# Patent

## Title
Surface proteins from gram-positive bacteria having highly conserved motifs and antibodies that recognize them

## Abstract

Isolated peptide sequences and proteins containing these sequences are provided which are useful in the prevention and treatment of infection caused by Gram-positive bacteria. The peptide sequences have been shown to be highly conserved motifs in the surface proteins of Gram-positive bacteria, and these consensus sequences include amino acid sequences such as LPXTG (SEQ ID NO:13), ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14), VEGAVVEKPVAEAYLKQN (SEQ ID NO:15), and EYAGVDIDLAKKIAK (SEQ ID NO:16). By virtue of the highly conserved regions, the sequences and the proteins including these sequences can be utilized to generate antibodies which can recognize these highly conserved motifs and the proteins containing them and thus be useful in the treatment or prevention of a wide range of infections caused by Gram-positive bacteria.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 10/140,372, filed May 8, 2002, now U.S. Pat. No. 6,790,448 which claimed the benefit of U.S. Provisional Patent Application No. 60/289,132, filed May 8, 2001, and incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates in general to surface-located proteins from gram-positive bacteria, and in particular to a group of proteins that contain highly conserved sequence motifs. In addition, the invention relates to polyclonal and monoclonal antibodies which can recognize these proteins and which can recognize the conserved motifs. Further, the invention relates to the use of the proteins, conserved motifs and antibodies generated thereto in compositions and methods used to treat or prevent infections and other pathogenic conditions caused by a wide-array of gram-positive bacteria.
BACKGROUND OF THE INVENTION
Bacterial surface proteins of gram-positive bacteria are known to be important during the infection process since they mediate bacterial attachment to host tissues, and/or interact with the host immune system. For example, in the gram-positive bacteria Staphylococcus aureus, several of these proteins have been well characterized and were found to bind extracellular matrix proteins such as collagen, fibronectin, fibrinogen, as well as immunoglobulin G. These binding proteins include fibronectin binding proteins such as disclosed in U.S. Pat. Nos. 5,175,096; 5,320,951; 5,416,021; 5,440,014; 5,571,514; 5,652,217; 5,707,702; 5,789,549; 5,840,846; 5,980,908; and 6,086,895; fibrinogen binding proteins such as disclosed in U.S. Pat. Nos. 6,008,341 and 6,177,084; and collagen binding proteins as disclosed in U.S. Pat. Nos. 5,851,794 and 6,288,214; all of these patents incorporated herein by reference.
Previous studies have shown that the collagen and fibronectin binding proteins have been shown to contribute to the virulence of S. aureus in animal models. In addition, immunization of mice with certain of these binding protein has been shown in some cases to provide protection from septic death due to S. aureus. However, in some cases, certain formulations based on bacterial proteins from specific gram-positive bacteria such as S. aureus were not always effective in treating patients, and moreover these formulations will generally be species specific and thus do not generally afford protection against infection from a variety of gram-positive bacteria. Accordingly, it is very important to develop ways of locating surface proteins which will be utilized effectively in methods of treating or preventing infection, and in particular it is highly desirable to develop methods of treatment which can be utilized in a broad-based application to treat or prevent a wide variety of infections caused by gram-positive bacteria.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide methods for isolating proteins from gram-positive bacteria which can be utilized in methods of treating or preventing a wide range of infections caused by gram-positive bacteria.
It is another object of the present invention to provide surface proteins from gram-positive bacteria that have highly conserved sequence motifs near their carboxyl termini which can be utilized to generate antibodies that will be protective against a wide variety of gram positive bacteria.
It is further an object of the present invention to provide a method of generating an immune response to a wide variety of gram-positive bacteria by administering an immunogenic amount of an isolated peptide sequence which is highly conserved in gram positive bacteria or by administering proteins which include these highly conserved sequence motifs.
It is a further object of the present invention to provide a vaccine for treating or preventing infection from gram-positive bacteria which comprises an isolated peptide sequence which is highly conserved in gram positive bacteria or a protein which includes one or more of these highly conserved sequence motifs in an amount effective to generate an immune response to said peptides or proteins.
It is still further an object to provide compositions for treating or preventing an infection from gram-positive bacteria which comprise an isolated peptide sequence which is highly conserved in gram positive bacteria or a protein which includes one or more of these highly conserved sequence motifs and a pharmaceutically acceptable vehicle, carrier or excipient.
It is still further an object of the present invention to provide isolated antibodies which recognize these highly conserved sequence motifs or proteins which contain said motifs, and to utilize these antibodies in treating or preventing infection caused by a broad range of gram-positive bacteria.
It is an additional object of the present invention to provide diagnostic kits which can utilize the conserved sequences, proteins, and/or antibodies in accordance with the invention in order to diagnose and identify infections caused by gram-positive bacteria.
These and other objects are provided by virtue of the present invention which comprises the identification, isolation, and/or purification of highly conserved amino acid sequences from gram positive bacteria and proteins which contain said sequences, and the use of these sequences and/or proteins to treat or prevent infections caused by a wide range of gram-positive bacteria. In addition, the invention comprises monoclonal and polyclonal antibodies which recognize these sequences and proteins, as well as vaccines and other pharmaceutical compositions which utilize these peptide sequences and proteins, and methods of eliciting an immune response against a broad range of gram positive bacteria by administering the peptides and/or proteins to a human or animal in an amount effective to generate an immune response. The sequences and proteins of the present invention can thus be used in methods or achieving passive or active immunity in patients so as to treat or prevent a wide range of infections caused by gram-positive bacteria.
These embodiments and other alternatives and modifications within the spirit and scope of the disclosed invention are described in, or will become readily apparent from, reference to the detailed description of the preferred embodiments provided herein below.



