Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-08-04
2001-10-09
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S192100, C424S234100, C424S243100, C530S300000, C530S350000
Reexamination Certificate
active
06299880
ABSTRACT:
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides and recombinant host cells transformed with the polynucleotides.
BACKGROUND OF THE INVENTION
Several cell surface associated proteins of the Staphylococci and Streptococci involved in microbial adhesion to different host tissues and considered to be important factors in bacterial pathogenesis have been identified in the last decade (see Patti, J. M., Allen, B. L., McGavin, M. J. and Hook, M., MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues [1994] Annu.Rev.Microbiol. 48, 585-617.).
The LPXTG motif has been identified as characteristic of surface proteins in Grain-positive bacteria (Navarre, W. W. and Schneewind,O. [1994] Molecular Microbiology 14(1) 115-121); Fischetti et al. [1990] Mol. Microbiol. 4 1603-5; Schneewind et al. [1993] EMBO J. 4803-4811).
Navarre, W. W. and Schneewind, O. [1994] demonstrate that the position of the LPXTG motif in a cell surface protein dictates that region of the protein which is anchored to the cell wall and the proportion of the N-terminal fragment which no longer resides in the cytoplasm.
Different approaches have been put forward to address such proteins from
Staphylococcus aureus
as antibacterial targets, e.g. fibronectin binding proteins (EP0294349, EP0397633, WO94/18327), fibrinogen binding protein (WO94/06830), collagen binding protein (WO92/07002) and bone sialoprotein binding protein (WO94/13310). The binding proteins or binding fragments thereof are used as antibacterial agents to block binding of the organism to host tissue, as vaccines to raise antibodies to the organism in the host animal or as antigens to raise therapeutic antibodies which can be used to block binding of the organism to host tissue.
Recently several novel approaches have been described which purport to follow global gene expression during infection (Chuang, S. et al. [1993] Global Regulation of Gene Expression in
Escherichia coli
J. Bacteriol. 175, 2026-2036, Mahan, M.J. et al. [1993] Selection of Bacterial Virulence Genes That Are Specifically Induced in Host Tissues SCIENCE 259, 686-688, Hensel, M. et al. [1995] Simultaneous Identification of Bacterial Virulence Genes by Negative Selection SCIENCE 269, 400-403). These new techniques have so far been demonstrated with gram negative pathogen infections and not with infections with gram positives presumably due to the much slower development of global transposon mutagenesis and suitable vectors needed for these strategies in these organisms, and in the case of that process described by Chuang, S. et al.[1993] the difficulty of isolating suitable quantities of bacterial RNA free of mammalian RNA derived from the infected tissue to furnish bacterial RNA labelled to sufficiently high specific activity. The present invention employs a novel technology to determine gene expression in the pathogen at different stages of infection of the mammalian host. A novel aspect of this invention is the use of a suitably labelled oligonucleotide probe which anneals specifically to the bacterial ribosomal RNA in Northern blots of bacterial RNA preparations from infected tissue. Using the more abundant ribosomal RNA as a hybridisation target greatly facilitates the optimisation of a protocol to purify bacterial RNA of a suitable size and quantity for RT-PCR from infected tissue.
A suitable oligonucleotide useful for applying this method to genes expressed in
Staphylococcus aureus
is
5′-gctcctaaaaggttactccaccggc-3′
Use of the technology of the present invention enables identification of bacterial genes transcribed during infection, inhibitors of which would have utility in anti-bacterial therapy. Specific inhibitors of such gene transcription or of the subsequent translation of the resultant mRNA or of the function of the corresponding expressed proteins would have utility in anti-bacterial therapy.
SUMMARY OF THE INVENTION
The present invention relates to a novel cell surface protein from
S. aureus
WCUH 29, characterised in that it comprises the amino acid sequence given in SEQ ID NO 1, or a fragment, analogue or derivative thereof.
The invention also relates to a polypeptide fragment of the cell surface protein, having the amino acid sequence given in SEQ ID NO 1, or a derivative thereof.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.
In particular the invention provides a polynucleotide having the DNA sequence given in SEQ ID NO 2.
The present invention also provides a novel protein from
Staphylococcus. aureus
WCUH29 obtainable by expression of a gene characterised in that it comprises the DNA sequence given SEQ ID NO 2, or a fragment, analogue or derivative thereof.
The invention also relates to novel oligonucleotides, including SEQ ID NOs 3 and 4, derived from the sequences SEQ ID NO 2.
The present invention includes variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptide characterised by the deduced amino acid sequence of SEQ ID NO 1.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is provided the use of a polypeptide of the invention for therapeutic or prophylactic purposes, for example, as an antibacterial agent or a vaccine.
In accordance with another aspect of the present invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunisation.
Further provided is a method for identifying compounds which bind to and inhibit an activity of the polypeptide of SEQ ID NO:1 comprising: contacting a cell expressing on the surface thereof a binding means for the polypeptide, said binding means being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said binding means, with a compound to be screened under conditions to permit binding to the binding means; and determining whether the compound binds to and activates or inhibits the binding by detecting the presence or absence of a signal generated from the interaction of the compound with the binding means.
Also provided is an antibody against the polypeptide of SEQ ID NO:1. Still further provided is an antagonist which inhibits the activity of the polypeptide of SEQ ID NO:1.
A method is also provided for the treatment of an individual having need to inhibit the polypeptide of SEQ ID NO:1 comprising: administering to the individual a therapeutically effective amount of an antagonist against the polypeptide of the invention.
Provided is a process for diagnosing a disease related to expression of the polypeptide of the invention comprising:determining a nucleic acid sequence encoding the polypeptide of SEQ ID NO:1.
A diagnostic process is provided comprising: analyzing for the presence of the polypeptide of SEQ ID NO:1 in a sample derived from a host.
Also provided is an antibody against the polypeptide of SEQ ID NO:1. Still further provided is an antagonist which inhibits the activity of the polypeptide of SEQ ID NO:1.
A method is also provided for the treatment of an individual having need to inhibit binding polypeptide of the invention comprising: administering to the individual a therapeutically effective amount of an antagonist against such polypeptide.
Provided is a process for diagnosing a disease related to expression of the polypeptide of the invention comprising:determining a nucleic acid sequence encoding the polypeptide of SEQ ID NO:1.
A diagnostic process is pr
Burnham Martin Karl Russell
Hodgson John Edward
Deibert Thomas S.
Gimmi Edward R.
King William T.
Navarro Mark
SmithKline Beecham Plc
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