The present invention is related to nucleic acids coding for adhesion factors of group B streptococcus, adhesion factors of group B streptococcus and uses thereof. More particularly, the present invention is related to a polypeptide being such adhesion factors and comprising an amino acid sequence, whereby the amino acid sequence is selected from the group comprising SEQ ID NO 11 to SEQ ID NO 20, and the use of such polypeptide for the manufacture of a vaccine.

Streptococcus agalactiae, or group B streptococcus (GBS), is a leading cause of infant mortality. GBS encompasses an estimated prevalence of several thousand cases per year resulting in an annual mortality rate in the United States between about 10% and 15% (Schuchat, 1998). Studies from the USA demonstrated a risk of 1-2 cases per 1000 live births (Zangwill, Schuchat, and Wenger, 1992) and incidence rates for different European countries vary between 0.24 and 1.26 per 1000 live births (Carstensen, Henrichsen, and Jepsen, 1985; Faxelius et al., 1988). In the United States, up to 30% of pregnant women carry GBS at least temporarily in the vagina or rectum without symptoms (Schuchat, 1998). Infants born to these women become colonized with GBS during delivery (Baker and Edwards, 1995). Aspiration of infected amniotic fluid or vaginal secretions allows GBS to gain access to the lungs. Common manifestations of this infection include bacteraemia, pneumonia, and meningitis (Spellerberg, 2000). Even infant survivors of GBS meningitis suffer from neurologic sequelae ranging from deafness, learning disabilities, as well as motor, sensory, and cognitive impairment (Baker and Edwards, 1995). Currently, antibiotic prophylaxis in parturients is the recommended approach for the prevention of neonatal disease by GBS (Baker, Halsey, and Schuchat, 1999); however, with the resurgence of antibiotic resistance in other streptococcal species, a similar plight in GBS may occur.
In addition to infant infections, GBS is also an important pathogen in the elderly and in immunocompromised persons, in which the incidence of invasive GBS disease is about 9 in 100,000 (Farley et al. 1993). Of these infections, the mortality rate can be as high as 30%.
An important GBS virulence determinant is the type-specific capsular polysaccharide, which prevents the deposition of host complement factor C3b and thereby inhibits opsonophagocytosis of the bacteria (Rubens et al. 1987). Nine distinct capsular serotypes, Ia, Ib, and II to VIII, have been identified so far in GBS (Wessels, 1997). Efforts are currently under way to develop a multivalent conjugate vaccine against GBS based on the capsule polysaccharides of the clinically relevant serotypes (Paoletti et al. 1999; Baker et al., 1999; Baker et al., 2000; Paoletti and Kasper, 2002). However, there are a number of technical difficulties to overcome with capsule-containing conjugate vaccines: multiple serotypes are needed, an appropriate protein conjugate needs to be identified and validated, and potential cross-reaction with human tissues needs to be addressed (Korzeniowska-Kowal, Witkowska, and Gamian, 2001). The use of cell surface proteins from GBS represents an attractive alternative to capsule polysaccharides for the development of a vaccine against these bacteria. The surface proteins Sip, Rib, α and β from GBS have already been shown to confer protective immunity in mice against GBS infections (Madoff et al. 1992; Larsson, Stalhammar-Carlemalm, and Lindahl, 1997; Larsson, Stalhammar-Carlemalm, and Lindahl, 1999; Brodeur et al., 2000). Also two unique surface proteins from a serotype V strain were shown in a mouse model to protect against GBS infection (Areschoug et al., 1999). Finally, antibodies against C5a peptidase from GBS were found to initiate macrophage killing of the bacteria (Cheng et al., 2001).
The interaction of GBS with its host is a complex process involving the colonization and penetration of epithelial and endothelial surfaces and the evasion of the immune defense (Spellerberg, 2000). In streptococci, fibrinogen binding has been shown to play a significant role in the adhesion to host surfaces (Courtney et al. 1994; Cheung et al., 1991; Ni et al. 1998; Pei and Flock, 2001) and the protection from the immune system (Courtney et al., 1997; Thern, Wastfelt, and Lindahl, 1998b; Ringdahl et al., 2000a; Thern, Wastfelt, and Lindahl, 1998a; Ringdahl et al., 2000b). Therefore, several studies have addressed the molecular basis of fibrinogen binding in streptococci of the serological groups A, C and G (Fischetti, 1989; Meehan, Nowlan, and Owen, 1998; Vasi et al. 2000).
Fibrinogen is a 330 kDa glycoprotein found in high concentrations in blood plasma (Fuss, Palmaz, and Sprague, 2001; Mosesson, Siebenlist, and Meh, 2001b; Mosesson, Siebenlist, and Meh, 2001a). It is a hexamer composed of each of two Aα-, Bβ-, and γ-chains linked together by disulfide bonds. Fibrinogen is a key player in haemostasis and mediates platelet adherence and aggregation at sites of injury. Furthermore, it is cleaved by thrombin to form fibrin, which is the major component of blood clots. Fibrinogen also plays a role in opsonophagocytosis. It has been shown to inhibit the binding of the activated complement factor C3b, thereby blocking the activation of the alternative complement pathway (Whitnack, Dale, and Beachey, 1984b, Whitnack and Beachey, 1985, Whitnack and Beachey, 1982; Whitnack. Dale, and Beachey, 1983; Whitnack, Dale, and Beachey, 1984a). The newborn's unique susceptibility for disseminated GBS infections has been associated with a relative complement deficiency (Mills, Bjorksten, and Quie, 1979; Edwards et al., 1983; Edwards, 1983). Fibrinogen binding of GBS may thus play an important role in the inhibition of the residual complement activity in the newborn (Noel, Katz, and Edelson, 1991).
In several studies, the interaction of GBS with human fibrinogen has been demonstrated (Schonbeck, Bjorck, and Kronvall, 1981; Lammler, Chhatwal, and Blobel, 1983, Chhatwal, Lammler, and Blobel, 1984b; Spellerberg et al., 2002). However, the molecular basis of fibrinogen binding in GBS remained unknown.
GBS has been demonstrated to bind to and invade epithelial and endothelial cells (Gibson et al., 1993; La Penta et al., 1997, Winram et al. 1998). Treatment of GBS with the protease trypsin abolishes the adhesive and invasive properties of the bacteria (Valentin-Weigand and Chhatwal, 1995; Winram, Jonas, Chi, and Rubens, 1998), indicating a proteinacious nature of the adhesins and invasins in GBS. As adhesins and invasins are located on the surface of the bacteria and are important for the virulence of GBS, they represent ideal targets for the development of a GBS vaccine.
The problem underlying the present invention was to provide means for the development of medicaments such as vaccines against bacterial infections. More particularly, the problem was to provide new adhesions factors of GBS which can be used for the manufacture of said medicaments.
The problem is solved in a first aspect by an isolated nucleic acid molecule, preferably encoding a fibrinogen-binding-polypeptide or such protein or a fragment thereof, comprising a nucleic acid sequence which is selected from the group comprising                a) a nucleic acid having at least 70% identity to a nucleic acid sequence which is selected from the group comprising SEQ ID NO 1 to SEQ ID NO 6,        b) a nucleic acid which is essentially complementary to the nucleic acid of a),        c) a nucleic acid comprising at least 15 sequential bases of the nucleic acid of a) or b),        d) a nucleic acid which anneals under stringent hybridisation conditions to the polynucleotide of a), b) or c), and        e) a nucleic acid which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in a), b), c) or d).
The problem is solved in a second aspect by an isolated nucleic acid molecule, preferably encoding an adhesion factor or a fragment thereof, comprising a nucleic acid sequence which is selected from the group comprising                a) a nucleic acid having at least 70% identity to a nucleic acid sequence set forth in SeqID NO 7, SeqID NO 8, SeqID NO 9 or SeqID NO 10.        b) a nucleic acid which is essentially complementary to the nucleic acid of a),        c) a nucleic acid comprising at least 15 sequential bases of the nucleic acid of a) or b),        d) a nucleic acid which anneals under stringent hybridisation conditions to the nucleic acid of a), b) or c), and        e) a nucleic acid which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in a), b), c) or d).
In an embodiment of both aspects of the present invention the identity is at least 80%, preferably at least 90%, more preferably 100%.
In a further embodiment of both aspects of the present invention the nucleic acid is DNA.
In a still further embodiment of both aspects of the present invention the nucleic acid is RNA.
In a preferred embodiment of both aspects of the present invention the nucleic acid molecule is isolated from a bacterium.
In a more preferred embodiment of both aspects of the present invention the bacterium is a species selected from the group comprising Streptococci, Staphylococci, and Lactococci.
In an even more preferred embodiment of both aspects of the present invention the bacterium is a species which is selected from the group comprising Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus mutans.
In a most preferred embodiment of both aspects of the present invention the bacterium is Streptococcus agalactiae.
In an embodiment of the first aspect of the present invention the nucleic acid molecule encodes a fibrinogen-binding-protein comprising at least one repeat of an amino acid motive comprising 16 amino acids.
In an embodiment of the second aspect of the present invention the nucleic acid molecule encodes an adhesion factor which interacts with epithelial cells.
In a preferred embodiment of the first aspect of the present invention the encoded fibrinogen-binding-protein comprises 19 repeats of the amino acid motive whereby the amino acid motive is any one of the ones specified or disclosed herein.
In a more preferred embodiment of the first aspect of the present invention the repeats are encoded by a polynucleotide selected from the group comprising SEQ ID NO 21 to SEQ ID NO 112.
In a third aspect the problem underlying the present invention is solved by an isolated nucleic acid molecule comprising a nucleic acid sequence, whereby the nucleic acid sequence is selected from the group comprising SEQ ID NO 21 to SEQ ID NO 21 to 112.
In a fourth aspect the problem underlying the present invention is solved by an isolated nucleic acid molecule encoding for a polypeptide whereby the polypeptide comprises an amino acid motive, whereby the amino acid motive is G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X (SEQ ID NO 222).
In a preferred embodiment of any of the aspects 1 to 4 of the present invention the nucleic acid is DNA, RNA or mixtures thereof, preferably the nucleic acid molecule is isolated from a genomic DNA.
In a fifth aspect the problem underlying the present invention is solved by a vector comprising a nucleic acid molecule according to any aspect of the present invention.
In a preferred embodiment the vector is adapted for recombinant expression of the polypeptide encoded by any of the nucleic acid molecules according to any aspect of the present invention.
In a sixth aspect the problem underlying the present invention is solved by a cell comprising the vector according to the present invention.