BRIEF DESCRIPTION OF THE DRAWING FIGURES
FIG. 1 is a depiction of ClustalW multiple sequence alignment of the amino acid sequences of the surface proteins in accordance with the invention which have been characterized as the cell division group (or group 1) from 6 Gram-positive bacteria, shown from top to bottom as saur, S. aureus; sepi, S. epidermidis; smut, S. mutans; spne, S. pneunomiae; efae; E. faecalis; and spyo, S. pyogenes, which are identified, respectively, as SEQ ID NOS 1–6. In the drawing figure, the dark-shaded regions represent highly conserved residues, and light-shaded regions represent relatively well-conserved residues.
FIG. 2 is a depiction of ClustalW multiple sequence alignment of the amino acid sequences of the surface proteins in accordance with the invention which have been characterized as the amino acid transporter group (or group 2) from 6 Gram-positive bacteria, shown from top to bottom as spyo, S. pyogenes; spne, S. pneunomiae; smut, S. mutans; efae, E. faecalis; saur, S. aureus; and sepi, S. epidermidis; and which are identified, respectively, as SEQ ID NOS 7–12. In the drawing figure, the dark-shaded regions represent highly conserved residues, and light-shaded regions represent relatively well-conserved residues.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the present invention, the present inventors have isolated novel surfaces proteins from gram positive bacteria that are characterized in that they contain highly conserved sequences which can be utilized in the identification and isolation of surface proteins from gram positive bacteria, and which can be used to generate antibodies which will recognize said highly conserved sequences and/or the surface proteins containing said sequences. In particular, these novel proteins containing their unique highly conserved sequences were obtained in accordance with the invention using an algorithm the present inventors devised for reviewing publicly available sequence information regarding Gram-positive bacteria so as to identify and/or isolate and purify highly conserved regions in the genome and the proteins which contain those highly conserved regions. In the identification and isolation process of the invention, numerous genomes from Gram-positive bacteria are selected, and in a suitable example, genomes of six Gram-positive bacteria, namely Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus Faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans, all of which are important human pathogens, were selected and subject to the present identification process. The genomes of four S. aureus strains were publicly available at the time of the analysis and were all included in the process to identify conserved regions of the genome and/or proteome.
In the specific example, the S. aureus genome sequences were obtained from the websites of The Institute for Genomic Research (TIGR) (strain COL), The Sanger Center (a methicillin resistant strain and a methicillin sensitive strain), and University of Oklahoma's Advanced Center for Genome Technology (OU-ACGT) (strain 8325). The genome sequences of E. faecalis (strain V583), S. epidermidis (strain RP62A) and S. pneumoniae Type 4 were obtained from TIGR, and the sequences of S. mutans and S. pyogenes (group A) were from OU-ACGT.
In one preferred process, the identification steps or “data mining” was performed using a combination of software developed by the inventors, Glimmer2 from TIGR and stand-alone BLAST from the National Center for Biotechnology Information. The system was set up on a Silicon Graphics machine running IRIX6.5. In the preferred process of the present invention, an algorithm is used which consists of the following steps: (1) process each sequence file which usually contains multiple contigs into individual files each of which consists one contig; (2) predict genes based on the sequencing; (3) add a unique identification tag to each predicted gene so that genes from different organisms can be put into one single database; (4) extract genes from each genome; (5) translate each gene into its amino acid sequences; (6) form a blast searchable database of the protein sequences; and (7) perform a blast search of the database to find proteins that contain the desired conserved motif such as the LPXTG (SEQ ID NO: 13) motif wherein X can be any amino acid.
After LPXTG-containing proteins were identified, they were collected into a subset and used to establish a separate blast searchable database. Each protein in this subset was blasted against each other as well as to the large protein database to identify LPXTG-containing proteins that are conserved among these organisms. In the analysis of the LPXTG-containing proteins, two groups were located as discussed further below. Members in each group exhibited substantial overall sequence homology with each other as can be seen from Tables 1 and 2.







