponding to two-thirds of the FimH amino terminal blocked the ability of uropathogenic
E. coli
to bind to cells of a human bladder epithelial cell line, and protected mice from infection in vivo (Langermann et al, 1997). In a more recent in vivo study, a vaccine based on the FimH-FimC chaperone complex immunogen protected cynomolgus monkeys from infection by an
E. coli
cystitis isolate (Langermann et al,
J Infect Dis
, 2000; 181:774).
Thankanel et al (1997) reported that a domain localized in the FimH adhesin of
Escherichia coli
Type 1 fimbriae is capable of receptor recognition. He further reported the use of a domain specific antibody to confer protection against urinary tract infection. In that report, mice actively immunized with sFimH
1-25
peptide (see Table 3, SEQ ID NO:2) exhibited significantly lower levels of bacterial bladder colonization when challenged by type-1 fimbriated
E. coli
. As expected from the conservation of FimH sequence among type 1-fimbriated bacteria, broad immunological cross-reactivity was reported for type-1 fimbriated. Antibodies generated against a peptide-carrier protein immunogen wherein the peptide is SEQ ID NO:2 displayed significant cross-reactivity to type-1 frimbriated urinary tract isolates
Klebsiella pneumoniae
Cl111,
K. pneumoniae
Cl120,
Enterobacter aerogenes, E. coli
Cl115,
E. coli
Cl116,
E. coli
Cl118,
E. coli
Cl121,
E. coli
Cl123,
E. coli
Cl124,
E. coli
Cl5, and
Serr
Ford Vanessa L.
Morgan & Finnegan , LLP
Smith Lynette R. F.
United Biomedical Inc.
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