Title:  IgG-binding protein from Staphylococcus and nucleotide sequence encoding this protein

United States Patent:  6,548,639

Issued:  April 15, 2003

Inventors:  Frykberg; Lars (Uppsala, SE); Jacobsson; Karin (Uppsala, SE)

Assignee:  Biostapro AB (Stockholm, SE)

Appl. No.:  554080

Filed:  May 12, 2000

PCT Filed:  November 11, 1998

PCT NO:  PCT/SE98/02036

PCT PUB.NO.:  WO99/24467

PCT PUB. Date: May 20, 1999

A recombinant DNA molecule coding for a protein expressed by a Staphylococcus aureus bacterium, comprising the nucleotide sequence SEQ ID NO:1 or a homologous sequence, or a partial or homologous sequence of SEQ ID NO:1 coding for a polypeptide fragment comprising at least 15 amino acid residues, is described. Further, a protein expressed by such a bacterium or a polypeptide fragment comprising at least 15 amino acid residues, comprising the amino acid sequence SEQ ID NO:2 binds IgG and apolipoprotein H. Examples of the polypeptide fragments comprise the SEQ ID NO:3 through 6. These proteins and polypeptide fragments may be coupled to an inert carrier or matrix. Vectors comprising such a DNA molecule or the corresponding RNA molecule, and antibodies specifically binding to a polypeptide having an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, are also disclosed. The DNA or RNA molecules, the vectors and the antibodies mentioned may all be used in different types of vaccines against Staphylococcal infections. Moreover, a method of isolating and/or purifying apolipoprotein H from a liquid medium, especially from serum, is described.

DESCRIPTION OF THE INVENTION

The present invention is based on cloning and nucleotide sequence determination of a complete gene (sbi) encoding a novel IgG-binding protein. The gene encodes a protein of 436 amino acids, denoted protein Sbi, with one IgG-binding domain that exhibits an immunoglobulin-binding specificity similar to protein A and without the typical Gram-positive cell wall anchoring sequence LPXTG (SEQ ID NO:7) (Schneewind et al, 1995) suggesting that the protein is not anchored in the cell wall. Analysis of other S. aureus strains shows that this gene is not unique for strain 8325-4. For instance, the Sbi-protein is highly expressed in strain Newman 4, which shows that the IgG-binding activity observed in S. aureus is not mediated only by protein A. In fact, this (sbi) gene is present in all tested strains of S. aureus.

Further, it has now been revealed that the Sbi protein of the invention binds apolipoprotein H, a major serum component, in addition to IgG. Hitherto, no bacterial protein binding to apolipoprotein H has been reported. Therefore, neither is this combination of the protein binding to these two serum components previously known. The portion of the protein which binds to IgG is located near the N- terminal of the protein, whereas the middle portion binds to apolipoprotein H. This enables the use of the protein, or an appropriate polypeptide fragment, in immobilised form for the isolation and/or purification of apolipoprotein H.

Thus, one aspect of the present invention is directed to a recombinant DNA molecule coding for a protein expressed by a bacterium of the genus Staphylococcus aureus, comprising the nucleotide sequence SEQ ID NO:1, defined in the sequence listing and the claims, or a homologous sequence to SEQ ID NO:1coding for said protein, or a partial or homologous sequence of the sequence SEQ ID NO:1 coding for a polypeptide fragment of said protein comprising at least 15 amino acid residues.

This recombinant DNA molecule may be inserted into plasmids, phages or phagemides for the expression/production of the protein or protein fragments.

Another aspect of the invention is directed to a protein expressed by a bacterium of the genus Staphylococcus aureus or a polypeptide fragment of said protein comprising at least 15 amino acid residues other than the 84 aa fragment at the position 38-121, which protein comprises the amino acid sequence SEQ ID NO:2, defined in the sequence listing and the claims, or a homologous sequence to the sequence SEQ ID NO:2 comprising a few mismatches in the amino acid sequence of SEQ ID NO:2, or polypeptide fragments of said homologous sequence comprising at least 15 amino acid residues.

The disclaimer of the 84 aa fragment at the position 38-121 of the SEQ ID NO:2 is made because, as already mentioned, it has been previously disclosed (Jacobsson & Frykberg, 1995).

It is well known in the art that there may be a few mismatches of amino acids residues in the amino acid sequence of a protein while the protein still retains its major characteristics. The mismatches may be replacements of one or several amino acids, deletions of amino acid residues or truncations of the protein. Such mismatches occur frequently in genetic variations of native proteins. It is believed that up to 15% of the amino acid residues may be replaced in a protein while the protein still retains its major characteristics. The protein of the invention comprises 436 amino acid residues, and therefore up to 66 mismatches would be acceptable. However, preferably there will be less than 20, more preferably less than 10, and most preferably less than 5 mitsmatches in the amino acid sequence of the protein of the invention.