In a preferred embodiment the cell is a host cell.
In a seventh aspect the problem underlying the present invention is solved by a polypeptide, preferably a fibrinogen-binding-polypeptide and/or an adhesion factor, comprising an amino acid sequence, whereby the amino acid sequence is encoded by a nucleic acid molecule according to any aspect of the present invention, and fragments of said polypeptide.
In an eighth aspect the problem underlying the present invention is solved by a polypeptide, preferably a fibrinogen-binding-polypeptide and/or an adhesion factor, comprising an amino acid sequence, whereby the amino acid sequence is selected from the group comprising SEQ ID NO 11 to SEQ ID NO 20.
In an embodiment of this aspect of the present invention the polypeptide, preferably a fibrinogen-binding-polypeptide and/or an adhesion factor, having an amino acid sequence according to any of SEQ ID NO 11 to 16 is a fibrinogen-binding protein.
In a further embodiment of this aspect of the present invention the polypeptide is an adhesion factor which interacts with epithelial cells. In an even more preferred embodiment the epithelial cells are human epithelial cells.
In a ninth aspect the problem underlying the present invention is solved by a polypeptide comprising an amino acid sequence, whereby the amino acid sequence is selected from the group comprising SEQ ID NO 113 to SEQ ID NO 205. In an embodiment the polypeptide comprises at least one of the amino acid sequence according to SEQ ID NO 113 to SEQ ID NO 225 in combination with at least one other amino acid sequence. More preferable this at least one other amino acid sequence is an amino acid sequence according to any of SEQ ID NO 113 to SEQ ID NO 205.
In a tenth aspect the problem underlying the present invention is solved by a polypeptide comprising an amino acid motive, whereby the polypeptide comprises an amino acid motive, whereby the amino acid motive is G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X (SEQ ID NO 222).
In an eleventh aspect the problem underlying the present invention is solved by a process for producing a polypeptide according to any aspect of the present invention comprising expressing the nucleic acid molecule according to any aspect of the present invention.
In a twelfth aspect the problem underlying the present invention is solved by a process for producing a cell which expresses a polypeptide according to any aspect of the present invention or a fragment thereof, comprising transforming or transfecting a suitable host cell with the vector according to the present invention such that the transformed or transfected cell expresses the polypeptide encoded by the polynucleotide contained in the vector.
In a thirteenth aspect the problem underlying the present invention is solved by a pharmaceutical composition, especially a vaccine, comprising a polypeptide or a fragment thereof, as defined in any aspect of the present invention or a nucleic acid molecule according to any aspect of the present invention.
In a preferred embodiment the pharmaceutical composition comprises an immunostimulatory substance, whereby the immunostimulatory substance is preferably selected from the group comprising polycationic polymers, immunostimulatory deoxynucleotides (ODNs), synthetic KLK peptides, neuroactive compounds, alumn, Freund's complete or incomplete adjuvants or combinations thereof.
In a preferred embodiment the immunostimulatory substance is a combination of either a polycationic anion and immunostimulatory deoxynucleotides or of synthetic KLK peptides and immunostimulatory deoxynucleotides.
In a more preferred embodiment the polycationic polymer is a polycationic peptide and/or whereby the neuroactive compound is human growth hormone.
In a fourteenth aspect the problem underlying the present invention is solved by the use of a polypeptide according to any aspect of the present invention or a fragment thereof for the manufacture of a medicament, especially for the manufacture of a vaccine against bacterial infection.
In a preferred embodiment the bacterial infection is a bacterial infection of Streptococcus agalactiae.
In a fifteenth aspect the problem underlying the present invention is solved by the use of molecules which inhibit the binding of a polypeptide according to any aspect of the present invention to fibrinogen for the manufacture of a medicament to prevent and treat bacterial infection. Preferably, the bacterial infection is a Streptococcus agalactiae infection.
In a further embodiment the molecules are selected from the group comprising fibrinogen receptor antibodies, fibrinogen receptor mimotopes and fibrinogen receptor antagonists binding to a polypeptide according to any aspect of the present invention.
In a sixteenth aspect the problem underlying the present invention is solved by the use of molecules which inhibit the binding of a polypeptide according to any aspect of the present invention to epithelial cells, preferably human epithelial cells.
In a seventeenth aspect the problem underlying the present invention is solved by an antibody, or at least an effective part thereof, which binds at least to a selective part of the polypeptide or a fragment thereof according to any aspect of the present invention.
In an embodiment the antibody is a monoclonal antibody.
In a further embodiment said effective part comprises Fab fragments.
In a still further embodiment the antibody is a chimeric antibody.
In a preferred embodiment the antibody is a humanized antibody.
In an eighteenth aspect the problem underlying the present invention is solved by a hybridoma cell line, which produces the antibody according to the present invention.
In a nineteenth aspect the problem underlying the present invention is solved by the use of the antibody according to the present invention for the preparation of a medicament for treating or preventing bacterial infections, especially Streptococcus agalactiae infections.
In a twentieth aspect the problem underlying the present invention is solved by an antagonist which reduces or inhibits the activity of the polypeptide or a fragment thereof according to any aspects of the present invention.
In a twenty-first aspect the problem underlying the present invention is solved by a method for identifying an antagonist capable of reducing or inhibiting the activity of the polypeptide or fragment thereof according to any aspect of the present invention comprising:                a) contacting an isolated or immobilized polypeptide according to any of the aspects of the present invention or a fragment thereof with a candidate antagonist under conditions to permit binding of said candidate antagonist to said polypeptide or fragment thereof, in the presence of a component capable of providing a detectable signal in response to the binding of the candidate antagonist to said polypeptide or fragment thereof; and        b) detecting the presence or absence of a signal generated in response to the binding of the antagonist to the polypeptide or fragment thereof, preferably the presence of a signal indicating a compound capable of inhibiting or reducing the activity of the polypeptide or fragment thereof.
In a twenty-second aspect the problem underlying the present invention is solved by a method for identifying an antagonist capable of reducing or inhibiting the activity of a polypeptide or a fragment thereof according to any the aspects of the present invention comprising:                a) providing the polypeptide according to any aspect of the present invention or a fragment thereof,        b) providing an interaction partner of the polypeptide according to any aspect of the present invention, preferably the antibody according to the present invention,        c) providing a candidate antagonist,        d) reacting the polypeptide, the interaction partner of the polypeptide and the candidate antagonist, and        e) determining whether the candidate antagonist inhibits or reduces the activity of the polypeptide.
In a twenty-third aspect the problem underlying the present invention is solved by a method for identifying an antagonist capable of reducing or inhibiting the interaction activity of the polypeptide according to the present invention or a fragment thereof to its interaction partner comprising:                a) providing the polypeptide according to the present invention or a fragment thereof,        b) providing an interaction partner to said polypeptide or a fragment thereof, preferably an antibody according to the present invention,        c) allowing interaction of said polypeptide or fragment thereof to said interaction partner to form an interaction complex,        d) providing a candidate antagonist,        e) allowing a competition reaction to occur between the candidate antagonist and the interaction complex, and        f) determining whether the candidate antagonist inhibits or reduces the interaction activities of the polypeptide or the fragment thereof with the interaction partner.
In a twenty-fourth aspect the problem underlying the present invention is solved by an antagonist identified or identifiable by a method according to the twenty-second or twenty-third aspect of the present invention.
In a twenty-fifth aspect the problem underlying the present invention is solved by a process for in vitro diagnosis of a disease related to expression of the polypeptide or a fragment thereof according to any aspect of the present invention comprising determining the presence of a polynucleotide sequence encoding said polypeptide or the presence of a polypeptide according to any aspect of the present invention.
In a twenty-sixth aspect the problem underlying the present invention is solved by a process for in vitro diagnosing a disease related to expression of the polypeptide according to the present invention or a fragment thereof, comprising determining the presence of a nucleic acid sequence encoding said polypeptide or a fragment thereof according to the present invention, or the presence of the polypeptide according to the present invention or a fragment thereof.
In a twenty-seventh aspect the problem underlying the present invention is solved by a process for in vitro diagnosis of a bacterial infection, preferably Streptococcus agalactiae infection, comprising the step of determining the presence of a nucleic acid molecule according to any aspect of the present invention, or of a polypeptide according to any aspect of the present invention.
In a preferred embodiment of the latter three aspects of the present invention the presence is determined in a sample which is preferably derived from a host organism.
In a twenty-eighth aspect the problem underlying the present invention is solved by an affinity device comprising a support material and immobilized to said support material a polypeptide according to any aspect of the present invention or a nucleic acid molecule according to any aspect according to the present invention.
In a twenty-ninth aspect the problem underlying the present invention is solved by the use of a polypeptide according to any aspect of the present invention for the isolation and/or purification and/or identification of an interaction partner of said polypeptide.
In a thirtieth aspect the problem underlying the present invention is solved by the use of any of the polypeptides according to any aspect of the present invention for the generation of a peptide binding to said polypeptide.
In a preferred embodiment the peptide is selected from the group comprising anticalines.
In a thirty-first aspect the problem underlying the present invention is solved by the use of a polypeptide according to any aspect of the present invention for the manufacture of a functional nucleic acid, whereby the functional nucleic acid is selected from the group comprising aptamers and spiegelmers.
In a thirty-second aspect the problem underlying the present invention is solved by the use of a nucleic acid molecule according to any aspect of the present invention for the manufacture of a functional ribonucleic acid, whereby the functional ribonucleic acid is selected from the group comprising ribozymes, antisense nucleic acids and siRNA.
In a thirty-third aspect the problem underlying the present invention is solved by the use of a polypeptide according to the present invention or a fragment thereof as an antigen.
In a thirty-fourth aspect the problem underlying the present invention is solved by the use of a nucleic acid according to any aspect of the present invention for the manufacture or generation of a functional nucleic acid, preferably a ribonucleic acid, wherein the functional ribonucleic acid is selected from the group comprising ribozymes, antisense nucleic acids and siRNA.
In a thirty-fifth aspect the problem underlying the present invention is solved by the use of the polypeptides according to the present invention or any fragment thereof for the generation or manufacture of an antibody.
As used herein the term SEQ ID NO X to SEQ ID NO Y is an abbreviation for any of the SEQ ID Nos comprised by X and Y including X and Y.
