Patent 6,881,410

The present invention provides bacterial and fungal ABC transporter proteins, immunogenic fragments thereof, neutralising agents specific thereto and binding agents specific thereto for therapeutic and diagnostic use, together with diagnostic test methods, methods of same and kits for performing same. Also provided are immunodominant conserved antigens from gram positive staphylococci, together with neutralising and binding agents specific thereto for use in therapy and diagnosis, and methods of same. Also provided are Staphylococcal holomogues of IstA and IstB and immunogenic fragments thereof, and their uses in methods of treatment and diagnosis of the human or animal body.

Description of the Invention

The present invention concerns the treatment and diagnosis of infections of bacteria and fungi, particularly of gram positive cocci, in particular enterococci and staphylococci.

The widespread use of antibiotics and other agents (for example penicillin vancomycin, methicillin, cephalosporin, tetracycline, chloramphenicol, glycopeptides and aminoglycosides) to treat bacterial infections has led to the rapid development of bacteria resistant to the agents (see for example CDC, 1993, JAMA, 270: 1796; IDCP, 1996, Infect Dis. Clin Pract., 5: 1-2; Spera, R. V. and Farber, B. F., 1994, Drugs, 48: 678-688) and many bacteria have multiple drug resistance. This proves a particular problem in clinical environments (Morris, J. G. et al., 1995, Ann. Intern. Med., 123: 250-259; Boyce, J. M. et al., 1994, J. Clin. Micro., 32: 1148-1153). Bacteria which are particularly problematic are Enterococci and Staphylococci.

Enterococci are the second most common hospital-acquired infections in the USA, causing intra-abdominal abcesses, endocarditis, infections of the urinary tract and soft tissues and septicaemia (with a high morality of 34-68%). They are now resistant to ampicillin, gentamicin and, increasingly, vancomycin. VRE (vancomycin-resistant enterococci) are currently untreatable and are responsible for 14% of sepsis in intensive care units. There has been a 20 fold increase in VRE between 1989 and 1993 (CDC National Nosocomial Infection Surveillance; WHO Report, 1996).

Staphylococccus aureus is one of the commonest causes of skin infection, septicaemia, osteomyelitis and endocarditis. 26% of S. aureus isolates in France (1989), and in the USA (1991) 38.3%, were multi-resistant MRSA (methicillin resistant S. aureus). The MRSA is resistant to all antibiotics except vancomycin and morality from MRSA septicaemia is 40%. However, the use of vancomycin is one of last resort since it may cause nephrotoxicity, ototoxicity, bone marrow toxicity and the red man syndrome. Transfer of vancomycin-resistance from a VRE to S. aureus has been demonstrated in the laboratory and clinically with certain species of staphylococcus. There is a significant possibility that this may occur with S. aureus in patients, and would lead to the creation of a bacterium untreatable by current therapies.

According to the present invention there is provided a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof for use in a method of treatment of the human or animal body. There is also provided a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof used in the manufacture of a medicament.

ABC transporters are well known (Faith, M. J. and Kolter, R., 1993, Microbiological Reviews, 57(4): 995-1017; Borenkamp, S. J. and St Geme, J. W. III, 1994, Infection and Immunity 62: 3320-3328). However, they have no known therapeutic application, nor has one been suggested. Similarly, agents which neutralise their activity have not been proposed for a therapeutic application.

Particular uses of the ABC transporter protein or immunogenic fragment include their use as immunogens, for example as vaccines.

Reference to ABC transporter proteins is to both importer and exporter proteins. Reference to immunogenic fragment is also reference to analogues of immunogenic fragments, particularly mimotopes (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).

A bacterium may be an entercoccus, for example selected from the group of E.faecium, E.faecalis, E.avium, E.gallinarium, E.durans, E.mundtii and E.casseflavus.

The experimental section (below) describes particular enteroccal ABC transporter proteins having weights of 97 and 54 kDa. Hence the ABC transporter protein may be an enteroccal protein selected from the group of 97 and 54 kDa immunodominant conserved antigens.