The polypeptide fragments of the protein of the invention should comprise at least 15 amino acid residues to be sure that the fragments are not found in other known proteins. These fragments may be used e.g. as probes, diagnostic antigens, and vaccine components, possibly coupled to carriers.

In an embodiment of this aspect of the invention a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:3, defined in the sequence listing and the claims. This polypeptide fragment lacks the signal sequence of the SEQ ID NO:1.

In another embodiment a polypeptide fragment of the protein according to the invention has an amino acid sequence SEQ ID NO:4, defined in the sequence listing and the claims. This polypeptide fragment binds apolipoprotein H.

In yet another embodiment a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:5, defined in the sequence listing and the claims. This 120 aa polypeptide fragment binds IgG. It was chosen for immunisation purposes, in stead of the known IgG binding 84 aa fragment, since once the whole amino acid sequence was deduced, it became evident there were sequence similarities suggesting two IgG binding domains.

In still another embodiment a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:6. This polypeptide fragment binds apolipoprotein H, and has been used for isolation and purification of said serum protein.

In a preferred embodiment of this aspect of the invention the protein or polypeptide according to the invention is coupled to an inert carrier or matrix. The carrier may be e.g. plastic surfaces, such as microplates, beads etc.; organic molecules such as biotin; proteins, such as bovine serum albumin; peptide linkers, polypeptides e.g. resulting in fusion proteins. The matrix may be particles used for chromatographic purposes, such as Sepharose.RTM..

A further aspect of the invention is directed to a vector selected from the group consisting of plasmids, phages or phagemides comprising a nucleotide sequence according to the invention.

These vectors may be used for the production of the proteins or polypeptides of the invention. They may also be used in vaccines.

Yet another aspect of the invention is directed to antibodies specifically binding to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, and SEQ ID NO:6. The specific binding of binding of an antibody to an amino acid sequence of the invention requires e.g an affinity constant of at least 10⁷ liters/mole, preferably at least 10⁹ liters/mole.

The antibodies of the invention may be monoclonal or polyclonal. They may be used in diagnostic tests, but preferably in vaccines for passive immunization.

Still another aspect of the invention is directed to the use of a protein or polypeptide according to the invention, optionally in immobilised form, as an immunising component in the production of a vaccine against Staphylococcus infections.

Another use aspect of the invention is directed to the use of a vector according to the invention for the production of a vaccine against Staphylococcal infections.

Yet another use aspect of the invention is directed to the use of antibodies according to the invention for the production of a vaccine for the passive immunisation of a mammal against Staphylococcus infections.

An additional aspect of the invention is directed to a vaccine against Staphylococcal infections comprising as an immunising component a protein or polypeptide according to the invention, optionally in immobilised form.

Another vaccine aspect of the invention is directed to a vaccine against Staphylococcal infections comprising a vector according to the invention.

A DNA molecule, or the corresponding RNA derived from the present sequence, as described in claim 1, may be used in a vector for vaccine purposes. Examples of suitable forms of administration include intravenous, percutaneous, and intramuscular administration.

Yet another vaccine aspect of the invention is directed to a vaccine for the passive immunisation of a mammal, especially a human being, against Staphylococcus infections comprising antibodies according to the invention.

One embodiment of the invention comprises the passive immunization of patients with an impaired immune defense or patient awaiting major surgery, such as patients in line for an organ transplantation or awaiting the insertion of a prosthetic device, such as a hip prosthesis or similar major surgical intervention. According to the present invention, a high dose of antibodies against the novel protein can be administered to any patient before or at the time of hospitalisation, in order to prevent Staphylococcus infection.

The vaccines may contain other ingredients selected with regard to the intended administration rout, and these ingredients are chosen by the vaccine manufacturer in collabaration with pharmacologists. Examples of administration routs include intravenous administration, percutaneous administration, oral and nasal administration.

A further aspect of the invention is directed to a method of prophylactic and/or therapeutic treatment of Staphylococcus infections in a mammal comprising administration to said mammal of an immunologically effective amount of a vaccine according to any one of the vaccines of the invention.

Still another aspect of the invention is directed to a method of isolating and/or purifying apolipoprotein H from a liquid medium, especially from serum, comprising chromatographic separation of apolipoprotein H from said liquid medium with an immobilised protein or polypeptide according to the invention as stationary phase.

In a preferred embodiment of the invention column chromatography is used for the isolation/purification of apolipoprotein H from blood serum. The protein or polypeptide of the invention is coupled to e.g. Sepharose.RTM. and is used as packing material for the column. The apolipoprotein H-containing serum is brought into contact with the immobilized protein or polypeptide and the apolipoprotein H is adsorbed. Finally, the apolipoprotein H is eluated from the column.

Claim 1 of 2 Claims

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