The present inventors have surprisingly found that the genomes of GBS comprises a variety of adhesion factors which share a common amino acid motive. This amino acid motive is responsible for the binding of the adhesion factor to fibrinogen. As used herein, an adhesion factor is a factor, preferable a peptide or a protein which mediates the binding of a microorganism to a substrate. Preferably, the microorganism is GBS. More preferably, the substrate is fibrinogen and a host cell, respectively. The adhesion factor as used herein can be an adhesin or an invasin. The common amino acid motive can be described as follows using the one letter code for amino acids:G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X  (SEQ ID NO 222).
As may be taken from the above sequence the amino acid motive comprises a total of 16 positions. Some of the positions have to be occupied by a distinct amino acid such as, e.g., position 1 or 3 or 4. Other positions such as positions 15 or 16 may be occupied by any amino acid, preferably by a naturally occurring amino acid. These positions are marked in the above sequence with an 'X'. Still further positions can be occupied by different amino acids. These different amino acids are indicated in the above motive, whereby the various amino acids are separated by '/'. Accordingly, at position 2 N, S or T may be present. Any permutations of the above sequence of amino acids can be realized by the one skilled in the art, which are thus within the scope of the present invention.
The present invention is thus related in one aspect to the above amino acid motive. More particularly, the present invention is related to any peptide or polypeptide which comprises this amino acid motive. It is to be understood that the terms peptide and polypeptide are used in a synonymous way if not indicated to the contrary.
Polypeptides, as used herein, include all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, unless otherwise indicated, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. Among the known modifications which may be present in polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modification and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be generally as a result of posttranslational event, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post-translational modification of the peptide, a methionine residue at the NH2-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variant of the protein of the invention. The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized recombinantly by expressing a polynucleotide in a host cell.
Any polypeptide comprising the amino acid motive is regarded as a polypeptide according to the present invention. As explained in greater detail in the examples, the present inventors have found that GBS comprises a number of adhesion factors which comprise not only one copy of the amino acid motive but a number thereof. Thus any polypeptide comprising a plurality or being composed of a plurality of the amino acid motive is a polypeptide according to the present invention. For example, the adhesion factor referred to herein as FbsA may comprise as little as one unit of the amino acid motive to as much as 19 copies thereof.
Other adhesion factors according to the present invention are those referred to herein as PabA, PabB, PabC and PabD. It is to be understood that the term polypeptides according to the present invention also comprise any fragment, derivative or analog thereof. Further preferred polypeptides according to the present invention are those the amino acid sequence of which corresponds to SEQ ID 11 to 20.
The fragment, derivative or analog of the polypeptide of the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Among the particularly preferred embodiments of the invention in this regard are polypeptides set forth in the Sequence Listing, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments. Additionally, fusion polypeptides comprising such polypeptides, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments, in addition to a heterologous polypeptide, are contemplated by the present invention. Such fusion polypeptides and proteins, as well as polynucleotides encoding them, can readily be made using standard techniques, including standard recombinant techniques for producing and expressing a recombinant polynucleic acid encoding a fusion protein.
Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.
Further particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragment, having the amino acid sequence of any polypeptide set forth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptide of the present invention. Also especially preferred in this regard are conservative substitutions. Most highly preferred polypeptides having an amino acid sequence set forth in the Sequence Listing without substitutions.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. Also the polypeptides according to the present invention are preferably isolated polypeptides.
The polypeptides of the present invention include any polypeptide set forth in the Sequence Listing (in particular a mature polypeptide) as well as polypeptides which have at least 70% identity to a polypeptide set forth in the Sequence Listing, preferably at least 80% or 85% identity to a polypeptide set forth in the Sequence Listing, and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide set forth in the Sequence Listing and still more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% similarity (still more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to a polypeptide set forth in the Sequence Listing and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 5 amino acids and more preferably at least 10, 15 or 16 or multiples thereof. Preferably, the multiples are multiples of a repeat of 16 amino acids, whereby the 16 amino acids correspond to the amino acid motive as disclosed herein.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
Also among preferred embodiments of this aspect of the present invention are polypeptides comprising fragments of the polypeptide having the amino acid sequence set forth in the Sequence Listing, and fragments of variants and derivatives of the polypeptides set forth in the Sequence Listing.
As used herein a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned S. agalactiae polypeptides and variants or derivatives thereof.
Such fragments may be "free-standing", i.e., not part of or fused to another amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of a polypeptide of the present invention comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro-polypeptide regions fused to the amino terminus of the fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from a polypeptide of the present invention.
Representative examples of polypeptide fragments of the invention, include, for example, in any selected polypeptide, fragments from about amino acid number 45-60, 61-76, 77-92, 93-108, 109-124, 125-140, 141-156, 157-172, 173-188, 189-204, 205-220, 221-236, 237-252, 253-268, 269-284, 285-300, 301-316, 317-332, 333-348, 410-414 of the amino acid sequences disclosed herein, or any of the repeats, either alone or in combination with one or several of the ones mentioned in the following tables 1 and 2, optionally combined with the signal peptide or the LPXTG (SEQ ID NO: 259) motif.
TABLE 1FbsA of GBS strain 6313FbsA of GBS strain 706 S21-35 signal peptide1-35 signal peptide45-60 repeat 1 (SEQ ID 113)45-60 repeat 1 (SEQ ID 132)61-76 repeat 2 (SEQ ID 114)61-76 repeat 2 (SEQ ID 133)77-92 repeat 3 (SEQ ID 115)77-92 repeat 3 (SEQ ID 134)93-108 repeat 4 (SEQ ID 116)93-108 repeat 4 (SEQ ID 135)109-124 repeat 5 (SEQ ID 117)109-124 repeat 5 (SEQ ID 136)125-140 repeat 6 (SEQ ID 118)125-140 repeat 6 (SEQ ID 137)141-156 repeat 7 (SEQ ID 119)141-156 repeat 7 (SEQ ID 138)157-172 repeat 8 (SEQ ID 120)157-172 repeat 8 (SEQ ID 139)173-188 repeat 9 (SEQ ID 121)173-188 repeat 9 (SEQ ID 140)189-204 repeat 10 (SEQ ID 122)189-204 repeat 10 (SEQ ID 141)205-220 repeat 11 (SEQ ID 123)205-220 repeat 11 (SEQ ID 142)221-236 repeat 12 (SEQ ID 124)221-236 repeat 12 (SEQ ID 143)237-252 repeat 13 (SEQ ID 125)237-252 repeat 13 (SEQ ID 144)253-268 repeat 14 (SEQ ID 126)253-268 repeat 14 (SEQ ID 145)269-284 repeat 15 (SEQ ID 127)269-284 repeat 15 (SEQ ID 146)285-300 repeat 16 (SEQ ID 128)285-300 repeat 16 (SEQ ID 147)301-316 repeat 17 (SEQ ID 129)301-316 repeat 17 (SEQ ID 148)317-332 repeat 18 (SEQ ID 130)378-382 LPXTG motif333-348 repeat 19 (SEQ ID 131)(SEQ ID NO: 259)410-414 LPXTG motif(SEQ IN NO: 259)
TABLE 2FbsA of GBS strain 33 H1AFbsA of GBS strain 176 H4A1-35 signal peptide1-35 signal peptide45-60 repeat 1 (SEQ ID 149)45-60 repeat 1 (SEQ ID 162)61-76 repeat 2 (SEQ ID 150)61-76 repeat 2 (SEQ ID 163)77-92 repeat 3 (SEQ ID 151)77-92 repeat 3 (SEQ ID 164)93-108 repeat 4 (SEQ ID 152)154-158 LPXTG motif109-124 repeat 5 (SEQ ID 153)(SEQ ID NO: 259)125-140 repeat 6 (SEQ ID 154)141-156 repeat 7 (SEQ ID 155)157-172 repeat 8 (SEQ ID 156)173-188 repeat 9 (SEQ ID 157)189-204 repeat 10 (SEQ ID 158)205-220 repeat 11 (SEQ ID 159)221-236 repeat 12 (SEQ ID 160)237-252 repeat 13 (SEQ ID 161)314-318 LPXTG motif(SEQ ID NO: 259)
TABLE 3FbsA of GBS strain O90RFbsA of GBS strain SS11691-35 signal peptide1-34 signal peptide45-60 repeat 1 (SEQ ID 165)45-60 repeat 1 (SEQ ID 175)61-76 repeat 2 (SEQ ID 166)61-76 repeat 2 (SEQ ID 176)77-92 repeat 3 (SEQ ID 167)77-92 repeat 3 (SEQ ID 177)93-108 repeat 4 (SEQ ID 168)93-108 repeat 4 (SEQ ID 178)109-124 repeat 5 (SEQ ID 169)109-124 repeat 5 (SEQ ID 179)125-140 repeat 6 (SEQ ID 170)125-140 repeat 6 (SEQ ID 180)141-156 repeat 7 (SEQ ID 171)141-156 repeat 7 (SEQ ID 181)157-172 repeat 8 (SEQ ID 172)157-172 repeat 8 (SEQ ID 182)173-188 repeat 9 (SEQ ID 173)173-188 repeat 9 (SEQ ID 183)189-204 repeat 10 (SEQ ID 174)189-204 repeat 10 (SEQ ID 184)267-270 LPXTG motif205-220 repeat 11 (SEQ ID 185)(SEQ ID NO: 259)221-236 repeat 12 (SEQ ID 186)237-252 repeat 13 (SEQ ID 187)253-268 repeat 14 (SEQ ID 188)269-284 repeat 15 (SEQ ID 189)285-300 repeat 16 (SEQ ID 190)301-316 repeat 17 (SEQ ID 191)317-332 repeat 18 (SEQ ID 192)333-348 repeat 19 (SEQ ID 193)349-364 repeat 20 (SEQ ID 194)365-380 repeat 21 (SEQ ID 195)381-396 repeat 22 (SEQ ID 196)397-412 repeat 23 (SEQ ID 197)413-428 repeat 24 (SEQ ID 198)429-444 repeat 25 (SEQ ID 199)445-460 repeat 26 (SEQ ID 200)461-476 repeat 27 (SEQ ID 201)477-492 repeat 28 (SEQ ID 202)493-508 repeat 29 (SEQ ID 203)509-524 repeat 30 (SEQ ID 204)586-590 LPXTG motif(SEQ ID NO: 259)
As used herein "about" includes the particularly recited ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
Preferred fragments of the invention include, for example, truncation polypeptides including polypeptides having an amino acid sequence set forth in the Sequence Listing, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out above also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally. Degradation forms of the polypeptides of the invention in a host cell are also preferred.
Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of the polypeptide of the present invention. Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions of the polypeptide of the present invention, and combinations of such fragments.
Preferred regions are those that mediate activities of the polypeptide of the present invention. Most highly preferred in this regard are fragments that have a chemical, biological or other activity of the polypeptide of the present invention, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Particularly preferred are fragments comprising a receptor activity for such as, e.g., fibrinogen in case of FbsA or the host cell in case of PabA, PabB, PabC und PabD that confer a function essential for the ability of S. agalactiae to cause disease in humans and/or that are able to mediate the adherence and/or invasion of S. agalactiae to or into epithelial cells, more preferably human epithelial cells. Further preferred polypeptide fragments are those that comprise or contain antigenic or immunogenic determinants in an animal, especially in a human. A host cell as used herein is a cell which is capable of uptaking of GBS in the natural host or in an internalization assay such as, e.g., the one as described in example 1.
The polypeptides according to the present invention may be used for the detection of the organism or organisms in a sample containing these polypeptides. Preferably such detection is for diagnosis, more preferable for the diagnosis of a disease, most preferably for the diagnosis of a disease related or linked to the presence or abundance of Gram-positive bacteria, especially bacteria selected from the group comprising streptococci, staphylococci and lactococci. More preferably, the microorganisms are selected from the group comprising Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus mutans.
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of the polypeptide of the present invention in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of the polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example, and to identify the infecting organism. Assay techniques that can be used to determine levels of a polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these, ELISAs frequently are preferred. An ELISA assay initially comprises preparing an antibody specific to the polypeptide, preferably a monoclonal antibody. In addition, a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, such as horseradish peroxidase enzyme.
The polypeptides according to the present invention may also be used for the purpose of or in connection with an array. More particularly, at least one of the polypeptides according to the present invention may be immobilized on a support. Said support typically comprises a variety of polypeptides whereby the variety may be created by using one or several of the polypeptides according to the present invention and/or polypeptides being different therefrom. The characterizing feature of such array as well as of any array in general is the fact that at a distinct or predefined region or position on said support or a surface thereof, a distinct polypeptide is immobilized. Because of this any activity at a distinct position or region of an array can be correlated with a specific polypeptide. The number of different polypeptides immobilized on a support may range from as little as 10 to several 1000 different polypeptides. The density of polypeptides per cm2 is in a preferred embodiment as little as 10 oligonucleotides per cm2 to at least 400 different polynucleotides per cm2 and more particularly at least 1000 different polypeptides per cm2.
The manufacture of such arrays is known to the one skilled in the art and, for example, described in U.S. Pat. No. 5,744,309. The array preferably comprises a planar, porous or non-porous solid support having at least a first surface. The polypeptides as disclosed herein, are immobilized on said surface. Preferred support materials are, among others, glass or cellulose. It is also within the present invention that the array is used for any of the diagnostic applications described herein. Apart from the polypeptides according to the present invention also the nucleic acid molecules according to the present invention may be used for the generation of an array as described above. This applies as well to an array made of antibodies, preferably monoclonal antibodies as, among others, described herein.
The isolated nucleic acid molecule according to the present invention, also referred to herein as the nucleic acid (molecule) according to the present invention, codes for the amino acid motive and the polypeptides according to the present invention. The nucleic acid molecule according to the present invention can in a first alternative be a nucleic acid (molecule) which has an identity of at least 70% to a nucleic acid molecule which has the nucleic acid sequence as specified in SEQ ID No.1 to 10. It is also within the present invention that the isolated nucleic acid molecule has a similarity of at least 70% of any sequence, which encodes any of the polypeptides of the present invention. Preferably, the identity is at least 80% and more preferably the identity is at least 90%. Identity may also be 95%, 96%, 97%, 98%, 99% or 99.5%.
Identity, as known in the art and used herein, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the mach between strings of such sequences. Identity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, the term is well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403 (1990)).
The nucleic acid according to the present invention can as a second alternative also be a nucleic acid which is at least essentially complementary to the nucleic acid described as the first alternative above. As used herein complementary means that a nucleic acid strand is base pairing via Watson-Crick base pairing with a second nucleic acid strand. Essentially complementary as used herein means that the base pairing is not occurring for all of the bases of the respective strands but leaves a certain number or percentage of the bases unpaired or wrongly paired. The percentage of correctly pairing bases is preferably at least 70%, more preferably 80%, even more preferably 90% and most preferably any percentage higher than 90%. It is to be noted that a percentage of 70% matching bases is considered as homology and the hybridization having this extent of matching base pairs is considered as stringent. Hybridization conditions for this kind of stringent hybridization may be taken from Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1987. More particularly, the hybridization conditions can be as follows:                Hybridization performed e.g. in 5×SSPE, 5×Denhardt's reagent, 0.1% SDS, 100 g/mL sheared DNA at 68° C.        Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.        High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.
Genomic DNA with a GC content of 50% has an approximate TM of 96° C. For 1% mismatch, the TM is reduced by approximately 1° C.
In addition, any of the further hybridization conditions described herein are in principle applicable as well.
The nucleic acid according to the present invention can as a third alternative also be a nucleic acid which comprises a stretch of at least 15 bases of the nucleic acid according to the first and second alternative of the nucleic acid molecule according to the present invention as outlined above. Preferably, the bases form a contiguous stretch of bases. However, it is also within the present invention that the stretch consists of two or more moieties which are separated by a number of bases.
The nucleic acid according to the present invention can as a fourth alternative also be a nucleic acid which anneals under stringent hybridisation conditions to any of the nucleic acids of the present invention according to the above outlined first, second, and third alternative. Stringent hybridisation conditions are typically those described herein.
Finally, the nucleic acid according to the present invention can as a fifth alternative also be a nucleic acid which, but for the degeneracy of the genetic code, would hybridise to any of the nucleic acids according to any of the nucleic acids of the present invention according to the first, second, third, and fourth alternative as outlined above. This kind of nucleic acid refers to the fact that preferably the nucleic acids according to the present invention code for the polypeptides according to the present invention and thus for adhesins and invasions, respectively. This kind of nucleic acid is particularly useful in the detection and thus diagnosis of the nucleic acid molecules according to the present invention and thus of the respective microorganisms such as GBS and any disease or diseased condition where this kind of microorganism is involved. Preferably, the hybridisation would occur or be preformed under stringent conditions as described in connection with the fourth alternative described above.
Polynucleotide(s) as used herein generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among other, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. The term polynucleotide also embraces short polynucleotides often referred to as oligonucleotide(s). "Polynucleotide" and "nucleic acid" or "nucleic acid molecule" are often used interchangeably herein.
Using the information provided herein and known, standard methods, such as those for cloning and sequencing and those for synthesizing polynucleotides and polypeptides (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), one can generate numerous unique fragments, both longer and shorter than the polynucleotides and polypeptides set forth in the Sequence Listing, of the S. agalactiae genome and the S. agalactiae coding regions, which are encompassed by the present invention. To be unique, a fragment must be of sufficient size to distinguish it from other known nucleic acid sequences, most readily determined by comparing any selected S. agalactiae fragment to the nucleotide sequences in computer databases such as GenBank. Such comparative searches are standard in the art. Many unique fragments will be S. agalactiae—specific. Typically, a unique fragment useful as a primer or probe will be at least about 20 to 25 nucleotides in length, depending upon the specific nucleotide content of the sequence. Additionally, fragments can be, for example, at least about 30, 40, 50, 60, 75, 80, 90, 100, 150, 200, 250, 300, 400, 500 or more nucleotides in length. The nucleic acid fragment can be single, double or triple stranded, depending upon the purpose for which it is intended.
Additionally, as discussed above and below, modifications can be made to the S. agalactiae polynucleotides and polypeptides that are encompassed by the present invention. For example, nucleotide substitutions can be made which do not affect the polypeptide encoded by the nucleic acid, and thus any polynucleotide which encodes the polypeptides of this invention is within the present invention. Additionally, certain amino acid substitutions (and corresponding nucleotide substitutions to encode them) can be made which are known in the art to be neutral (Robinson W. E. Jr. and Mitchell, W. m., AIDS 4: S141-S162 (1990)). Such variations may arise naturally as allelic variations (e.g. due to genetic polymorphism) or may be produced by human intervention (e.g. by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutations. Minor changes in amino acid sequences are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'l Biomed. Res. Found., Washington D.C.). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Likewise, such amino acid changes result in a different nucleic acid encoding the polypeptides and proteins. Thus, alternative polynucleotides, which are within the parameters of the present invention, are contemplated by such modifications.
Furthermore, some of the polynucleotide sequences set forth in the Sequence Listing are open reading frames (ORFs), i.e. coding regions of S. agalactiae. The polypeptide encoded by each open reading frame can be deduced, and the molecular weight of the polypeptide thus calculated using amino acid residue molecular weight values well known in the art. Any selected coding region can be functionally linked, using standard techniques such as standard subcloning techniques, to any desired regulatory sequence, whether a S. agalactiae regulatory sequence or a heterologous regulatory sequence, or to a heterologous coding sequence to create a fusion protein, as further described herein.
Polynucleotides of the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
The coding sequence which encodes a S. agalactiae polypeptide of this invention may be identical to the coding sequence of a polynucleotide set forth in the sequence listing. It also may be a polynucleotide with a different sequence which, as a result of the redundancy (degeneracy) of the genetic code, encodes a S. agalactiae polypeptide set forth in the sequence listing.
Polynucleotides of the present invention which encode a S. agalactiae polypeptide as disclosed herein, including those set forth in the sequence listing may include, but are not limited to, the coding sequence for a mature polypeptide, by itself; the coding sequence for a mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro-protein sequence; the coding sequence of a mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals, for example), ribosome binding, mRNA stability elements, and additional coding sequence which encode additional amino acids, such as those which provide additional functionalities. Thus, for instance, a polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide (SEQ ID NO: 260), such as the tag provided in the pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), for instance, hexa-histidine (SEQ ID NO: 260) provides for convenient purification of the fusion protein. The HA tag may also be used to create fusion proteins and corresponds to an epitope derived of influenza hermagglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated genetic elements.
In accordance with the foregoing, the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly a polypeptide having a S. agalactiae amino acid sequence set forth in the Sequence Listing. The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
The present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having a deducted S. agalactiae amino acid sequence set forth in the Sequence Listing. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or on-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Preferred are polynucleotides encoding a variant, analog, derivative or fragment, or a variant, analogue or derivative of a fragment, which have a S. agalactiae sequence as set forth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid(s) is substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the S. agalactiae polypeptides set forth in the Sequence Listing. Also especially preferred in this regard are conservative substitutions.
Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding a polypeptide according to the present invention and more particularly those polypeptides having an amino acid sequence set forth in the Sequence Listing, and polynucleotides which are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% or at least 85% identical over their entire length to a polynucleotide encoding a S. agalactiae polypeptide according to the present invention and more particularly those polypeptides set forth in the Sequence Listing, including complementary polynucleotides. In this regard, polynucleotides at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% identical over their entire length to the same are particularly preferred, and among these particularly preferred polypeptides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these, those with at least 98% and at least 99% are particularly highly preferred, with at least 99% or 99.5% being the more preferred.
Preferred embodiments in this respect, moreover, are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA set forth in the Sequence Listing.
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. Stringent conditions are typically selective conditions. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. For a specific sequence, stringent conditions can be determined empirically according to the nucleotide content, as is known in the art and also exemplified herein. For example, a typical example of stringent conditions is hybridization of a 48mer having 55% GC content at 42° C. in 50% formamide and 750 mM NaCl followed by washing at 55° C. in 15 mM NaCl and 0.1% SDS.
As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the polynucleotides of the present invention. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 20, at least 25 or at least 30 bases, and may have at least 50 bases. Particularly preferred probes will have at least 30 bases, and will have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.
For example, the coding region of the polynucleotide of the present invention may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine to which members of the library the probe hybridizes.
The polynucleotides and polypeptides of the present invention may be employed as reagents and materials for development of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays, inter alia.
The polynucleotides of the present invention that are oligonucleotides can be used in the processes herein as described, but preferably for PCR, to determine whether or not the S. agalactiae genes identified herein in whole or in part are present and/or transcribed in infected tissue such as blood. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained. For this and other purposes the arrays comprising at least one of the nucleic acids according to the present invention as described herein, may be used.
The polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
The present invention additionally contemplates polynucleotides functionally encoding fusion polypeptides wherein the fusion polypeptide comprises a fragment of a S. agalactiae polypeptide and one or more polypeptide(s) derived from another S. agalactiae polypeptide or from another organism or a synthetic polyamino acid sequence. Such polynucleotides may or may not encode amino acid sequences to facilitate cleavage of the S. agalactiae polypeptide from the other polypeptide(s) under appropriate conditions.
In sum, a polynucleotide of the present invention may preferably encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
Isolated as used herein means separated "by the hand of man" from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living organism in its natural state is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
The nucleic acids according to the present invention may be chemically synthesized. Alternatively, the nucleic acids can be isolated from various microorganisms by methods known to the one skilled in the art. Appropriate sources are, e.g. Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans and Streptococcus pneumoniae.
The nucleic acids according to the present invention may be used for the detection of nucleic acids and organisms or samples containing these nucleic acids. Preferably such detection is for diagnosis, more preferable for the diagnosis of a disease, most preferably for the diagnosis of a disease related or linked to the present or abundance of S. agalactiae.
S. agalactiae bacteria, which have infected eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, may be detected at the DNA level by a variety of techniques. By selecting regions of nucleic acids that vary among strains of S. agalactiae, preferred candidates for distinguishing a specific strain of S. agalactiae can be obtained. Furthermore, by selecting regions of nucleic acids that vary between S. agalactiae and other organisms, preferred candidates for distinguishing S. agalactiae from other organisms can be obtained. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324: 163-166 (1986) prior to analysis. RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid forming part of the polynucleotide of the present invention can be used to identify and analyze for its presence and/or expression. Using PCR, characterization of the strain of S. agalactiae present in a mammal, and especially a human, may be made by an analysis of the genotype of the prokaryote gene. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridising amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished form mismatched duplexes by Rnase A digestion or by differences in melting temperatures.
Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer can be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
Genetic characterization based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualised by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g. Myers et al., Science, 230: 1242 (1985)).
Sequence changes at specific locations also may be revealed by nuclease protection assays, such as Rnase and S1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, Rnase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, e.g., restriction fragment length polymorphisms (RFLP) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
Cells carrying mutations or polymorphisms in the gene of the present invention may also be detected at the DNA level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA or cDNA may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers complementary to the nucleic acid encoding the polypeptide of the present invention can be used to identify and analyse mutations. The primers may be used to amplify the gene isolated from the individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be diagnosed.
The invention provides a process for diagnosing disease, arising from infection with S. agalactiae, comprising determining from a sample isolated or derived from an individual an increased level of expression of a polynucleotide having the sequence of a polynucleotide set forth in the Sequence Listing. Expression of polynucleotide can be measured using any one of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, Rnase protection, Northern blotting, other hybridisation methods and the arrays described herein.
The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention. Introduction of polynucleotides, into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONONG: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Polynucleotide constructs in cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector, a single or double-stranded RNA or DNA viral vector. Plasmids generally are designated herein by a lower case p preceded and/or followed by capital letters and/or number, in accordance with standard naming conventions that are familiar to those of skill in the art. Starting plasmids disclosed herein are either commercially available, publicly available, or can be constructed from available plasmids by routine application of well known, published procedures, given the teachings herein. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.
Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic cells, are well known and employed routinely by those of skill in the art.
A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include, among other, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs.
In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG or others such as GUG and UUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as transcription factors, repressor binding sites and termination, among other.
Vectors for propagation and expression generally will include selectable markers and amplification regions, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)
Representative examples of appropriate cells which host said vectors include bacterial cells, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
The following vectors, which are commercially available, are provided by way of example. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, Pkk233-3, pDR540, pRIT5 available from Pharmacia, and pBR322 (ATCC 37017). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, PXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available, such as pKK232-8 and pCM7. Promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.
Among known prokaryotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lacl and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.
Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus "(RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.
Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site will be 5′ to the AUG that initiates translation of the polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal in constructs for use in eukaryotic hosts. A transcription termination signal appropriately disposed at the 3′ end of the transcribed region may also be included in the polynucleotide construct.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide.
These signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N- or C-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, regions may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize or purify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another protein or part thereof. In drug discovery, for example, proteins have been fused with antibody Fc portions for the purpose of high-throughout screening assays to identify antagonists. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well known to those skilled in the art.
Mammalian expression vectors may comprise expression sequences, such as an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences that are useful or necessary for expression.
The polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding protein may be employed to regenerate the active conformation when the polypeptide is denatured during isolation and or purification.
The polypeptides according to the present invention can be produced by chemical synthesis as well as by biotechnological means. The latter comprise the transfection or transformation of a host cell with a vector containing a nucleic acid according to the present invention and the cultivation of the transfected or transformed host cell under conditions, which are known to the ones skilled in the art. The production method may also comprise a purification step in order to purify or isolate the polypeptide to be manufactured. In a preferred embodiment the vector is a vector according to the present invention.
In a further aspect the present invention relates to an antibody directed to any of the polypeptides, derivatives or fragments thereof according to the present invention. The present invention includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. It is within the present invention that the antibody may be chimeric, i.e. that different parts thereof stem from different species or at least the respective sequences are taken from different species.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a non-human. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique known in the art, which provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Köhler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985); U.S. Pat. No. 5,545,403; U.S. Pat. No. 5,545,405; U.S. Pat. No. 5,654,403; U.S. Pat. No. 5,792,838; U.S. Pat. No. 5,316,938; U.S. Pat. No. 5,633,162; U.S. Pat. No. 5,644,036; U.S. Pat. No. 5,858,725.
Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
Alternatively, phage display technology could be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fab or from naïve libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al; (1991) Nature 352, 624-628).
If two antigen binding domains are present, each domain may be directed against a different epitope—termed 'bispecific' antibodies.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
Thus, among others, antibodies against the polypeptide of the present invention may be employed to inhibit and/or treat infections, particularly bacterial infections and especially infections arising from S. agalactiae.
Polypeptide derivatives include antigenically, epitopically or immunologically equivalent derivatives which form a particular aspect of this invention. The term "antigenically equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent derivative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof can be used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein, for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively, a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof, may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
Preferably the antibody or derivative thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized", wherein the complementarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest at al., (1991) Biotechnology 9, 266-273.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscle (Wolff et al., (1992) Hum. Mol. Genet. 1, 363; Manthorpe et al., (1963) Hum. Gene Ther. 4, 419) delivery of DNA complexed with specific protein carriers (Wu et al., (1989) J. Biol. Chem. 264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef (1986) PNAS 83, 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., (1989) Science 243, 375), particle bombardment (Tang et al., (1992) Nature 356, 152; Eisenbraun et al., (1993) DNA Cell. Biol. 12, 791) and in vivo infection using cloned retroviral vectors (Seeger et al., (1984) PNAS 81, 5849).
In a further aspect the present invention relates to a peptide binding to any of the polypeptides according to the present invention, and a method for the manufacture of such peptides whereby the method is characterized by the use of the polypeptides according to the present invention and the basic steps are known to the one skilled in the art.
Such peptides may be generated by using methods according to the state of the art such as phage display or ribosome display. In case of phage display, basically a library of peptide is generated, such as in form of phages, and this kind of libraries is contacted with the target molecule, in the present case the polypeptides according to the present invention. Those peptides binding to the target molecule are subsequently removed, preferably as a complex with the target molecule, from the respective reaction. It is known to the one skilled in the art that the binding characteristics, at least to a certain extend, depend on the particularly realized experimental set-up such as the salt concentration and the like. After separating those peptides binding to the target molecule with a higher affinity or a bigger force, from the non-binding members of the library, and optionally also after removal of the target molecule from the complex of target molecule and peptide, the respective peptide(s) may subsequently be characterised. Prior to the characterisation optionally an amplification step is realized such as, e.g. by propagating the peptide coding phages. The characterisation preferably comprises the sequencing of the target binding peptides. Basically, the peptides are not limited in their lengths, however, preferably peptides having a lengths from about 8 to 20 amino acids are preferably obtained in the respective methods. The size of the libraries may be about 102 to 1018, preferably 108 to 1015 different peptides, however, is not limited thereto.
A particular form of target binding polypeptides are the so-called "anticalines" which are, among others, described in German patent application DE 197 42 706.
In a further aspect the present invention relates to functional nucleic acids interacting with any of the polypeptides according to the present invention, and a method for the manufacture of such functional nucleic acids whereby the method is characterized by the use of the polypeptides according to the present invention and the basic steps are known to the one skilled in the art. The functional nucleic acids are preferably aptamers and spiegelmers.
