JP2008517900A - Staphylococcus aureus Isd protein-based antiinfectives - Google Patents

Abstract

  The present invention provides novel anti-infective agents that act on Staphylococcus aureus (S. aureus) iron-regulated surface determinants IsdA, IsdB, and IsdC.

Description

This application claims priority from US Provisional Application No. 60 / 621,921 filed Oct. 25, 2004, the entire contents of which are incorporated herein by reference.

Background Iron is an absolute requirement for the growth of most microorganisms, with exceptions considered to be Lactobacillus (Archibald (1983) FEMS Microbiol. Lett. 19: 29-32) and Borrelia burgdorferi (original language: Borrelia burgdorferi) (Posey and Gherardini
(2000) Science 288: 1651-1653). Iron is a nutrient that often limits growth, despite being the fourth most abundant element in the crust. In an aerobic environment and at physiological pH, iron exists in the trivalent iron (Fe ³⁺ ) state and forms insoluble hydroxide and oxyhydroxide precipitates. Mammals overcome iron limitations by having high affinity iron-binding glycoproteins such as transferrin and lactoferrin that serve to solubilize and deliver iron to host cells (Weinberg (1999) Emerg. Infect. Dis. 5: 346-352). As a result, extracellular free iron is further restricted so that the concentration of free iron in the human body is 10 ^-18 M, several orders of magnitude lower than that required to support productive bacterial infection. (Braun et al., (1998) Bacterial iron transport:
mechanisms, genetics, and regulation, p. 67-145. In A. Sigel and H. Sigel (ed.), Metal Ions in Biological Systems,
vol. 35.Iron transport and storage in microorganisms, plants, and animals.
Marcel Dekker, Inc., New York). Iron isolation is an important innate defense against bacterial infections (Skaar and
Schneewind (2004) Microbes and Infect. 6: 390-397).

In order to overcome the iron limitation, bacteria have evolved several different mechanisms to acquire this essential nutrient. For example, members of the Pasteurella family appear to express receptors that recognize iron-loaded transferrin and lactoferrin (Gray-Owen and Schryvers, (1996) Trends Microbiol. 4: 185-91). However, one of the most common iron-acquisition mechanisms is the cognate cell envelope receptor that actively internalizes the low-molecular-weight, high-affinity iron chelator called siderophore and the trivalent iron-siderophore complex. Through the use of. Many siderophores can successfully compete with transferrin and lactoferrin for host iron. Indeed, the expression of an iron-siderophore uptake system is expressed by septic E. coli. E. coli (Williams (1979)
Infect. Immun. 26: 925-932), Vibrio anguillarum (Original: Vibrio anguillarum) (Crosa et
al. (1980) Infect. Immun. 27: 897-902), Erwinia chrysanthemi (Original: Erwinia chrysanthemi) (Enard et
al. (1988) J. Bacteriol. 170: 2419-2426) and Pseudomonas aeruginosa (original language: Pseudomonas aeruginosa) (Meyer et
al. (1996) Infect. Immun. 64: 518-523).

Staphylococcus aureus (Original: Staphylococcus aureus) (S. aureus) is sstABCD (Morrissey et al. (2000)
Infect. Immun. 68: 6281-6288), sirABC (Heinrichs et al. (1999)
J. Bacteriol. 181: 1436-1443), fhuCBG (Sebulsky et al. (2000) J. Bacteriol. 182: 4394-4400), and sbn (Dale et
al., (2004) Infect. Immun. 72: 29-37) Has several different iron-regulated ABC transporters, including those encoded by the operon. Although the transported substrate is unknown for this sst and sir system, the fhuCBG gene is coordinated with fhuD1 and fhuD2 (Sebulsky and Heinrichs (2001) J. Bacteriol. 183: 4994-5000), iron (III) -Involved in the acquisition of hydroxamate complexes. Many coagulase negative Staphylococcus (CoNS) and S. cerevisiae. Several members of Staphylococcus, including the Aureus strain, produce siderophores. Two of these siderophores, staphyloferrin A (Konetschny-Rapp et al.
(1990) Eur. J. Biochem. 191: 65-74; Meiwes et
al. (1990) FEMS Microbiol. Lett. 67: 201-206) and staphyloferrin B (Dreschel et al. (1993)
BioMetals. 6: 185-192; Haag et al.
(1994) FEMS Microbiol. Lett. 115: 125-130) is of the polycarboxylate class, but the third aureochelin (Courcol et al. (1997)
Infect. Immun. 65: 1944-1948) has not been chemically characterized. In addition, a further siderophore encoded by the sbn operon is It appears to be specific for Aureus and is S. cerevisiae compared to other CoNS strains. It appears to be a key determinant of Aureus fungal power (Dale et al., (2004) Infect. Immun. 72: 29-37).

