Methods of immunization by administering fibrinogen binding...

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

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C530S350000, C530S387100, C424S130100, C424S164100, C424S184100, C514S002600

Reexamination Certificate

active

06299879

ABSTRACT:

DESCRIPTION
TECHNICAL FIELD
The present invention relates to fibrinogen binding proteins. Further the invention relates to pharmaceutical compositions and method for treatment.
The object of the present invention is to obtain fibrinogen binding proteins.
A further object is to obtain said protein by means of genetic engineering technique by using, e.g. a plasmid comprising a nucleotide sequence coding for said protein.
BACKGROUND OF THE INVENTION
Clumping of
Staphylococcus aureus
in plasma has been suggested as a potential virulence factor.
1-5
Several mechanisms can be responsible for this aggregation. A fibronectin-binding protein has been suggested to cause aggregation of staphylococci in fibronectin at the concentration found in sera.
5,6
The presence of protein A causes staphylococci to aggregate in normal human sera, which frequently contain specific immunoglobulins directed against staphylococcal antigens.
7
Due to a high cell surface hydrophobicity, many staphylococcal strains auto-regulate under isotonic conditions.
8
It is believed that clumping of staphylococci in fibrinogen is caused by the so called clumping factor or fibrinogen-binding protein, situated on the staphylococcal cell surface.
1,9
Fibrinogen has also been suggested to mediate adhesion of
S. aureus
to cultured human endothelial cells
10
and to catheters in vitro and in vivo.
11,12
It has been disputed whether clumping factor is distinct from coagulase
1
or if it is a cell-bound form of coagulase.
13, 14
Staphylococcus aureus
coagulases can be grouped into eight different serotypes
15
and the existence of multiple molecular forms of coagulases has been suggested,
16
although most investigators believe that lower molecular weight subspecies in coagulase preparations are due to proteolytic degradation of a larger protein.
17
Staphylococcal coagulases have been shown to induce polymerization of fibrinogen to fibrin by binding, and thereby activating, prothrombin. The coagulase-prothrombin complex causes the release of fibrinopeptides from fibrinogen in a manner similar to that described for thrombin in physiological blood clotting.
18
Fibrinogen precipitation and network formation can also be induced non-enzymatically, e.g. by exposing fibrinogen to various highly positively charged molecules like protamine, which interacts with specific negatively charged sites on the D-domain of fibrinogen.
19
We have recently described staphylococcal components that interact with fibrinogen and which can be purified from
S. aureus
culture supernatants.
13
These are an 87 kDa coagulase and a 19 kDa fibrinogen-binding protein. The 87 and 19 kDa fibrinogen-binding proteins are essentially extracellular proteins, but can to some extent be found on the staphylococcal cell surface. Thus, these proteins can give rise to the clumping phenomenon both by inducing coagulation and by direct fibrinogen-binding.
In this report we show that there are at least three distinct fibrinogen-binding proteins produced by
S. aureus
strain Newman, and that two of these proteins are coagulases.
Results
SDS-PAGE analysis of fibrinogen binding-proteins produced at different times during staphylococcal cell growth
Staphylococcus aureus
strain Newman was grown in BHI or LB and samples were taken every hour for 14 h. Culture supernatants were applied onto fibrinogen-Sepharose and the eluted material was analysed on Coomassie blue-stained SDS-PAGE gels.
FIG. 1