Aptamers are D-nucleic acids, which are either single stranded or double stranded and which specifically interact with a target molecule. The manufacture or selection of aptamers is, e.g., described in European patent EP 0 533 838. Basically the following steps are realized. First, a mixture of nucleic acids, i.e. potential aptamers, is provided whereby each nucleic acid typically comprises a segment of several, preferably at least eight subsequent randomised nucleotides. This mixture is subsequently contacted with the target molecule whereby the nucleic acid(s) binds to the target molecule, such as based on an increased affinity towards the target or with a bigger force thereto, compared to the candidate mixture. The binding nucleic acid(s) are/is subsequently separated from the remainder of the mixture. Optionally, the thus obtained nucleic acid(s) is amplified using, e.g. polymerase chain reaction. These steps may be repeated several times giving at the end a mixture having an increased ratio of nucleic acids specifically binding to the target from which the final binding nucleic acid is then optionally selected. These specifically binding nucleic acid(s) are referred to as aptamers. It is obvious that at any stage of the method for the generation or identification of the aptamers samples of the mixture of individual nucleic acids may be taken to determine the sequence thereof using standard techniques. It is within the present invention that the aptamers may be stabilized such as, e.g., by introducing defined chemical groups which are known to the one skilled in the art of generating aptamers. Such modification may for example reside in the introduction of an amino group at the 2′ position of the sugar moiety of the nucleotides. Aptamers are currently used as therapeutical agens. However, it is also within the present invention that the thus selected or generated aptamers may be used for target validation and/or as lead substance for the development of medicaments, preferably of medicaments based on small molecules. This is actually done by a competition assay whereby the specific interaction between the target molecule and the aptamer is inhibited by a candidate drug whereby upon replacement of the aptamer from the complex of target and aptamer it may be assumed that the respective drug candidate allows a specific inhibition of the interaction between target and aptamer, and if the interaction is specific, said candidate drug will, at least in principle, be suitable to block the target and thus decrease its biological availability or activity in a respective system comprising such target. The thus obtained small molecule may then be subject to further derivatisation and modification to optimise its physical, chemical, biological and/or medical characteristics such as toxicity, specificity, biodegradability and bioavailability.
Spiegelmers and their generation or manufacture is based on a similar principle. The manufacture of spiegelmers is described in international patent application WO 98/08856. Spiegelmers are L-nucleic acids, which means that they are composed of L-nucleotides rather than D-nucleotides as aptamers are. Spiegelmers are characterized by the fact that they have a very high stability in biological systems and, comparable to aptamers, specifically interact with the target molecule against which they are directed. In the process of generating spiegelmers, a heterogeneous population of D-nucleic acids is created and this population is contacted with the optical antipode of the target molecule, in the present case for example with the D-enantiomer of the naturally occurring L-enantiomer of the polypeptides according to the present invention. Subsequently, those D-nucleic acids are separated which do not interact with the optical antipode of the target molecule. But those D-nucleic acids interacting with the optical antipode of the target molecule are separated, optionally determined and/or sequenced and subsequently the corresponding L-nucleic acids are synthesized based on the nucleic acid sequence information obtained from the D-nucleic acids. These L-nucleic acids, which are identical in terms of sequence with the aforementioned D-nucleic acids interacting with the optical antipode of the target molecule, will specifically interact with the naturally occurring target molecule rather than with the optical antipode thereof. Similar to the method for the generation of aptamers it is also possible to repeat the various steps several times and thus to enrich those nucleic acids specifically interacting with the optical antipode of the target molecule.
In a further aspect the present invention relates to functional nucleic acids interacting with any of the nucleic acid molecules according to the present invention, and a method for the manufacture of such functional nucleic acids whereby the method is characterized by the use of the nucleic acid molecules and their respective sequences according to the present invention and the basic steps are known to the one skilled in the art. The functional nucleic acids are preferably ribozymes, antisense oligonucleotides and siRNA.
Ribozymes are catalytically active nucleic acids, which preferably consist of RNA which basically comprises two moieties. The first moiety shows a catalytic activity whereas the second moiety is responsible for the specific interaction with the target nucleic acid, in the present case the nucleic acid coding for the polypeptides according to the present invention. Upon interaction between the target nucleic acid and the second moiety of the ribozyme, typically by hybridisation and Watson-Crick base pairing of essentially complementary stretches of bases on the two hybridising strands, the catalytically active moiety may become active which means that it catalyses, either intramolecularly or intermolecularly, the target nucleic acid in case the catalytic activity of the ribozyme is a phosphodiesterase activity. Subsequently, there may be a further degradation of the target nucleic acid which in the end results in the degradation of the target nucleic acid as well as the protein derived from the said target nucleic acid. Ribozymes, their use and design principles are known to the one skilled in the art, and, for example described in Doherty and Doudna ((2001) Ribozym structures and mechanism. Annu. Rev. Biophys. Biomolstruct. 30, 457-475) and Lewin and Hauswirth (Ribozyme Gene Therapy: Applications for molecular medicine. 2001 7: 221-8).
The activity and design of antisense oligonucleotides for the manufacture of a medicament and as a diagnostic agent, respectively, is based on a similar mode of action. Basically, antisense oligonucleotides hybridise based on base complementarity, with a target RNA, preferably with a mRNA, thereby activate RNase H. RNase H is activated by both phosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupled DNA, however, is rapidly degraded by cellular nucleases with the exception of phosphorothioate-coupled DNA. These resistant, non-naturally occurring DNA derivatives do not inhibit RNase H upon hybridisation with RNA. In other words, antisense polynucleotides are only effective as DNA RNA hybride complexes. Examples for this kind of antisense oligonucleotides are described, among others, in U.S. Pat. No. 5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on the nucleic acid sequence of the target molecule which in the present case are the nucleic acid molecules for the polypeptides according to the present invention, either from the target protein from which a respective nucleic acid sequence may in principle be deduced, or by knowing the nucleic acid sequence as such, particularly the mRNA, suitable antisense oligonucleotides may be designed based on the principle of base complementarity.
Particularly preferred are antisense-oligonucleotides, which have a short stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNA bases is required for activation of bacterial RNase H and a minimum of 5 bases is required for mammalian RNase H activation. In these chimeric oligonucleotides there is a central region that forms a substrate for RNase H that is flanked by hybridising "arms" comprised of modified nucleotides that do not form substrates for RNase H. The hybridising arms of the chimeric oligonucleotides may be modified such as by 2′-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonate or phosphoramidate linkages in said arms. Further embodiments of the antisense oligonucleotide useful in the practice of the present invention are P-methoxyoligonucleotides, partial P-methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.
Of particular relevance and usefulness for the present invention are those antisense oligonucleotides as more particularly described in the above two mentioned US patents. These oligonucleotides contain no naturally occurring 5′□3′-linked nucleotides. Rather the oligonucleotides have two types of nucleotides: 2′-deoxyphosphorothioate, which activate RNase H, and 2′-modified nucleotides, which do not. The linkages between the 2′-modified nucleotides can be phosphodiesters, phosphorothioate or P-ethoxyphosphodiester. Activation of RNase H is accomplished by a contiguous RNase H-activating region, which contains between 3 and 5 2′-deoxyphosphorothioate nucleotides to activate bacterial RNase H and between 5 and 10 2′-deoxyphosphorothioate nucleotides to activate eucaryotic and, particularly, mammalian RNase H. Protection from degradation is accomplished by making the 5′ and 3′ terminal bases highly nuclease resistant and, optionally, by placing a 3′ terminal blocking group.
More particularly, the antisense oligonucleotide comprises a 5′ terminus and a 3′ terminus; and from 11 to 59 5′□3′-linked nucleotides independently selected from the group consisting of 2′-modified phosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriester nucleotides; and wherein the 5′-terminal nucleoside is attached to an RNase H-activating region of between three and ten contiguous phosphorothioate-linked deoxyribonucleotides, and wherein the 3′-terminus of said oligonucleotide is selected from the group consisting of an inverted deoxyribonucleotide, a contiguous stretch of one to three phosphorothioate 2′-modified ribonucleotides, a biotin group and a P-alkyloxyphosphotriester nucleotide.
Also an antisense oligonucleotide may be used wherein not the 5′ terminal nucleoside is attached to an RNase H-activating region but the 3′ terminal nucleoside as specified above. Also, the 5′ terminus is selected from the particular group rather than the 3′ terminus of said oligonucleotide.
The nucleic acids as well as the polypeptides according to the present invention may be used as or for the manufacture of vaccines. Preferably such vaccine is for the prevention or treatment of diseases caused by, related to or associated with GBS. In so far another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, which comprises inoculating the individual with the polypeptide of the invention, or a fragment or variant thereof, adequate to produce antibody to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus infections.
Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises, through gene therapy or otherwise, delivering a nucleic acid functionally encoding the polypeptide, or a fragment or a variant thereof, for expressing the polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibodies or a cell mediated T cell response, either cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.
A further aspect of the invention relates to an immunological composition which, when introduced into a host capable of having induced within it an immunological response, induces an immunological response in such host, wherein the composition comprises recombinant DNA which codes for and expresses an antigen of the polypeptide of the present invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.
The polypeptide of the invention or a fragment thereof may be fused with co-protein, which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein, which will have immunogenic and protective properties. This fused recombinant protein preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al., Science 273: 352 (1996).
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with S. agalactiae. Such fragments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. This approach can allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of S. agalactiae infection in mammals, particularly humans.
The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused e.g. by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.
The present invention also includes a vaccine formulation, which comprises the immunogenic recombinant protein together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
It is also within the present invention that the vaccine comprises apart from the polypeptide and/or nucleic acid molecule according to the present invention other compounds, which are biologically or pharmaceutically active. Preferably, the vaccine composition comprises at least one polycationic peptide. The polycationic compound(s) to be used according to the present invention may be any polycationic compound, which shows the characteristic effects according to the WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues (WO 97/30721). Especially preferred are substances like polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% of basic amino acids in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.
These polycationic compounds may be produced chemically or recombinantly or may be derived from natural sources.
Cationic (poly)peptides may also be anti-microbial with properties as reviewed in Ganz et al., 1999; Hancock, 1999. These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly (WO 02/13857). Peptides may also belong to the class of defensins (WO 02/13857). Sequences of such peptides can, for example, be found in the Antimicrobial Sequences Database under the following internet address:                http://www.bbcm.univ.trieste.it/˜tossi/pag2.html
Such host defense peptides or defensives are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.