TABLE 1







Percentage amino acid sequence similarities among the cell division


group (group 1) from 6 Gram-positive bacteria.














E. faecalis
S. epidermidis
S. pneumoniae
S. pyogenes
S. mutans
S. aureus

















E. faecalis
100
55
66
41
67
56


S. epidermidis

100
52
41
53
90


S. pneumoniae


100
45
78
51


S. pyogenes



100
44
41


S. mutans




100
52


S. aureus





100
















TABLE 2







Percentage amino acid sequence similarities among the amino acid


transporter group (group 2) from 6 Gram-positive bacteria.














S. pneumoniae
S. pyogenes
S. mutans
S. epidermidis
S. aureus
E. faecalis

















S. pneumoniae
100
81
91
51
51
69


S. pyogenes

100
81
48
48
68


S. mutans


100
51
51
67


S. epidermidis



100
87
49


S. aureus




100
49


E. faecalis





100









In addition, after multiple sequence alignment, there are stretches of completely identical sequences in each group, as shown in FIGS. 1 and 2. Moreover, a homology search with known genes indicated that the first group (SEQ ID NOS 1–6 of FIG. 1) appeared to be a novel group of proteins that belonged to a family of cell division proteins, while the second group (SEQ ID NOS 7–12 of FIG. 2) appeared to be characterized as a family of amino acid transporters. However, none of the proteins in the two groups has been described for the organisms that were analyzed, and therefore they are novel for these bacteria.
In addition, each protein in the two groups was examined for the presence of signal peptide through the Signal mail server at Center for Biological Sequence Analysis, the Technical University of Denmark. Each was predicted to contain a signal peptide at the proper position, which appeared to confirm that these are surface proteins. In general, cell division proteins and amino acid transporters are important proteins for bacteria survival in vitro and in vivo. The fact that these proteins exhibit such high-level sequence conservation among the organisms suggests that they perform conserved functions, and it is clear that similar surface proteins are present in other Gram-positive bacteria which will also be characterized by the conserved regions in accordance with the present invention.
In addition to the sequence motif LPXTG which was discussed above, the present inventors uncovered 3 additional novel peptide sequences motifs that were conserved in the proteins identified using the method as described above. In particular, these conserved regions have the amino acid sequences identified as “SA-1”: ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14); “SA-2”: VEGAVVKPVAEAYLKQN (SEQ ID NO:15), and “SA-3”: EYAGVDIDLAKKIAK (SEQ ID NO:16). The peptide sequences were selected from 3 regions in a Staphylococcus aureus protein that belongs to one ABC transporter group. Each region is highly conserved among the 6 Gram-positive bacteria examined (Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumoniae, and Staphylococcus aureus). Also, in order to increase the chance that the sequences will be exposed on the surface, we limited the selection of the sequences to hydrophilic regions using the method of Kyte and Doolittle.
In accordance with the/present invention, these specific peptides may be obtained in any of a number of suitable ways well known in the art to generate peptides, and similarly, proteins containing these peptides may be obtained through physical isolation and/or separation methods from actual bacteria, or through conventional methods of protein synthesis. In the present case, one suitable method for preparing the peptides of the invention is through synthesis using an Advanced Chem Tech 396 multiple peptide synthesizer, using Fmoc chemistry and activation with HBTU. After cleavage from the resin, peptides can be purified by reverse-phase chromatography on a Waters Delta-Pak C18 column, eluted with gradient of acetonitrile in 0.1% trifluoroacetic acid/water. The purity of the peptides obtained in this fashion has been further confirmed by mass spectrometry analysis, and the peptide-KLH conjugation with EDC. The carrier protein KLH and the peptides (1:1 by weight) were coupled using EDC (Pierce) for 2 hours at room temperature. The reaction mixture is subjected to a desalting column pre-equilibrated with the purification buffer (0.083 M sodium phosphate, 0.9 M NaCl, pH 7.2). The conjugated peptides were eluted with the purification buffer and 0.5 ml fractions were collected. Each fraction was measured for its absorbance at 280 nm and the fractions containing the conjugate were pooled.
Accordingly, in accordance with the present invention, there are provided isolated amino acid sequences, namely ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14); VEGAVVEKPVAEAYLKQN (SEQ ID NO:15), and EYAGVDIDLAKKIAK (SEQ ID NO:16), which are highly conserved regions in surface proteins from Gram-positive bacteria which can be utilized to generate antibodies that can recognize these sequences and which thus can be utilized in methods of treating or preventing a wide range of Gram-positive bacteria that will have proteins containing these sequences. In addition, it is contemplated that proteins from Gram-positive bacteria that contain these conserved sequences may also be isolated and/or purified, and may also be used to generate antibodies which recognize these proteins and which can be utilized in methods of treating or preventing infection caused by Gram-positive bacteria.
