Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2001-05-24
2004-07-27
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S185100, C424S190100, C424S242100, C530S350000
Reexamination Certificate
active
06767545
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel composition and method for treatment and prevention of infection by
Pseudomonas aeruginosa.
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BACKGROUND OF THE INVENTION
Pseudomonas aeruginosa
is a significant opportunistic pathogen that causes a variety of life-threatening infections in immunosuppressed or immunocompromised patients [1-4]. Individuals who are at risk of developing
P. aeruginosa
infections include cystic fibrosis patients, burn patients, severe neutropenic patients (e.g., cancer patients receiving chemotherapy) and intensive care unit patients receiving respiratory support. The cost of these infections is high, >60,000 lives per year in North America and about $5 billion/year in health care costs.
The first step in the Pseudomonas infection process appears to be the attachment to the host cell. This attachment is mediated by pili on the surface of the bacterium [2, 5, 6].
P. aeruginosa
uses several adhesins to mediate attachment to mucosal surfaces, but analysis of the binding properties of the adhesins [1, 7, 8] and binding competition studies [9] indicate that the pilus is the dominant adhesin responsible for initiating infections [1].
P. aeruginosa
pili have a structure resembling a hollow tube of about 5.2 nm in outer diameter, 1.2 nm in central channel diameter, and an average length of 2.5 &mgr;m [10-12]. The pilus of
P. aeruginosa
is composed of multiple copies of a 13-17 kDa monomeric protein subunit called pilin, which are capable of self-assembling into pili.
The C-terminal region of the pilin monomer contains the epithelial cell binding domain [5, 12], and is semiconserved in seven different strains of this bacterium [13, 14]. This semiconserved region has also been shown to bind to a minimal structural carbohydrate receptor sequence, &bgr;-GalNAc(1-4)&bgr;Gal, found in glycosphingolipids, specifically asialo-GM1 and asialo-GM2 [15, 16]. There is evidence that pili binding to a host cell is mediated multivalent binding of C-terminal binding domains in each pili to epithelial-cell receptors, with such binding serving to mobilize receptors on the cells. This, in turn, may be responsible to cytokine, e.g., IL-8 production by the host cells and consequent inflammatory response.
The C-terminal disulfide-bridged 17-residue region of the PAK pilin is known to be important in raising antibodies that block binding of both bacteria or their pili to epithelial cells [6, 17, 18]. Both monoclonal antisera generated from
P. aeruginosa
pili or polyclonal antisera generated from synthetic peptides representing the receptor binding domain of the pathogen have been shown to be efficacious in preventing infection [19].
The ability of antibodies produced against the C-terminal pilin-peptide domain to effectively inhibit Pseudomonas infection has been demonstrated (see, for example, U.S. Pat. No. 5,468,484), and the use of the pilin-peptide domain for use in vaccination against Pseudomonas infection has also been demonstrated, e.g., U.S. Pat. Nos. 5,445,818, 5,494,672, and 5,612,036.
It would also be desirable to directly treat an existing Pseudomonas infection, or to treat an individual at risk of Pseudomonas infection prophylactically. Although intact pili have been proposed for this purpose, this method is limited by the fact that isolated, self-assembled pili have the ability to provoke a strong inflammatory response. Alternatively, the C-terminal pilin peptide has been proposed for this purpose, but this approach is limited by the relatively weak binding of the peptide to the host-cell receptor sites.
SUMMARY OF THE INVENTION
The invention includes, in one aspect, a composition for use in treating or preventing infection by
Pseudomonas aeruginosa
. The composition comprises a
P. aeruginosa
pilin protein having an N-terminal peptide region modified to prevent self assembly of the peptide. The peptide may be formulated in a pharmaceutically acceptable carrier, such as an aerosolizable liquid or particle vehicle, or an injectable solution.
In one general embodiment, the modified N-terminal peptide region lacks an N-terminal portion of native
P. aeruginosa
, preferably the first 15 up to the first 40 amino acids residues of native
P. aeruginosa
, more preferably the first 25 up to the first 30 amino acids.
In another general embodiment, the N-terminal region is modified, e.g., by amino acid substitutions, to prevent or inhibit alpha-helix formation in the N-terminal region, thereby preventing the pilin peptide from self-assembling.
In still another embodiment, the N-terminal region of the pilin peptide is replaced by a peptide moiety capable of forming a coiled-coil heterodimer or homodimer structure with an oppositely charged or identical alpha-helix forming peptide moiety, as represented by a so-called leucine zipper peptide. The modified pilin peptide can form dimeric structures which have higher binding affinity to host cells than the corresponding monomer, by virtue of divalent binding, but which are less inflammatory than intact pili, due to t
Glasier Linda M. G.
Hazes Bart
Irvin Randall T.
Parimi Sastry A.
Read Randy J.
Governors of the University of Alberta
Graser Jennifer E.
Parimi Sastry A.
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