Especially preferred for use as polycationic substances in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application WO 02/13857, incorporated herein by reference), especially antimicrobial peptides derived from mammal cathelicidin, preferably from human, bovine or mouse.
Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are cathelin or related or derived substances from cathelin. For example, mouse cathelin is a peptide, which has the amino acid sequence NH2-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO: 261). Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino acids which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without further immunactivating substances.
Another preferred polycationic substance to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application WO 02/32451, incorporated herein by reference).
The pharmaceutical composition of the present invention may further comprise Immunostimulatory nucleic acid(s). Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acid derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine di-nucleotides (CpG) in a certain base context (e.g. described in WO 96/02555). Alternatively, also nucleic acids based on inosine and cytidine as e.g. described in the WO 01/93903, or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and PCT/EP 02/05448, incorporated herein by reference) may preferably be used as immunostimulatory nucleic acids for the present invention. Preferably, the mixtures of different immunostimulatory nucleic acids may be used according to the present invention.
It is also within the present invention that any of the aforementioned polycationic compounds is combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones as described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857 and PCT/EP 02/05448 and the Austrian patent application A 1924/2001, incorporated herein by reference.
In addition or alternatively such vaccine composition may comprise apart from the polypeptide/nucleic acid molecules according to the present invention a neuroactive compound. Preferably, the neuroactive compound is human growth factor as, e.g. described in WO 01/24822. Also preferably, the neuroactive compound is combined with any of the polycationic compounds and/or immunostimulatory nucleic acids as aforementioned.
In a further aspect the present invention is related to a pharmaceutical composition. Such pharmaceutical composition is, for example, the vaccine described herein. Also a pharmaceutical composition is a pharmaceutical composition which comprises any of the following compounds or combinations thereof: the nucleic acids according to the present invention, the polypeptides according to the present invention, the vector according to the present invention, the cells according to the present invention, the antibody according to the present invention, the functional nucleic acids according to the present invention and the binding peptides such as the anticalines according to the present invention, any agonists and antagonists screened as described herein. In connection therewith any of these compounds may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications. In general, the compositions are administered in an amount of active agent of at least about 10 μg/kg body weight. In most cases they will be administered in one or more doses in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10 μg/kg to about 1 mg/kg body weight, daily. For administration particularly to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg and typically around 1 mg/kg. For example, a dose may be 1 mg/kg daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like. The physician in any event will determine the actual dosage, which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical application, for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
The pharmaceutical composition may be administered in conjunction with an in-dwelling device. In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, etc.
The composition of the invention may be administered by injection to achieve a systematic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent Streptococcus infections.
Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation.
In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.
Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of 1 μg/ml to 10 mg/ml for bathing of wounds or indwelling devices.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks.
With the indicated dose range, no adverse toxicological effects should be observed with the compounds of the invention, which would preclude their administration to suitable individuals.
The antibodies described above may also be used as diagnostic reagents to detect the presence of bacteria containing the polypeptides according to the present invention.
In a further embodiment the present invention relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. The ingredient(s) can be present in a useful amount, dosage, formulation or combination. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
In connection with the present invention any disease related use as disclosed herein such as, e.g. use of the pharmaceutical composition or vaccine, is particularly a disease or diseased condition which is caused, linked or associated with Gram-positive bacteria, more particularly bacteria selected from the group comprising Streptococci, Staphylococci and Lactococci. More preferably, the microorganisms are selected from the group comprising S. agalactiae, S. pyogenes, S. pneumoniae and S. mutans. In connection therewith it is to be noted that S. agalactiae comprises several strains including those disclosed herein. Also, the disease may be particularly a disease occurring in any patient selected from the group comprising people with chronic illness such as diabetes mellitus and liver failure, pregnant women, the fetus and the newborn. A disease related, caused or associated with the bacterial infection to be prevented and/or treated according to the present invention includes in neonates sepsis, pneumonia and meningitis, and in adults sepsis and soft tissue infections. Pregnancy-related infections are sepsis, amnionitis, urinary tract infection and stillbirth.
In a still further embodiment the present invention is related to a screening method using any of the polypeptides or nucleic acids according to the present invention. Screening methods as such are known to the one skilled in the art and can be designed such that an agonist or an antagonist is screened. Preferably an antagonist is screened which in the present case inhibits or prevents the binding of any polypeptide according to the present invention to an interaction partner. Such interaction partner can be a naturally occurring interaction partner or a non-naturally occurring interaction partner. Preferable the interaction partner is fibrinogen or a fragment thereof in case of FbsA or any host cell in case of PabA, PabB, PabC, and PabD, including epithelial cells, preferably human epithelial cells.
The invention also provides a method of screening compounds to identify those, which enhance (agonist) or block (antagonist) the function of polypeptides or polynucleotides of the present invention, such as its interaction with a binding molecule. The method of screening may involve high-throughput.
For example, to screen for agonists or antagonists, the interaction partner of the polynucleotide and nucleic acid, respectively, according to the present invention, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, may be prepared from a cell that expresses a molecule that binds to the polypeptide of the present invention. The preparation is incubated with labelled polypeptide in the absence or the presence of a candidate molecule, which may be an agonist or antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labelled ligand. Molecules, which bind gratuitously, i.e., without inducing the functional effects of the polypeptide, are most likely to be good antagonists. Molecules that bind well and elicit functional effects that are the same as or closely related to the polypeptide are good agonists.
The functional effects of potential agonists and antagonists may by measured, for instance, by determining activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of the polypeptide of the present invention or molecules that elicit the same effects as the polypeptide Reporter systems that may be useful in the regard include but are not limited to colorimetric labelled substrate converted into product, a reporter gene that is responsive to changes in the functional activity of the polypeptide, and binding assays known in the art.
Another example of an assay for antagonists is a competitive assay that combines the polypeptide of the present invention and a potential antagonist with membrane bound binding molecules, recombinant binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. The polypeptide can be labelled such as by radioactivity or a colorimetric compound, such that the number of polypeptide molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds to the same sites on a binding molecule without inducing functional activity of the polypeptide of the invention.
Potential antagonists include a small molecule, which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56:560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description of these molecules).
Preferred potential antagonists include derivatives of the polypeptides of the invention.
As used herein the activity of a polypeptide according to the present invention is its capability to bind to any of its interaction partner or the extent of such capability of its binding to its or any interaction partner.
In a particular aspect, the invention provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the invention may be used: i) in the prevention of adhesion of S. agalactiae to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; ii) to block protein mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshire et al., Infect. Immun. 60:2211 (1992)). iii) to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial proteins which mediate tissue damage; iv) to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
Each of the DNA coding sequence provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein upon expression can be used as a target for the screening of antibacterial drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The antagonists and agonists may be employed, for instance, to inhibit diseases arising from infection with Streptococcus, especially S. agalactiae, such as sepsis.
In a still further aspect the present invention is related to an affinity device such affinity device comprises at least a support material and any of the polypeptides according to the present invention, which is attached to the support material. Because of the specificity of the polypeptides according to the present invention for their target cells or target molecules or their interaction partners, the polypeptides allow a selective removal of their interaction partner(s) from any kind of sample applied to the support material provided that the conditions for binding are met. The sample may be a biological or medical sample, including but not limited to, fermentation broth, cell debris, cell preparation, tissue preparation, organ preparation, blood, urine, lymph liquid, liquor and the like.
The polypeptide may be attached to the matrix in a covalent or non-covalent manner. Suitable support material is known to the one skilled in the art and can be selected from the group comprising cellulose, silicon, glass, aluminium, paramagnetic beads, starch and dextrane.
The present invention is further illustrated by the following figures, examples and the sequence listing from which further features, embodiments, and advantages may be taken. It is to be understood that the present examples are give by way of illustration only and not by way of limitation of the disclosure.
The figures to which it might be referred to in the specification are described in the following in more detail.
FIG. 1 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the serotype III GBS strain 6313. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 2 shows a Southern blot analysis to determine the presence of the fbsA gene in different clinical isolates of GBS. Chromosomal DNA from different GBS strains belonging to serotypes Ia, Ib, II, III, IV, and V, respectively was digested with HindIII and, after size separation and blotting onto nylon membrane, hybridised with a digoxigenin-labelled fbsA-specific DNA probe.
FIG. 3 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the serotype Ia GBS strain 706 S2. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 4 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the serotype Ib GBS strain 33H1A. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 5 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the serotype II GBS strain 176H4A. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 6 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the capsule GBS mutant O90R. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 7 shows the DNA sequence of the fbsA-encoding region and the deduced FbsA protein from the serotype V GBS strain SS1169. The putative ribosomal binding site (RBS) is underlined and the potential transcriptional terminator is indicated by antiparallel arrows. Within the deduced FbsA protein, letters in bold and italic indicate the putative signal peptide sequence and letters in bold and underlined mark the cell wall-anchor motif LPKTG (SEQ ID NO: 262). Repeats in FbsA are numbered and marked by arrows.
FIG. 8 shows a schematic comparison of the FbsA proteins from the GBS strains 6313 (serotype III), 706 S2 (serotype Ia), 33H1A (serotype Ib), O176H4A (serotype II), O90R (derived from serotype Ia) and SS1169 (serotype V), respectively. Indicated are the locations of the signal peptide (black box), the wall-spanning region (WSR; boxes with vertical bars), the cell wall anchor motif (LPKTG) (SEQ ID NO: 262), and the membrane-spanning region (MSR; boxes with diagonal bars). The number of individual repeats is indicated for each protein. Grey boxes represent a repeat with the sequence motif 'GNVLERRQRDAENRSQ' (SEQ ID NO: 113), boxes with horizontal bars represent repeats with an R14K substitution and dotted boxes show the location of repeats with both an A11V and R14K substitution. Repeats that carry an E12D substitution are indicated below the FbsA proteins from GBS strains 33H1A and SS1169. Above FbsA from 33H1A, a repeat carrying a single A11V substitution is indicated.
FIG. 9 shows a Western blot analysis of truncated FbsA derivatives to identify the fibrinogen-binding domain in FbsA. Hexahistidyl-tagged (SEQ ID NO: 260) fusion proteins, representing the mature FbsA protein (FbsA-19), the N-terminal repeat-containing region (FbsA-N) or the C-terminal part (FbsA-C) of FbsA were separated by SDS-PAGE, blotted onto nitrocellulose and tested for their binding to human fibrinogen. The fibrinogen binding activity of the three proteins encoded by different constructs are indicated below the schematic FbsA drawing.