In accordance with the invention, the antibodies generated by immunization with either the conserved sequences described above or proteins containing these sequences may be either monoclonal or polyclonal, and may be prepared in any of a number of conventional ways well known to those of ordinary skill in the art. For example, monoclonal antibodies in accordance with the present invention may be produced, e.g., using the method of Kohler and Milstein (Nature 256:495–497, 1975), or other suitable ways known in the field, and in addition can be prepared as chimeric, humanized, or human monoclonal antibodies in ways that would be well known in this field. Still further, monoclonal antibodies may be prepared from a single chain, such as the light or heavy chains, and in addition may be prepared from active fragments of an antibody which retain the binding characteristics (e.g., specificity and/or affinity) of the whole antibody. By active fragments is meant an antibody fragment which has the same binding specificity as a complete antibody which recognizes and binds to the peptide sequences or the proteins of the present invention, and the term “antibody” as used herein is meant to include said fragments. Additionally, antisera prepared using monoclonal or polyclonal antibodies in accordance with the invention are also contemplated and may be prepared in a number of suitable ways as would be recognized by one skilled in the art.
As indicated above, antibodies which recognize the conserved sequences, or proteins containing these sequences, as set forth above, may be prepared in a number of suitable ways that would be well known in the art, such as the well-established Kohler and Milstein method described above which can be utilized to generate monoclonal antibodies. In one such method, mice are injected intraperitoneally once a week for a prolonged period with a antigen comprising a purified recombinant peptide or protein in accordance with the invention, followed by a test of blood obtained from the immunized mice to determine reactivity to the purified antigen. Following identification of mice suitably reactive to the antigen, lymphocytes isolated from mouse spleens may be fused to mouse myeloma cells to produce hybridomas positive for the antibodies against the peptides and/or proteins of the invention which are then isolated and cultured, following by purification and isotyping.
In order to generate monoclonal antibodies in accordance with the invention, it is thus preferred that these be generated using recombinantly prepared peptide sequences or proteins using conventional methods well known in the art. For example, one such method employs the use of E. coli expression vector pQE-30 as an expression vector for cloning and expressing recombinant proteins and peptides. In this method, PCR is used to amplify DNA coding for the peptide sequences of the invention, and a suitable E. coli expression vector such as PQE-30 (Qiagen) is used to allow for the expression of a recombinant fusion protein having the appropriate sequences. The cells containing these fusion proteins may be harvested, and the peptides of the invention may be eluted suing suitable buffer solutions. The peptides can then be subject to further purification steps, e.g., put through an endotoxin removal process, and the appropriate peptides obtained in this fashion may then be utilized to elicit an immune response and generate antibodies in accordance with the invention.
As indicated above, although production of antibodies using recombinant forms of the peptides or proteins of the invention is preferred, antibodies may be generated from natural isolated and purified proteins or peptides as well, and monoclonal or polyclonal antibodies can be generated using the natural peptides or proteins or active regions in the same manner as described above to obtain such antibodies. Still other conventional ways are available to generate the antibodies of the present invention using recombinant or natural purified peptides or proteins or its active regions, as would be recognized by one skilled in the art.
As would be recognized by one skilled in the art, the antibodies of the present invention may also be formed into suitable pharmaceutical compositions for administration to a human or animal patient in order to treat or prevent an infection caused by Gram-positive bacteria. Pharmaceutical compositions containing the antibodies of the present invention, or effective fragments thereof, may be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such as saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the patient and the patient's condition, and a variety of modes of administration would be suitable for the compositions of the invention, as would be recognized by one of ordinary skill in this art. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.
For topical administration, the composition is formulated in the form of an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). Wound or surgical dressings, sutures and aerosols may be impregnated with the composition. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.
Additional forms of antibody compositions, and other information concerning compositions, methods and applications with regard to other surface proteins will generally also be applicable to the present invention, including those antibodies and compositions as disclosed, for example, in U.S. Pat. No. 6,288,214 (Hook et al.), incorporated herein by reference. Similarly, other forms of antibody compositions, and other information concerning compositions, methods and applications with regard to other surface proteins and peptides which will also be applicable to the present invention are disclosed in U.S. Ser. No. 09/810,428, filed Mar. 19, 2001, incorporated herein by reference; and U.S. Ser. No. 09/386,962, filed Aug. 31, 1999, incorporated herein by reference.
The antibody compositions of the present invention may also be administered with a suitable adjuvant in an amount effective to enhance the immunogenic response against the conjugate. For example, suitable adjuvants may include alum (aluminum phosphate or aluminum hydroxide), which is used widely in humans, and other adjuvants such as saponin and its purified component Quil A, Freund's complete adjuvant, RIBBI adjuvant, and other adjuvants used in research and veterinary applications. Still other chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol. 147:410–415 (1991) and incorporated by reference herein, encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739–1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as NovasomeTM lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be useful.
In any event, the antibody compositions of the present invention will thus be useful for treating or preventing infections caused by gram-positive bacteria and/or in reducing or eliminating the binding of gram-positive bacteria to host cells and/or tissues.