The 97 and 54 kDa enteroccal immunodominant conserved antigens are not novel per se (Clark, I. M. and Burnie, J. P., 1993, Serodiagn. Immunother. Infect Disease, 5: 85-92; Sulaiman, A. et al., 1996, Eur. J. Clin. Microbiol Infect Dis., 15:826-829). However, they have not been previously identified or suggested as being ABC transporter proteins, nor have they been suggested for a therapeutic application, nor have agents which neutralise them.

An immunogenic fragment may comprise an ATP binding site or a part thereof. Specific (therapeutically useful) epitopes have been determined to be displayed by the ATP binding sites of ABC transporters, and so they or neutralising agents specific to them may be used therapeutically. Similarly, agents which bind to them may be used diagnostically.

An enteroccal immunogenic fragment may have the sequence of SEQ ID NO: 3, and is displayed by the ABC transporters of e.g. E.faecium and F.faecalis.

An E.faecium immunogenic fragment may have the sequence of SEQ ID NO: 4.

A number of E.faecium specific epitopes have been found, and so the immunogenic fragment may have the sequence of any one of SEQ ID NOs: 5-8.

Also provided according to the present invention are neutralising agents specific to a bacterial or fungal ABC transporter protein or immunogenic fragment thereof for use in a method of treatment of the human or animal body.

A neutralising agent may be used in the manufacture of a medicament.

Neutralising agents are well known and may include antibodies and antigen binding fragments thereof (Harlow, E. and Lane, D., "Antibodies--A Laboratory Mannual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1988), ribozymes and antisense nucleic acid chains. Other neutralising agents will be readily apparent to one skilled in the art.

Also provided according to the present invention is a method of treatment of the human or animal body comprising treating a patient with a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof or a neutralising agent specific thereto according to the present invention.

Medicaments according to the present invention may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient (Remington's Pharmaceutical Sciences and US Pharmacopea, 1984, Mack Publishing Company, Easton, Pa., USA).

Also provided according to the present invention is a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof for use in a method of diagnosis of the human or animal body. The ABC transporter or immunogenic fragment thereof may be used in a method of diagnosis.

The bacterium may be an enterococcus, for example selected from the group of E.faecium, E.faecalis, E.avium, E.gallinarium, E.durans, E.mundtii and E.casseflavus.

The ABC transporter protein may be an enterocccal protein selected from the group of 97 and 54 kDa immunodominant conserved antigens.

An immunogenic fragment may comprise an ATP binding site or a part thereof.

The bacterium may be an enterocous, the immunogenic fragment having the sequence of SEQ ID NO: 3. The bacterium may be E.faecium, the immunogenic fragment having the sequence of SEQ ID NO: 4

The bacterium may be E.faecium, the immunogenic fragment having the sequence of any one of SEQ ID NOs: 5-8.

Also provided according to the present invention is a binding agent specific to a bacterial or fungal ABC transporter protein or immunogenic fragment thereof according to the present invention for use in a method of diagnosis of the human or animal body.

Also provided according to the present invention is a binding agent according to the present invention used in the manufacture of a diagnostic test kit.

Binding agents include any agent capable of detecting the protein or immunogenic fragment and are well known, and include, for example, antibodies and antigen binding fragments thereof.

Also provided according to the present invention is a method of diagnosis of the human or animal body comprising the use of a bacterial or fungal ABC transporter or an immunogenic fragment thereof or a binding agent specific thereto according to the present invention.

Also provided is a diagnostic test method for a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof according to the present invention comprising the steps of:

i) reacting a binding agent according to the present invention with a sample from a patient;

ii) detecting a reaction between the binding agent and antigen; and

iii) correlating the detection of the reaction with the presence of the protein or an immunogenic fragment thereof.

A sample from a patient may for example be patient dialysate or serum.

The binding agent may comprise an antibody, the diagnostic test method comprising the steps of:

i) reacting an antibody according to the present invention with a sample from a patient;

ii) detecting an antibody-antigen binding reaction; and

iii) correlating the detection of the antibody-antigen binding reaction with the presence of the protein or an immunogenic fragment thereof.

