Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-31
2004-08-17
Minnifield, N. M. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S069100, C435S069300, C435S320100, C435S243000, C435S253400, C424S244100
Reexamination Certificate
active
06777547
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates in general to proteins from group A Streptococci (GAS) that can bind collagen, and in particular to collagen-binding proteins designated Cpa1 and Cpa49, and the nucleic acid sequences coding for those proteins, which have been isolated from
Streptococcus pyogenes
and which can be used in methods to inhibit collagen binding and thus treat or prevent infectious diseases caused by group A Streptococcus bacteria.
BACKGROUND OF THE INVENTION
The Streptococci bacteria are a pathogenic genera of microorganisms which have been associated with a wide variety of infectious disorders including suppuration, abscess formation, a variety of pyogenic infections, and septicemia. In particular,
Streptococcus pyogenes
(a group A streptococci, or GAS) is a prominent pathogen which causes skin and mucous membrane infections, as well as deep-seated connective tissue infections and severe, sometimes fatal, septicemia. Like many other pathogens, in order to infect the human host successfully, GAS must have the ability to adjust the expression of its virulence factors according to the varying conditions of different anatomical sites.
In GAS, the expression of several virul nce factors is positively regulated at th I v I of transcription by the Mga regulator. See Perez-Casal t al. (1991); Ch n t al., 1993; Podbielski et al. (1995) and (1996). Regulated genes include M and M-related proteins (phagocytosis resistance, eukaryotic cell interactions), fibronectin-related proteins (serum opacity factor), Spe&bgr; (protease) and c5a peptidase (inactivation of complement factor c5a). Recent evidence has demonstrated that, in addition to iron levels, pH, CO
2
, and temperature (see Caparon et al., 1992; Podbielski et al., 1992; Okada et al., 1993; McIver et al., 1995) and activity of the Mga regulator is associated with logarithmic and late logarithmic growth phase. See McIver et al. (1997).
Another regulator in Streptococcus is RofA, a positive transcriptional regulator of the fibronectin-binding protein (prtF) (see Fogg et al., 1994 and 1997) that promote bacterial attachment to the host extracellular matrix (see Hanski et al., 1992; and Van Heyningen et al., 1993). In contrast to Mga-controlled genes, RofA positively regulates prtF transcription as well as its own transcription in response to increased levels of O
2
. By a potentially independent mechanism, transcription of prtF is also induced in response to intracellular superoxide levels (see Gibson et al., 1996).
These data have suggested differential expression of eukaryotic cell-binding proteins such as RofA-dependent prtF and Mga-dependent emm in response to O
2
and CO
2
partial pressures. These observations have led to the proposal that these regulators may influence the expression of proteins important for the attachment of GAS in different in vivo environments such as superficial Langerhans cells or subsurface keratinocytes (Okada t al. 1994; 1995). As has been observed with regard to other bacterial species, the attachment of bact ria to host cells or implanted biomaterials is generally initiated through “extracellular matrix prot ins,” or ECM's, which generally refer to such general families of macromolecules, collagens, structural glycoproteins, proteoglycans and elastins, including fibronectin, and fibrinogen, that provide support and modulate cellular behavior. However, the precise role of the bacteria's ability to bind to these extracellular matrix proteins and the knowledge of how to best utilize this information in order to prevent streptococcal infection has not yet been fully determined.
Moreover, outside of the two regulators RofA and Mga, very little is known with regard to environmentally dependent virulence gene expression in GAS, and thus there has been very limited information with regard to the regulation and inhibition of the extracellular matrix proteins that are responsible for the attachment and infection caused by GAS. In light of the extremely severe nature of the bacterial infections caused by the Streptococcal bacteria, it is extremely important to make a determination of which specific proteins are responsible for attachment to the surface of targeted cells, and to be able to use this information in order to develop vaccines and other biological agents which can be used to treat or prevention the severe infections associated with group A streptococci.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide isolated proteins (adhesins) from group A streptococci which can bind to intercellular matrix proteins such as collagen so as to be useful in dev loping m thods of inhibiting collag n binding and attachment of streptococcal bacteria to cells.
It is a further object of the present invention to provide isolated streptococcal surface proteins that are able to inhibit adhesion to the immobilized extracellular matrix or host cells present on the surface of implanted biomaterials.
It is a further object of the present invention to provide a vaccine which can be used in treating or preventing infection by group A streptococcal bacterial such as Streptococcus pyogenes.
It is still further an object of the present invention to generate antisera and antibodies to the collagen binding proteins from GAS which can also be useful in developing methods of treatment which can inhibit binding of the streptococcal bacteria to host cells or to implanted biomaterials and thus be employed in order to treat or prevent Streptococcal infection.
It is a further object of the present invention to provide improved materials and methods for detecting and differentiating collagen-binding proteins in streptococcal organisms in clinical and laboratory settings.
It is a further object of the invention to provide nucleic acid sequences which code for the collagen binding proteins in GAS which can also be useful in producing the collagen-binding proteins of the invention and in developing probes and primers specific for identifying and characterizing these proteins.
These and other objects are provided by virtue of the present invention which comprises isolated collagen binding prot ins from group A streptococcal bact ria such as
Streptococcus pyogenes
along with their amino acid and nucleic acid sequ nces. Two of the specific proteins isolated in accordance with the invention are designated Cpa1 and Cpa49 which are obtained from the collagen binding region in
Streptococcus pyogenes,
and the sequences for these proteins are those as shown in SEQ ID NOS. 2 and 4, respectively. The nucleic acid sequences coding for Cpa1 and Cpa49 are shown in SEQ ID NOS. 1 and 3, respectively. The isolated proteins of the present invention have been observed to bind to collagen, and thus can be utilized in methods of treating or preventing streptococcal infection through the inhibition of the ability of the bacteria to bind to collagen.
In another aspect of the present invention, there is also provided antisera and antibodies generated against the collagen binding proteins of the present invention which also can be utilized in methods of treatment which involve inhibition of the attachment of the Cpa proteins to collagen. In particular, specific polyclonal antiserum against Cpa has been generated which has been shown to react with Cpa in Western immunoblots and ELISA assays and which interferes with Cpa binding to collagen. This antiserum can thus be used for specific agglutination assays to detect bacteria which express Cpa on their surface. The antiserum apparently does not cross-react with bacteria which express the fibronectin-binding protein F1 on their surface despite the fact that a portion of protein F1 exhibits sequence homologies to Cpa1 to Cpa49.
Accordingly, in accordance with the invention, antisera and antibodies raised against the Cpa1 and Cpa49 proteins, or portions thereof, may be employed in vaccines, and other pharmaceutical compositions containing the prot ins for therapeutic purposes are also provided herein. In addition, diagnostic kits contain
Minnifield N. M.
Schulman B. Aaron
Stites & Harbison PLLC
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