In accordance with the present invention, isolated and/or purified conserved amino acid sequences such as SEQ ID NOS 14–16 are provided which can be utilized in methods of treating or preventing a Gram-positive bacterial infection. Accordingly, in accordance with the invention, nucleic acids are provided which encode the peptide sequences of the invention and which encode the proteins which contain these conserved sequences, or degenerates thereof.
As indicated above, in accordance with the present invention, methods are provided for preventing or treating a Gram-positive bacterial infection which comprise administering an effective amount of an antibody to the peptides or proteins identified above in amounts effective to treat or prevent the infection. In addition, the antibodies in accordance with the invention are particularly effective against a wide range of Gram-positive bacteria because they can recognize conserved peptide sequences, and/or proteins containing these sequences therein, which will be found in the wide range of gram-positive bacteria that commonly cause infection in human or animal patients.
Accordingly, in accordance with the invention, administration of the antibodies of the present invention in any of the conventional ways described above (e.g., topical, parenteral, intramuscular, etc.), and will thus provide an extremely useful method of treating or preventing Gram-positive bacterial infections in human or animal patients. By effective amount is meant that level of use, such as of an antibody titer, that will be sufficient to either prevent adherence of the gram-positive bacteria, or to inhibit binding of the bacteria to host cells, and thus will be useful in the treatment or prevention of a gram-positive bacterial infection. As would be recognized by one of ordinary skill in this art, the level of antibody titer needed to be effective in treating or preventing a particular Gram-positive infection will vary depending on the nature and condition of the patient, and/or the severity of the pre-existing infection.
In addition to the use of the present antibodies to treat or prevent Gram-positive bacterial infection, the present invention contemplates the use of these antibodies in a variety of ways, including the detection of the presence of gram-positive bacteria to diagnose a bacterial infection, whether in a patient or on medical equipment which may also become infected. In accordance with the invention, a preferred method of detecting the presence of such infections involves the steps of obtaining a sample suspected of being infected by one or more Gram-positive bacteria species or strains, such as a sample taken from an individual, for example, from one's blood, saliva, tissues, bone, muscle, cartilage, or skin. While adequate diagnostic tests can be performed using the sample itself, it is also possible to perform more complex tests which utilize the DNA of the sample. In these diagnostic tests, the cells can then be lysed, and the DNA extracted, precipitated and amplified. Following isolation of the sample, diagnostic assays utilizing the antibodies of the present invention may be carried out to detect the presence of Gram-positive bacteria, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and ELISA assays. In general, in accordance with the invention, a method of diagnosing a Gram-positive bacterial infection is contemplated wherein a sample suspected of being infected with such bacteria has added to it an antibody in accordance with the present invention, and a Gram-positive bacterial infection will be indicated by antibody binding to the appropriate proteins or peptides in the sample.
Accordingly, antibodies in accordance with the invention may be used for the specific detection of gram-positive bacterial or surface proteins, for the prevention of infection from Gram-positive bacteria, for the treatment of an ongoing infection, or for use as research tools. The term “antibodies” as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the peptides and/or proteins of the present invention, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies as set forth above. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art.
Any of the above described antibodies may be labeled directly with a detectable label for identification and quantification of Gram-positive bacteria. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).
Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.
Further, when administered as pharmaceutical compositions to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo, the antibodies of the present invention may be useful in those cases where there is a previous bacterial infection because of the ability of this antibody to further restrict and inhibit binding of Gram-positive bacteria to binding proteins such as fibrinogen or fibrin and thus limit the extent and spread of the infection. In addition, the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be “humanized” by transplanting the complimentarity determining regions of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al., Nature 321:522–525 (1986) or Tempest et al. Biotechnology 9:266–273 (1991) or “veneered” by changing the surface exposed murine framework residues in the immunoglobulin variable regions to mimic a homologous human framework counterpart as described, e.g., by Padlan, Molecular Imm. 28:489–498 (1991), or European Patent application 519,596, these references incorporated herein by reference. Even further, when so desired, the monoclonal antibodies of the present invention may be administered in conjunction with a suitable antibiotic to further enhance the ability of the present compositions to fight bacterial infections.