FIG. 10 shows the competitive inhibition of fibrinogen binding to GBS 6313 by the purified fusion proteins FbsA-19, FbsA-9 and Bsp, respectively. FbsA-9 differs from FbsA-19 in that it contains only 9 repeats in its repeat domain. The binding assay was performed with 125I-labelled fibrinogen in the presence of different concentrations of each fusion protein. Each experiment was performed at least in triplicate.
FIG. 11 shows a spot membrane analysis of fibrinogen binding by synthetic peptides derived from the repeat unit of FbsA. Fibrinogen binding was tested with peptides carrying the FbsA repeat motif 'GNVLERRQRDAENRSQ' (SEQ ID 113) and with peptides containing the scrambled sequence 'GLSQNRDVRENQRARE'. (SEQ ID 205) Synthetic peptides, which differed from the repeat motif in that single amino acids had been replaced by alanine, were probed for fibrinogen binding. Beside the spot membrane, the sequence of each synthetic peptide is listed. Bold and underlined letters indicate amino acid substitutions within the repeat motif.
FIG. 12 shows a spot membrane analysis of the fibrinogen binding repeat unit. Synthetic peptides were tested for fibrinogen binding, in which each of the amino acids of the fibrinogen-binding repeat was replaced by each of the 20 amino acids. The vertical letters, printed in bold, represent the FbsA-derived fibrinogen binding sequence 'GNVLERRQRDAENRSQ' (SEQ ID NO: 113). The horizontal letters represent those amino acids that were introduced in the synthetic peptides instead of the original amino acid in the respective position.
FIG. 13 shows the competitive inhibition of fibrinogen binding to GBS 6313 by synthetic peptides. The binding assay was performed with 125I-labelled fibrinogen in the presence of different concentrations of the peptides pep_FbsA (SEQ ID 211), carrying an FbsA-derived repeat unit, and pep_R6A, possessing an R6A substitution within the repeat unit. Each experiment was performed at least in triplicate.
FIG. 14 shows eukaryotic cell adherence (A) and invasion (B) of GBS strains 6313, 706 S2, and O90 μl and their respective fbsA deletion mutants. The values represent the result of at least four independent experiments performed in triplicate. Error bars are indicated.
FIG. 15. shows a peptide ELISA of FbsA peptides with human sera. The 5 biotinylated peptides (wild type <1>: GNVLERRQRDAENRSQ SEQ ID No. 113; alanine mutant peptides: <2> GAVLERRQRDAENRSQ SEQ ID No. 207, <3> GNALERRQRDAENRSQ SEQ ID No. 208, <4> GNVLEARQRDAENRSQ SEQ ID No. 211, <5> GNVLERAQRDAENRSQ SEQ ID No. 212; see also FIG. 11) were coated on Streptavidin-coated ELISA plates and analysed using 5 sera from patients infected with GBS. The patient sera were applied in a dilution of 1:200 and 1:1,000. IgG (A) and IgA (B) antibodies were detected with secondary anti-human antibodies coupled to Horse Radish Peroxidase and ABTS as substrate.
FIG. 16 shows the DNA sequence of the pabA/B-encoding region and the deduced PabA (nt 319-2964) and PabB (nt3087-5111) proteins from GBS 6313. Putative ribosomal binding sites (RBS) are underlined. Letters in bold and italics indicate the putative signal peptides of the deduced PabA and PabB proteins and letters in bold and underlined mark the region with high identity to the cell wall anchor motif from Gram positive bacteria.
FIG. 17 shows the DNA sequence of the pabC/D-encoding region and the deduced PabC (nt 487-2394) and PabD (nt 2461-3006) proteins from GBS 6313. Putative ribosomal binding sites (RBS) are underlined. Letters in bold and italics indicate the putative signal peptides of the deduced PabC and PabD proteins.
FIG. 18 shows a scanning electron microscopy of A549 cells incubated for two hours with latex beads coated with PabA, PabB, PabC, PabD, respectively. BSA-coated latex beads were used as a control.
FIG. 19 shows the adherence of GBS 6313 to and invasion of A549 cells in the presence of 100 μg/ml of PabA, PabB, PabC or PabD fusion proteins. The adherence of GBS 6313 to A549 cells (A) and its internalization into these cells (B) was arbitrarily set to 100% and the results obtained in the presence of the different fusion proteins was related to these values. Each experiment was performed at least three times in triplicate.
FIG. 20 shows eukaryotic cell adherence and internalization by GBS 6313 and its pabA and pabB deletion mutants. The adherence of GBS 6313 to A549 cells (A) and its internalization into these cells (B) was arbitrarily set to 100% and the results obtained with the GBS mutants 6313ΔpabA and 6313ΔpabB were related to these values. Each experiment was performed at least three times in triplicate.
FIG. 21 shows the testing of anti-PabA, anti-PabB, and anti-PabD antisera for their sensitivity in detecting their respective antigens. Serial dilutions of the fusion proteins PabA, PabB, and PabD were spotted onto nitrocellulose and probed with a 1:1000 dilution of the mice sera against the respective proteins. Bound antibodies were labelled with an anti-mouse-HRP conjugate and visualized by chemiluminescence.
FIG. 22 shows a Western blot analysis of culture supernatant of different S. agalactiae strains and their isogenic fbsA deletion mutants for the presence of fibrinogen binding proteins. 15 μg of proteins from concentrated culture supernatant of the different S. agalactiae strains and their fbsA deletion mutants was size separated by SDS-PAGE, blotted onto nitrocellulose and tested for the interaction with human fibrinogen. Bound fibrinogen was detected by incubating the blot with rabbit anti-fibrinogen antibodies followed by an incubation with goat anti-rabbit antibodies coupled to horseradish peroxidase. For the detection of fibrinogen-antibody complexes, chemiluminescence was used.
FIG. 23 shows the binding of different S. agalactiae strains and their fbsA deletion mutants to immobilized fibrinogen. Similar cell numbers of the different strains were incubated with fibrinogen, which was immobilized to Terasaki plates. The number of bacteria bound to fibrinogen was related to the number of input bacteria into the assay.
FIG. 24 shows the adherence and internalization of different S. agalactiae strains and their isogenic fbsA mutants into the lung epithelial cell line A549. Similar numbers of bacteria were used to infect A549 cells and the number of bacteria adherent to (A) and internalized by A549 cells (B) was related to the number of input bacteria.
FIG. 25 shows the adherence and internalization of the S. agalactiae strains 6313 and 6313ΔfbsA into the fibroblast cell line HEL299. HEL299 cells were infected with S. agalactiae at an MOI of 10:1 and the cell adherent and internalized bacteria were related to the number of input bacteria.
FIG. 26 shows the influence of FbsA protein on the adherence of S. agalactiae to A549 cells. The adherence assay was performed in the presence of different amounts of purified FbsA fusion protein and the number of cell adherent bacteria was related to the number of input bacteria.
FIG. 27 shows the binding of FbA-coated latex beads to human A549 cells. Latex beads were either coated with BSA (A) or FbsA fusion protein (B-D) and the interaction of the coated beads with the lung epithelial cell line A549 was analyzed by scanning electron microscopy.
FIG. 28 shows the transcriptional organization of the pabC-encoding region in S. agalactiae. The names on top of the figure indicate the genes to which the primer pairs annealed during PCR with total RNA (A), RT-PCR with total RNA (B) or PCR with chromosomal DNA (C) from S. agalactiae 6313.
FIG. 29 shows the PCR-analysis of GBS strains for the presence of pabC and pabD genes. The following strains were used for the PCR: 1, S. agalactiae 1137 (Ia); 2, S. agalactiae A90/14 (Ib); 3, S. agalactiae 6313 (III); 4, S. agalactiae 4416 S3 (III); 5, S. agalactiae 4357 (V); 6, S. agalactiae 4327 (V).
FIG. 30 shows the comparison of the amino acid sequences of the PabC proteins from S. agalactiae 6313, S. agalactiae NEM316, and S. agalactiae 2003V_R.
FIG. 31 shows (A) the restriction map of the pabC-encoding region in S. agalactiae and (B) the Western blot analysis of PabC and Gbs0851 fusion proteins for fibrinogen-binding. The fusion proteins were size-separated by SDS-PAGE, transferred onto a nitrocellulose membrane and tested for fibrinogen binding by Western blotting. Bound fibrinogen was detected with rabbit anti-fibrinogen antibodies, followed by peroxidase-labelled goat anti-rabbit antibodies, and visualized by chemiluminescence. PabC and Gbs0851: full-length fusion proteins; PabC-N: N-terminal 388 amino acids of PabC; PabC-C: C-terminal 222 amino acids of PabC. (C) Identification of the FbsA and PabC-binding sites within human fibrinogen by Western blot analysis. Human fibrinogen was size separated by SDS-PAGE and either Coomassie stained (left lane) or transferred onto nitrocellulose and tested for FbsA- or PabC-binding by Western blotting. Bound fusion proteins were detected with mouse anti-HisTag antibodies, followed by peroxidase-conjugated goat anti-mouse IgG fab fragments and visualized by chemiluminescence.
FIG. 32 shows the binding of recombinant PabC fusion proteins to immobilized fibrinogen in a capture ELISA assay. Microtiter wells were coated with a fixed amount of human fibrinogen, followed by the addition of increasing concentrations of the different PabC fusion proteins. Bound fusion protein was detected with mouse anti-HisTag antibodies and peroxidase-conjugated goat anti-mouse IgG fab fragments. Colour development was initiated by the addition of tetramethyl-benzidine substrate and stopped with H2SO4. The absorbance of the microtiter wells was read at 450 nm. Values represent the means of three independent experiments, each performed in triplicate.
FIG. 33 shows the adherence (A) and invasion (B) of the lung epithelial cell line A549 by the S. agalactiae strains 6313 pAT32, ΔpabC pAT32 and ΔpabC pATpabC, respectively. Bacterial adherence and invasion were calculated as follows: Adherence=number of adherent bacteria/total number of bacteria in the assay×100. Invasion=number of internalized bacteria/total number of bacteria in the assay×100. Each experiment was performed at least three times in triplicate. (C) Eukaryotic cell adherence and invasion (D) of S. agalactiae 6313 in the presence of different amounts of PabC and Bsp fusion proteins. Bacterial adherence and invasion were calculated as described in the legend of FIG. 32. Each experiment was performed at least three times in triplicate.

Nucleic acids coding for adhesion factor of group B