However, the most abundant source of iron in the human body is sequestered by heme-containing proteins (blood proteins). Heme is a cyclic molecule containing one iron atom bonded to four cyclic nitrogen atoms of porphyrin. Blood proteins are responsible for many cellular functions, and hemoglobin and myoglobin are the most abundant heme-containing proteins in mammals. Because iron is essential for the survival of bacterial pathogens, bacteria have acquired many several mechanisms for collecting iron from free heme and blood proteins. In particular S. Aureus can acquire iron from heme or blood proteins through the iron-regulated surface determinant (Isd) system. The Isd system contains cell surface proteins that can bind to and transport heme across the cell wall of bacterial cells, and at least one cytoplasmic protein that can extract iron from heme (Mazmanian et al., (2003 )
Science 299: 906-909; Clarke et al., (2004)
Mol. Microbiol. 51: 1509-1519). In addition, Isd proteins, particularly IsdA, include but are not limited to fibrinogen and fibronectin (Clarke et al.,
(2004) Mol. Microbiol. 51: 1509-1519), transferrin (Taylor and Heinrichs (2002) Mol. Microbiol. 43: 1603-1614) and hemin (Mazmanian et al., (2003) Science 299: 906-909) It seems to bind to a wide range of extracellular matrix proteins, including

S. Aureus can range from small skin and wound infections to more severe sequelae such as endocarditis, osteomyelitis and sepsis (Archer (1998) Clin. Infect. Dis. 26: 1179-1181). It is an epidemic human pathogen that causes illness. S. The ability of Aureus to invade and establish in many tissues is due to several fungal factors, including fibronectin-, elastin-, and collagen-binding proteins that support tissue adhesion, as well as multiple factors that cause tissue destruction and bacterial dissemination. Of exotoxin and protease. The ability of this bacterium to acquire iron during in vivo growth appears to be important for its pathogenesis, and several research groups are involved in binding and / or transporting the host iron compound Characterizing several different genes (Mazmanian et al., (2003)
Science 299: 906-9; Modun et al.,
(1998) Infect. Immun. 66: 3591-3596; Taylor
and Heinrichs (2002) Mol. Microbiol. 43: 1603-1614).

  Originally, penicillin was the worst S.C. Even Aureus infection could be used to treat. However, S. The emergence of Aureus penicillin resistant strains The effectiveness of penicillin in treating Aureus infection has diminished, and most S. cerevisiae encountered today with nosocomial infections. Aureus strains do not respond to penicillin. S. Aureus penicillin-resistant strains produce beta-lactamases that destroy antibiotic activity by converting penicillin to penicillic acid. In addition, this beta-lactamase coding gene is often episomal, typically transmitted on plasmids, and is often one of several genes on episomal factors that collectively provide multidrug resistance.

  Methicillin introduced in the 1960s Overcame the problem of Aureus resistance to penicillin. In these compounds, the part responsible for antibiotic activity in penicillin is preserved, and the other part of the penicillin as a good substrate for inactivating lactamase is altered or altered. However, including aminoglycosides, tetracyclines, chloramphenicol, macrolides and lincosamides, Methicillin resistance has emerged in this organism, along with resistance to many other antibiotics that are effective against Aureus. Indeed, the common S. aureus methicillin resistant strain is multidrug resistant. Methicillin resistance Aureus (MRSA) has become one of the most important nosocomial pathogens in the world and poses serious problems in infection control. Today, many strains are multi-resistant to virtually all antibiotics, the exception being vancomycin-type glycopeptide antibiotics. S. The drug resistance of Aureus infection presents significant therapeutic problems that will be exacerbated unless new therapeutics are developed. In this way, S.I. Although there is an urgent medical need for new and effective therapeutics to treat Aureus infection, it has not been met.

SUMMARY OF THE INVENTION The present invention is at least partially described by It is based on the identification and characterization of Isd (iron-regulated surface determinants) proteins IsdA, IsdB, and IsdC, which are part of the Isd system involved in the internalization of iron from heme and blood proteins in Aureus. IsdA, IsdB, and IsdC are S.I. It is expressed on the surface of Aureus cells but is important for growth and survival under iron restriction in vivo. Consequently, IsdA, IsdB, and IsdC proteins are attractive vaccine targets that, if inhibited, would impair bacterial growth in vivo. In addition, the IsdA, IsdB, and IsdC proteins are available from S. cerevisiae. It is an attractive drug target that can be used in screening assays to identify Aureus specific antibiotics.