Medical devices or polymeric biomaterials to be coated with the antibodies, proteins and active fragments described herein include, but are not limited to, staples, sutures, replacement heart valves, cardiac assist devices, hard and soft contact lenses, intraocular lens implants (anterior chamber or posterior chamber), other implants such as corneal inlays, kerato-prostheses, vascular stents, epikeratophalia devices, glaucoma shunts, retinal staples, scleral buckles, dental prostheses, thyroplastic devices, laryngoplastic devices, vascular grafts, soft and hard tissue prostheses including, but not limited to, pumps, electrical devices including stimulators and recorders, auditory prostheses, pacemakers, artificial larynx, dental implants, mammary implants, penile implants, cranio/facial tendons, artificial joints, tendons, ligaments, menisci, and disks, artificial bones, artificial organs including artificial pancreas, artificial hearts, artificial limbs, and heart valves; stents, wires, guide wires, intravenous and central venous catheters, laser and balloon angioplasty devices, vascular and heart devices (tubes, catheters, balloons), ventricular assists, blood dialysis components, blood oxygenators, urethral/ureteral/urinary devices (Foley catheters, stents, tubes and balloons), airway catheters (endotracheal and tracheostomy tubes and cuffs), enteral feeding tubes (including nasogastric, intragastric and jejunal tubes), wound drainage tubes, tubes used to drain the body cavities such as the pleural, peritoneal, cranial, and pericardial cavities, blood bags, test tubes, blood collection tubes, vacutainers, syringes, needles, pipettes, pipette tips, and blood tubing.
It will be understood by those skilled in the art that the term “coated” or “coating”, as used herein, means to apply the antibody or active fragment, or pharmaceutical composition derived therefrom, to a surface of the device, preferably an outer surface that would be exposed to a gram-positive bacterial infection. The surface of the device need not be entirely covered by the protein, antibody or active fragment.
In another embodiment of the invention, the antibodies may also be used as a passive vaccine which will be useful in providing suitable antibodies to treat or prevent a gram-positive bacterial infection. As would be recognized by one skilled in this art, such a vaccine may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle. However, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration, and the carrier is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.
The preferred dose for administration of an antibody composition in accordance with the present invention is that amount which will be effective in preventing or treating a gram-positive bacterial infection, and one would readily recognize that this amount will vary greatly depending on the nature of the infection and the condition of a patient. As indicated above, an “effective amount” of antibody or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. As pointed out below, the exact amount of the antibody or a particular agent that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Accordingly, the “effective amount” of any particular antibody composition will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The compositions may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, Mo.).
When used with suitable labels or other appropriate detectable biomolecule or chemicals, the monoclonal antibodies described herein are useful for purposes such as in vivo and in vitro diagnosis of gram-positive bacterial infections or detection of gram-positive bacteria. Laboratory research may also be facilitated through use of such antibodies. Various types of labels and methods of conjugating the labels to the antibodies of the invention are well known to those skilled in the art, such as the ones set forth below.
For example, the antibody can be conjugated (directly or via chelation) to a radiolabel such as, but not restricted to, 32P, 3H, 14C, 35S, 125I, or 131I. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. Bioluminescent labels, such as derivatives of firefly luciferin, are also useful. The bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light. Fluorogens may also be used to label proteins. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.
The location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren and Nelson (Mol. Cell. Biol., 7: 1326–1337, 1987).
As indicated above, the antibodies of the present invention, or active portions or fragments thereof, are particularly useful for fighting or preventing bacteria infection in patients or on in-dwelling medical devices to make them safer for use. In short, the antibodies of the present invention are thus extremely useful in treating or preventing Gram-positive infections in human and animal patients and in medical or other in-dwelling devices.
In accordance with the invention, a diagnostic kit is also provided which utilizes an antibody of the invention as set forth above, and in one typical example, this kit may comprise an antibody of the invention which can recognize a conserved peptide region as set forth above or a protein containing said region, means for introducing the antibody to a sample suspected of containing gram-positive bacteria, and means for identifying gram-positive bacteria that are recognized by said antibody.
In accordance with the present invention, the peptides and proteins as described above may also be utilized in the development of vaccines for immunization against Gram-positive infections, and thus a method of eliciting an immune response in a human or animal is also provided wherein an immunogenic amount of a peptide or protein in accordance with the invention is administered to a human or animal. In the preferred embodiment, vaccines in accordance with the invention are prepared using methods that are conventionally used to prepare vaccines, and the preferred vaccine comprises an immunogenic amount of the peptides or proteins as described above along with a pharmaceutically acceptable vehicle, carrier or excipient. As would be recognized by one of ordinary skill in the art, these vaccines may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle, however, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable vehicle, carrier or excipient in order to facilitate administration, and said carrier or other materials is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.