Also provided is a diagnostic test method for antibody specific against a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof according to the present invention comprising the steps of:

i) reacting an ABC transporter protein or an immunogenic fragment thereof according to the present invention with patient antisera;

ii) detecting an antibody-antigen binding reaction; and

iii) correlating the detection of the antibody-antigen binding reaction with the presence of antibody specific against the bacterial or fungal ABC transporter or an immunogenic fragment thereof.

Also provided is a kit of parts for performing a diagnostic test according to the present invention.

Also provided is a method of diagnosis comprising the use of a bacterial or fungal ABC transporter protein or an immunogenic fragment thereof or a binding agent specific thereto according to the present invention.

Also provided according to the present invention is a Staphylococcal homologue of IstA or IstB or an immunogenic fragment thereof. The staphylococcus may for example be S. aureus, coagulase negative staphylococci, S. epidermidis; S. haemolyticus or S. hyicus.

The homologues have been found to be 69 and 37 kDa immunodominant conserved antigens. By immunodominant conserved antigen is meant an antigen which elicits an immunogenic response in at least 50% of infected patients.

The Staphylococcus may be S.aureus, the homologue being of IstA and having the sequence of SEQ ID NO: 10, or an immunogenic fragment thereof. An immunogenic fragment of a S.aureus homologue of IstA may have the sequence of any one of SEQ ID NOs: 12-14.

The Staphylococcus may be S.aureus, the homologue being of IstB and having the sequence of SEQ ID NO: 11, or an immunogenic fragment thereof. An immunogenic fragment of a S.aureus homologue of IstB may have the sequence of SEQ ID NO: 15.

Also provided is a neutralising agent specific against a Staphylococcal homologue of IstA or IstB or an immunogenic fragment thereof according to the present invention.

Also provided is a binding agent specific to a Staphylococcal homologue of IstA or IstB or an immunogenic fragment thereof according to the present invention.

A Staphylococcal homologue of IstA or IstB or an immunogenic fragment thereof, or a neutralising agent specific thereto or a binding agent specific thereto according to the present invention may be for use in a method of treatment or diagnosis of the human or animal body.

Also provided is a method of treatment or diagnosis of the human or animal body comprising the use of a Staphylococcal homologue of IstA or IstB or an immunogenic fragment thereof, or a neutralising agent specific thereto or a binding agent specific thereto according to the present invention.

The present inventors have now succeeded in isolating two immunodominant conserved enterococcal antigens and two immunodominant conserved staphylococcal antigens and have found that, surprisingly, antibody specific to these antigens may be used to provide an effective therapy for enterococcal and staphylococcal infection.

The antigen may be for use in a method of treatment or diagnosis of the human or animal body.

The antigen may be for use as an immunogen. Hence the antigen may be used to stimulate an immunogenic response in patients in order to protect them against infection from the bacteria. The antigen may for example be for use as a vaccine.

The present invention also provides antibody or an antigen binding fragment thereof specific to an antigen according to the present invention. The antibody or an antigen binding fragment thereof may be for use in the diagnosis or treatment of infections of the enterococcus or staphylococcus. In the case of bacteria having multiple drug resistance, in particular those for which there is no current drug therapy, the use of antibody specific to the bacteria provides a novel and highly effective form of treatment for the infection.

The role that antibodies play in enterococcal and staphylococcal infection has not yet been fully defined (Moellering, R. C., 1995, In: Mandell, G. L., Bennett, J. E. and Dolin, R (eds.), Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, Fourth Edn., Churchill Livingstone, 1826-1835). Aitchison, E. J. et al. (1986, J. Med. Microbiol., 21: 161-167) investigated the surface components of an endocarditis associated isolate of E.faecalis using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and Western blotting. A major envelope protein antigen of molecular weight 53 kDa was defined in E. faecalis (and not in E. faecium). and other common antigens of E. faecalis had molecular weights of 65, 63, 56, 49.5. 30 and 21 kDa. They also found that growth of E. faecalis in serum to mimic in vivo growth conditions in endocarditis patients altered the antigenic pattern, with only two major antigens of molecular weights 56 and 53 kDa reacting with sera from endocarditis patients. They suggested that these antigens may be of diagnostic potential. Burnie, J. P. et al. (1987, J. Clin. Pathol., 40: 1149-1158) explored the role of immunoblotting in the diagnosis of culture-negative and enterococcal endocarditis. They found that in E. faecalis endocarditis there was a strong IgM response to E.faecalis bands of 112, 88-90 and 45-47 kDa, and a strong IgG response to 88-90 and 45-47 kDa bands. The 112 kDa antigen of E. faecalis was later used to develop an indirect enzyme-linked immmunosorbent assay (ELISA) for the diagnosis of E.faecalis endocarditis (Burnie, J. P. and Clark, I., 1989, J. Immunol. Methods, 123: 217-225). The three patients with E. faecium infection showing a strong IgG response to bands of 82-90 kDa.