In one aspect, the invention features an Isd protein-based vaccine. In certain exemplary embodiments, the Isd-based vaccine comprises an IsdA polypeptide and a pharmaceutically acceptable carrier. In certain embodiments, the IsdA polypeptide is SEQ ID
Contains the full-length amino acid sequence of NO: 3. In another embodiment, IsdA
The polypeptide is a peptide consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO: 3. In another embodiment, the Isd vaccine comprises an IsdB polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the IsdB polypeptide comprises the full length amino acid sequence of SEQ ID NO: 6. In another embodiment, the IsdB polypeptide has SEQ ID
NO: A peptide consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids out of 6. In another embodiment, the Isd vaccine comprises an IsdC polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the IsdC polypeptide comprises the full length amino acid sequence of SEQ ID NO: 9. In another embodiment, the IsdC polypeptide is a peptide consisting of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO: 9. The vaccine composition may be formulated as an injectable formulation and may further include an adjuvant.

  In another aspect, the invention features novel antibiotics that inhibit iron uptake into Staphylococcus aureus (S. aureus), including antibodies, antisense nucleic acids, and siRNA. The invention features antibodies against IsdA, IsdB and / or IsdC. In some embodiments, the antibody against the IsdA polypeptide is raised against the full-length recombinant amino acid sequence of SEQ ID NO: 3, or at least 5, 10, 15, 20, 25 of SEQ ID NO: 3. , 30, 35, 40, 45, or 50 amino acids. Antibodies against the IsdB polypeptide are directed against the full-length recombinant recombinant amino acid sequence of SEQ ID NO: 6, or at least 5, 10, 15, 20, 25, 30, 35, 40 of SEQ ID NO: 6. , 45, or 50 amino acids. The antibody against the IsdC polypeptide is against the full length recombinant amino acid sequence of SEQ ID NO: 9, or at least 5, 10, 15, 20, 25, 30, 35, 40 of SEQ ID NO: 9. May be generated for peptides consisting of 45 or 50 amino acids. The antibody against the Isd polypeptide may be monoclonal or polyclonal. Antibodies against Isd polypeptides may be formulated into injectable formulations and administered as antibacterial treatments.

  In a further aspect, the invention features screening assays that identify agents that inhibit or interfere with the expression level and / or function of any of the Isd proteins. In certain exemplary embodiments, the invention features screening assays for agents that inhibit IsdA expression and / or function. In one embodiment, the assay is a binding assay and an agent that interferes with its biochemical function by binding to the isd gene product is a candidate S. cerevisiae. An aureus-specific antibody. In another embodiment, the assay is an expression assay and the agent that decreases the level of expression of the Isd polypeptide is a candidate S. cerevisiae. An aureus-specific antibody.

  In a further aspect, the Isd protein is not limited to S. cerevisiae. It may be expressed on gram-positive bacteria including Aureus, Corynebacterium diphtheria (original: Corynebacterium diphtheriae), Listeria monocytogenes (original: Listeria monocytogenes), and Bacillus anthracis (original: Bacillus anthracis). Thus, using vaccines and inhibitors that target the Isd protein, as described herein, could treat a number of pathogenic gram-positive bacterial strains that cause disease in mammals.

  Additional features and advantages of the disclosed invention will be discussed below in connection with the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION Overview As discussed here, iron internalization through heme uptake is a pathogenic feature that can be attenuated by knocking out isd genes such as isdA, isdB, and isdC. Furthermore, as described herein, the Isd protein bound to heme is S. cerevisiae in the host. It appears to play an additional role in promoting Aureus survival. Isd protein bound to heme appears to act as an oxidative buffer that protects cells from the adverse effects of free radicals. Therefore, the mutant without expression of Isd protein is vulnerable to hydrogen peroxide, whereas wild-type S. cerevisiae. Aureus can survive even in higher concentrations of hydrogen peroxide.

  The Isd protein as described here is S. coli in vivo. It is essential for Aureus infection. Highly expressed during Aureus infection. S. As Aureus enters the host, it will encounter an iron-restricted environment, after which Iad protein expression is upregulated. In iron-restricted hosts, Isd expression is expressed in S. cerevisiae. It is likely that Aureus will remain up-regulated while collecting iron. In particular, IsdA is immunodominant as described here. This is because a 1: 4000 dilution of serum from a covalescent patient (ie, a patient infected with S. aureus) reacted positively with 4 micrograms of purified IsdA protein on a Western immunoblot. Thus, Isd protein is an attractive target for vaccine development. If the antigenic peptide of Isd protein is used as a vaccine target, It will generate an effective immune response against Aureus. Furthermore, methods for inhibiting the function of an Isd protein using an Isd-specific antibody, antisense RNA, siRNA or a small molecule inhibitor are described in S.A. It would be an effective way to attenuate the ability of Aureus.