The present invention thus provides for the identification and isolation of proteins having the signature conserved regions as set forth above, as well as the vaccines, antibodies and other forms of the invention as set forth above, and the invention will be particularly useful in developing and administering treatment regimens which can be used to fight or prevent infections caused by Gram-positive bacteria.
The following example is provided which exemplifies aspects of the preferred embodiments of the present invention. However, it will be appreciated by those of skill in the art that the techniques disclosed in the example which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Moreover, those of skill in the art will also appreciate that in light of the present specification, many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention
EXAMPLE
Identification and Isolation of Conserved Sequences and Proteins Containing them
Gram-positive bacteria have a group of surface-located proteins that contain a unique sequence motif near the carboxyl termini. The motif consists of amino acid residues LPXTG (X being any amino acids) that is necessary for anchoring the protein to the bacterial cell wall by a transamidase called sortase. These bacterial surface proteins are thought to be important during the infection processes since they may mediate bacterial attachment to host tissues, and/or interact with the host immune system. They are potential candidates for active and/or passive immunization, as well as targets for new types of antibiotics. In Staphylococcus aureus, several of these proteins have been well characterized and were found to bind extracellular matrix proteins such as collagen, fibronectin, fibrinogen, as well as immunoglobulin G. The collagen and fibronectin binding proteins were shown to contribute to the virulence of S. aureus in animal models. In addition, immunization of mice with the collagen binding protein provided protection from septic death due to S. aureus, indicating that it may be used as a vaccine. LPXTG containing proteins that bind host proteins were also found in other gram-positive organisms such as Enterococcus faecalis and streptococci.
In this study we devised an algorithm for mining publicly available genome sequences of Gram-positive bacteria for LPXTG containing genes. We chose the genomes of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus Faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans, all of which are important human pathogens. The genomes of four S. aureus strains were publicly available at the time of the analysis and were all included in the data mining process. The S. aureus genome sequences were obtained from the websites of The Institute for Genomic Research (TIGR) (strain COL), The Sanger Center (a methicillin resistant strain and a methicillin sensitive strain), and University of Oklahoma's Advanced Center for Genome Technology (OU-ACGT) (strain 8325). The genome sequences of E. faecalis (strain V583), S. epidermidis (strain RP62A) and S. pneumoniae Type 4 were obtained from TIGR, and the sequences of S. mutans and S. pyogenes (group A) were from OU-ACGT. Data mining was performed using a combination of software developed by us, Glimmer2 from TIGR and stand-alone BLAST from the National Center for Biotechnology Information. The system was set up on a Silicon Graphics machine running IRIX6.5. The algorithm consists the following steps: 1) process each sequence file which usually contains multiple contigs into individual files each of which consists one contig, 2) predict genes, 3) add unique identification tag to each predicted gene so that genes from different organisms can be put into one single database, 4) extract genes from each genome, 5) translate each gene into amino acid sequence, 6) form a blast searchable database of the protein sequences, and 7) blast search the database to find proteins that contain the LPXTG motif.
After LPXTG containing proteins were identified, they were collected into a subset and used to establish a separate blast searchable database. Each protein in this subset was blasted against each other as well as to the large protein database to identify LPXTG-containing proteins that are conserved among these organisms. Two groups were found. Members in each group exhibited substantial overall sequence homology with each other (see Tables 1 and 2 above). In addition, after multiple sequence alignment, there are stretches of completely identical sequences in each group (see FIGS. 1 and 2). Homology search with known genes indicated that the first group belongs to a family of cell division proteins, while the second group belongs to a family of amino acid transporters. However, none of the proteins in the two groups has been described for the organisms that we analyzed, and therefore they are novel for these bacteria.
Each protein in the two groups was examined for the presence of signal peptide through the Signal mail server at Center for Biological Sequence Analysis, the Technical University of Denmark. Each was predicted to contain a signal peptide at the proper position, indicating that these are most likely surface proteins. Cell division proteins and amino acid transporters are important proteins for bacteria survival in vitro and in vivo. The fact that these proteins exhibit such high-level sequence conservation among the organisms suggests that they perform conserved functions. We envision that similar surface proteins are present in other Gram-positive bacteria. In fact we have identified 3 novel peptide sequences from the conserved proteins. The peptide sequences were selected from 3 regions in a Staphylococcus aureus protein that belongs to one ABC transporter group. Each region is highly conserved among the 6 Gram-positive bacteria examined (Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumoniae, and Staphylococcus aureus).
Also, in order to increase the chance that the sequences will be exposed on the surface, we limited the selection of the sequences to hydrophilic regions using the method of Kyte and Doolittle. The sequences are listed below:










