Staphylococcal ABC transporter protein

Details Staphylococcal ABC transporter protein Staphylococcal ABC transporter protein

2000-09-29

2003-09-30

Duffy, Patricia A. (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

15 to 23 amino acid residues in defined sequence

C530S327000, C530S328000, C530S329000, C530S330000, C530S350000, C424S190100

Reexamination Certificate

active

06627730

ABSTRACT:

The present invention concerns the treatment and diagnosis of Staphylococcal infections, particularly those of
Staphylococcus aureus,
and provides a protein, epitopes of same, and antibodies and other binding and neutralising agents specific against same.
Multiple drug resistance (MDR) is an increasing problem amongst gram-positive bacteria (Banergee, S. N. et al. 1991, Am. J. Med. 91: 865-895; Shaberg, D. R. et al., 1991, Am. J. Med. suppl., 88: 72-75; Gaynes, R. P. et al., 1994, Infect. Dis. Clin. Pract., 6: 452-455), particularly in hospitals. In particular, methicillin-resistant
Staphylococcus aureus
(MRSA) and coagulase-negative staphylococci (CNS), particularly methicillin-resistant CNS, prove problematic, being resistant to all penicillins and cephalosporins. Resistance to other agents such as quinolones is widespread (Malabarta, A. et al., 1997, Eur. J. Med. Chem., 32: 459-478; Lewis, K., 1994, TIBS, 19: 119-123; Traub, W. H. et al., 1996, Chemotherapy, 42: 118-132). Treatment is typically effected using vancomycin or teicoplanin. However, resistance to these agents is spreading and so new therapies are needed.
WO 98/01154 discloses the use of bacterial and fungal ABC transporter proteins and neutralising agents specific against same in methods of treatment and diagnosis of the human or animal body. Enterococcal ABC transporter proteins having apparent molecular weights of 97 and 54 kDa are identified as being therapeutically useful, and various epitopes are also identified. Staphylococcal homologues of the IstA and IstB proteins of
Bacillus thuringiensis
(Menou et al., 1990, J. of Bacteriology, 173: 6689-6696) are also identified, the homologues having apparent molecular weights of 69 and 37 KDA and being immunodominant conserved antigens. Also identified are epitopes of same.
A Staphylococcal ABC transporter protein having an apparent molecular weight of 67 KDA has now been successfully isolated and purified by the present inventor from an epidemic MRSA strain, and has the coding sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. These sequences are partially identified by the
S. aureus
NCTC 8325 genome sequencing project as contig 1184, contig 1177 and contig 1158 containing amino-terminal sequence data. This protein has not previously been suggested to be an ABC transporter protein, and no diagnostic or therapeutic uses have previously been suggested for it. The protein has a calculated true molecular weight of 60.1 kDa, although post-translational modifications result in its being identified in experiments as having an apparent molecular weight of 67 kDa.
The role of the protein is neither suggested nor disclosed by the IstA and IstB homologues of WO 98/01154 since they have different sequences and different molecular weights. Additionally, the samples from which the IstA and IstB homologues were isolated were peritoneal dialysates rather than the blood and wound cultures used for the present invention (below), and such a purification method could not have led to the present invention since the previously used dialysis step caused a change in the relative proportions of antibody in the dialysate when compared to serum. Similarly, other known prior art does not suggest the role of the protein, nor does it suggest it to have a diagnostic or therapeutic use.
Thus according to the present invention there is provided a Staphylococcal ABC transporter protein having the sequence of SEQ ID NO: 2 or a partially modified form thereof or an immunogenic fragment thereof for use in a method of treatment or diagnosis of the human or animal body.
Immunogenic fragments of the protein include any fragment of the protein which elicit an immune response, and include epitopes (i.e. peptides carrying epitopes). Similarly, analogues (mimotopes) of epitopes may be readily created, the mimotopes having different sequences but displaying the same epitope and thus the term “immunogenic fragments” also encompasses immunogenic analogues of the fragments e.g. mimotopes. Epitopes may be readily determined and mimotopes readily designed (Geysen, H. M. et al., 1987, Journal of Immunological Methods, 102: 259-274; Geysen, H. M. et al.,1988, J. Mol. Recognit., 1(1):32-41; Jung, G. and Beck-Sickinger, A. G., 1992, Angew. Chem. Int. Ed. Eng., 31: 367-486).
The scope of the present invention does not extend to other non-Staphylococcal ABC transporter proteins, such as those of WO 98/01154. However, the invention does extend to encompass forms of the protein which have been insubstantially modified (i.e. which have been partially modified), particularly forms of the protein which display the same immunogenic properties as the protein itself.
By “partial modification” and “partially modified” is meant, with reference to amino acid sequences, a partially modified form of the molecule which retains substantially the properties of the molecule from which it is derived, although it may of course have additional functionality. Partial modification may, for example, be by way of addition, deletion or substitution of amino acid residues. Substitutions may be conserved substitutions. Hence the partially modified molecule may be a homologue of the molecules from which it was derived. It may, for example, have at least 70% homology with the molecule from which it was derived. It may for example have at least 80, 90 or 95% homology with the molecule from which it was derived. An example of a homologue is an allelic mutant. Similarly nucleotide sequences encoding the molecule or amino acid sequences may be partially modified to code for any such modifications to an amino acid sequence or molecule. Nucleotide sequences may also of course be modified such that they still code for the same amino acid residues but have a different nucleotide sequence.
The Staphylococcus may be
S.
aureus or it may for example be a coagulase-negative Staphylococcus,
S. epidermidis, S. haemolyticus, S. hyicus
or
S. saprophyticus.
An immunogenic fragment may comprise an ATP binding site or a part thereof. Peptides carrying (i.e. displaying) a number of epitopes of the ABC transporter protein have also been identified (below) and thus an immunogenic fragment of the protein may comprise the sequence of SEQ ID NO: 3,4,5,9,10,11 or 12. The epitopes of SEQ ID NOs: 3,4 and 5 are displayed by peptides having the sequences of SEQ ID NOs: 6,7 and 8 respectively, and thus an immunogenic fragment may comprise the sequence of SEQ ID NO: 6,7 or 8. In particular, experiments have shown that peptides having SEQ ID NOs: 6 and 7 which display epitopes having SEQ ID NOs: 3 and 4 are of particular therapeutic use. Peptides having the sequences of SEQ ID NOs: 13 and 14 have also been found to carry epitopes, antibody against which is therapeutic in an animal model (see experiments below) and thus an immunogenic fragment may have the formula of SEQ ID NO: 13 or 14. An additional epitope having the sequence of SEQ ID NO: 17 has also been found, and a peptide having the sequence of SEQ ID NO: 18 carrying same elicits the generation of polyclonal antisera specific against the 67 kDa antigen. Thus an immunogenic fragment may have the sequence of either one of SEQ ID NOs: 17 or 18.
The Staphylococcal ABC transporter protein, displaying epitopes including those described above, therefore provides a therapeutic and diagnostic opportunity—the protein and immunogenic fragments thereof may be used in therapy, both prophylactically (e.g. as immunostimulants such as vaccines) and for treatment of a Staphylococcal infection.
Binding agents and neutralising agents (such as antibodies) specific against the ABC transporter protein, immunogenic fragments thereof or partially modified forms thereof may also be used both diagnostically and therapeutically. Binding agents have a target to which they are specific, and in the case of a binding agent being an antibody, the target is an antigen. An example of a therapeutic medicament is antibody specific against the ABC transporter protein, and this may be employed in immunotherapy, for example pass

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