shows fibrinogen-binding proteins from culture supernatants of staphylococci grown in LB under low aeration conditions. Under these conditions, an 87 kDa protein was produced in large amounts, mainly during the first 7 h and a 60 kDa protein appeared after 5-6 h and was produced in large amounts after 9 h of growth. Under high aeration conditions, the 87 kDa protein was produced in lower amounts and the switch to production of the 60 kDa protein accurred after only 3 h resulting in a higher production of 60 kDa protein compared to when less air was supplied to the culture. Using a rich medium like BHI, and the same high aeration conditions, this switch again accurred after 7 h (data not shown). In all cultures, the 87 kDa protein was produced mainly during the exponential growth phase and the 60 kDa protein mainly during the post-exponential growth phase. The switch from production of the 87 kDa protein to production of the 60 kDa protein reflected the nutritional status, rather than the optical density of the culture. A 19 kDa protein was produced constitutively during these 14 h of growth (FIG.
1
).
SDS-PAGE, affinity- and immuno-blot analysis of affinity purified proteins
Staphylococcus aureus
grown in BHI for 3-4 h produced the 87 and 19 kDa proteins but no detectable 60 kDa protein. Such culture supernatants were applied onto fibrinogen-Sepharose in order to purify the 87 and 19 kDa proteins. Similarly, culture supernatants from
S. aureus
grown in LB for 6-8 h, containing predominantly the 60 kDa protein but also the 87 and 19 kDa proteins, were used to purify the 60 kDa protein. The crude material was first passed over fibrinogen-Sepharose, in order to eliminate the 87 and 19 kDa proteins, and the effluent (containing the 60 kDa protein which also bound to fibrinogen-Sepharose, but to a lower extent than the 87 and 19 kDa proteins) was applied onto prothrombin-Sepharose. The 87 and 19 kDa proteins did not bind to prothrombin-Sepharose. Eluted material from affinity purifications was subjected to SDS-PAGE and affinity-blot analysis (FIG.
2
). These blots were probed with fibrinogen or prothrombin, followed by rabbit antifibrinogen or rabbit antiprothrombin sera which had been pre-incubated with
S. aureus
culture supernatants in order to absorb naturally occuring antistaphylococcal antibodies. It could thus be shown that the 87 and 19 kDa proteins bound only to fibrinogen and not to prothrombin, while the 60 kDa protein bound both fibrinogen and prothrombin. Controls were performed by incubating filters with only pre-absorbed primary antibody, omitting fibrinogen and prothrombin (data not shown). In these controls, no 87, 60 or 19 kDa proteins were detected. By using a dilution series both of antigen and fibrinogen or prothrombin, it was shown that the binding reactions were specific and not the result if contaminating blood proteins in the fibrinogen and prothrombin preparations. For example, 10 ng/ml of fibrinogen could detect 0.1 ng of the 87 or 60 kDa proteins in these affinity-blots. When 10 ng/ml of prothrombin was used in these tests, 0.1 ng 60-kDa protein could be detected, while a concentration of 10 &mgr;g/ml of prothrombin could not detect a 1 ng 87-kDa band (data not shown).
The anti-19 serum recognized not only the 19 kDa protein but also the 87 kDa protein and a 35 kDa protein (FIG.
3
). Furthermore, there was a close resemblance between blots incubated with fibrinogen followed by antifibrinogen antibody and blots incubated with anti-19 serum.
Antibodies to the 60 kDa protein seem to occur naturally among several mammalian species (e.g. rabbit, goat and man; data not shown). The anti-19 serum, as well as pre-immune serum from the same rabbit, showed some reactivity towards this 60 kDa protein. However, pre-absorption with 19 kDa protein completely abolished binding to the 19 and 35 kDa bands, but not to the 60 kDa band, while antiserum pre-absorbed with 60 kDa protein reacted with the 19 and 35 kDa bands but not with the 60 kDa band (FIG.
4
).
Peptide mapping
Proteins were purified by a combination of affinity chromatography and preparative SDS-PAGE. The purity of these preparations was confirmed on silver stained SDS-PAGE gels (FIG.
5
). Dimerisation of the 19 kDa protein into a 35 kDa protein could be detected on the silver stained gels. On affinity-blots, using fibrinogen and antifibrinogen antibodies, not only the 35 kDa dimer, but also bands of higher molecular weight were detected. Upon digestion with &agr;-chymotrypsin, the dimerisation of the 19 kDa protein was disrupted, but the 19 kDa band was left intact. This protease did not ha

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