SA-1:
ALKTG KIDII ISGMT STPER KK
(SEQ ID NO:14)



 


SA-2:
VEGAV VEKPV AEAYL KQN
(SEQ ID NO:15)


 


SA-3:
EYAGV DIDLA KKIAK
(SEQ ID NO:16)






The peptides were synthesized in an Advanced Chem Tech 396 multiple peptide synthesizer, using Fmoc chemistry and activation with HBTU. After cleavage from the resin, peptides were purified by reverse-phase chromatography on a Waters Delta-Pak C18 column, eluted with gradient of acetonitrile in 0.1% trifluoroacetic acid/water. The purity of the peptides was further confirmed by mass spectrometry analysis.
The peptide-KLH conjugation with EDC: The carrier protein KLH and the peptides (1:1 by weight) were coupled using EDC (Pierce) for 2 hours at room temperature. The reaction mixture is subjected to a desalting column pre-equilibrated with the purification buffer (0.083 M sodium phosphate, 0.9 M NaCl, pH 7.2). The conjugated peptides were eluted with the purification buffer and 0.5 ml fractions were collected. Each fraction was measured for its absorbance at 280 nm and the fractions containing the conjugate were pooled.
The use of the conserved conjugated peptides and polypeptides: The principle, methods and applications described above for the three conjugated peptides are applicable and will be applied to proteins in the second group of highly homologous surface proteins. This evidenced that: 1) antibodies raised against these proteins will be able to recognize a wide range of Gram-positive bacteria and may be used as a basis for a broad spectrum passive immunization protocol; 2) protective, therapeutic, or diagnostic antibodies raised against these proteins could recognize conserved epitopes present on different species of Gram-positive bacteria; 3) a single mAb recognizing the conserved peptides could be used to protect against all Gram-positive bacterial infections; 4) these proteins may be used as a basis for a broad spectrum vaccine; and 5) these proteins may be used as novel targets for designing new types of antimicrobial agents.

## Claims

1. An isolated antibody capable of binding an amino acid sequence selected from the group consisting of ALKTGKIDIIISGMTSTPERKK (SEQ ID NO: 14), VEGAVVEKPVAEAYLKQN (SEQ ID NO: 15), and EYAGVDIDLAKKIAK (SEQ ID NO: 16).

2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody according to claim 1, wherein the antibody is a polyclonal antibody.

4. A pharmaceutical composition comprising the isolated antibody according to claim 1 and a pharmaceutically acceptable vehicle, carrier or excipient.

5. Isolated antisera containing an antibody according to claim 1.