The antibody may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it may be an immunoglobulin M antibody or an immunoglobulin G antibody. The antibody or fragment may be of animal, for example, mammalian origin and may be for example of murine, rat, sheep or human origin. It may be a natural antibody or a fragment thereof, or, if desired, a recombinant antibody fragment i.e. an antibody or antibody fragment which has been produced using recombinant DNA techniques.

Particular recombinant antibodies or antibody fragments include, (1) those having an antigen binding site at least part of which is derived from a different antibody, for example those in which the hypervariable or complementarity determining regions of one antibody have been grafted into the variable framework regions of a second, different antibody (as described in, for example, European Patent Specification No 239400); (2) recombinant antibodies or fragments wherein non-Fv sequences have been substituted by non-Fv sequences from other, different antibodies (as described in, for example, European Patent Specification Nos 171469, 173494 and 194276); or (3) recombinant antibodies or fragments possessing substantially the structure of a natural immunoglobulin but wherein the hinge region has a different number of cysteine residues from that found in the natural immunoglobulin but wherein one or more cysteine residues in a surface pocket of the recombinant antibody or fragment is in the place of another amino acid residue present in the natural immunoglobulin (as described in, for example, International Patent Application Nos PCT/GB88/00730 and PCT/GB88/00729).

The antibody or antibody fragment may be of polyclonal, monoclonal or recombinant origin. It may be specific for at least one epitope.

Antigen binding antibody fragments include, for example, fragments derived by proteolytic cleavage of a whole antibody, such as F(ab')2, Fab' or Fab fragments, or fragments obtained by recombinant DNA techniques, for example Fv fragments (as described, for example, in International Patent Application No PCT/GB8810747).

The antibodies according to the invention may be prepared using well-known immunological techniques employing the protein expressed during infection as antigen. Thus, for example, any suitable host may be injected with the protein and the serum collected to yield the desired polyclonal antibody after appropriate purification and/or concentration (for example by affinity chromatography using the immobilised protein as the affinity medium). Alternatively splenocytes or lymphocytes may be recovered from the protein injected host and immortalised using for example the method of Kohler et al. (1976, Eur. J. Immunol., 6: 511), the resulting cells being segregated to obtain a single genetic line producing monoclonal antibodies. Antibody fragments may be produced using conventional techniques, for example, by enzymatic digestion with pepsin or papain. Where it is desired to produce recombinant antibodies according to the invention these may be produced using, for example, the methods described in European Patent Specification Nos 171469, 173494, 194276 and 239400.

Antibodies according to the invention may be labelled with a detectable label or may be conjugated with an effector molecule, for example a drug e.g. an antibacterial agent or a toxin or an enzyme, using conventional procedures and the invention extends to such labelled antibodies or antibody conjugates.

Such antibody may for example be expressed in transgenic animals, for example transgenic sheep and may be achieved using existing transgenic expression systems.

According to the present invention there is also provided methods of treatment and diagnosis of infections due to streptococci and enterococci comprising the use of an immunodominant conserved antigen according to the present invention or an antibody specific thereto or an antigen binding fragment thereof.

Claim 1 of 10 Claims

What is claimed is:

1. A method of treatment of a human or animal patient having a bacterial infection, comprising treating said patient with an antibody or antibody binding fragment specific to an immunogenic fragment of an ABC transporter protein of said bacterium such that said bacterial infection is treated, wherein the bacterium is E. faecium and the immunogenic fragment has the sequence of SEQ ID NO : 6, 7 or 8, and said antibody or antibody binding fragment binds to at least one sequence of SEQ ID NO 6, 7 or 8.

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