  The Isd protein described here is S. cerevisiae. It is expressed on Aureus. In a further embodiment, the Isd protein will be expressed in other Gram positive bacteria. Non-limiting examples of Gram-positive pathogens that express Isd protein include S. aureus, Corynebacterium diphtheria, Listeria monocytogenes, and Bacillus anthracis. Thus, as described herein, vaccines and inhibitors that target the Isd protein could be used to treat other pathogenic Gram-positive bacterial strains that cause disease in mammals.

2. Definitions For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  The articles “one” (original word: “a”) and “one (original word:“ an ”) are used here to refer to one or more (ie at least one) of the grammatical objects of this article. For example, “one element” means one element or two or more elements.

  The term “adjuvant” as used herein refers to a substance that elicits an enhanced immune response when used in combination with a specific antigen.

  The term “agent” as used herein refers to a compound, a mixture of compounds, a biological macromolecule (eg, a nucleic acid, antibody, protein or portion thereof, such as a peptide), or a bacterial, plant, fungal or animal (especially mammalian) cell. Or it is used to refer to an extract made from tissue. Agents can be identified by screening assays described below. Such agents may be inhibitors or antagonists of Isd mediated iron uptake in Staphylococcus aureus. Because of the activity of such an agent, it is a “therapeutic agent” that is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject. It will be suitable.

  The term “antagonist” or “inhibitor” refers to an agent that downregulates (eg, suppresses or inhibits) at least one biological activity of a protein. An antagonist will be a compound that inhibits or reduces the interaction between one protein and another molecule, such as a target peptide or enzyme substrate. An antagonist may also be a compound that downregulates the expression of a gene or reduces the amount of expressed protein present.

As used herein, the term “antibody” specifically binds an antigen or antigen-binding portion of any immunoglobulin or immunoglobulin (eg, IgG, IgD, IgA, IgM and IgE), ie, an antigen. ("Immunoreacts"), refers to a polypeptide containing an antigen binding site. An antibody may comprise at least one heavy (H) chain and at least one light (L) chain interconnected by at least one disulfide bond. The term “V _H ” refers to the heavy chain variable region of an antibody. The term “V _L ” refers to the light chain variable region of an antibody. In exemplary embodiments, the term “antibody” specifically covers monoclonal and polyclonal antibodies. “Polyclonal antibody” refers to an antibody obtained from the serum of an animal immunized with one or more antigens. “Monoclonal antibody” refers to an antibody produced by a single clone of hybridoma cells. Techniques for producing monoclonal antibodies include, but are not limited to, hybridoma technology (see Kohler & Milstein (1975) Nature 256: 495-497); trioma technology; human B cell hybridoma technology (Kozbor, et al. ( 1983) Immunol. Today 4:72), EBV hybridoma technology (Cole, et al., 1985 In: Monoclonal
Antibodies and Cancer Therapy, see Alan R. Liss, Inc., pp. 77-96) and phage display.

  Polyclonal or monoclonal antibodies can be further manipulated or modified to produce chimeric or humanized antibodies. “Chimeric antibodies” are encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin segments belonging to different species. For example, a substantial portion of a variable (V) segment of a mouse monoclonal antibody-derived gene, such as obtained as described herein, could be joined to a substantial portion of a human constant (C) segment. Such chimeric antibodies are more likely to be less antigenic to humans than mouse monoclonal antibodies.

The term “humanized antibody” (HuAb) as used herein has a framework region that is substantially identical to a human framework (ie, at least 85%) while having a CDR from a non-human antibody. A chimeric antibody wherein any constant region has a polypeptide sequence identity of at least about 85-90% and preferably about 95% to a human immunoglobulin constant region. To tell. See, for example, PCT publication WO 90/07861 and European Patent No. 0451216. All of these HuAbs, except perhaps those that are CDRs, are substantially identical to corresponding portions of one or more natural human immunoglobulin sequences. The term “framework region” as used herein refers to the immunoglobulin light and heavy chain variable regions, Kabat, et al. (1987) Sequences of Proteins of Immunologic.
Interest, 4 ^th Ed.,
US Dept. Health
and as defined in Human Services, refers to a portion that is relatively conserved (ie, other than CDR) between different immunoglobulins within a single species. The human constant region DNA sequence can be isolated from a variety of human cells according to known techniques, but is preferably isolated from immortalized B cells.
Variable regions or CDRs for making humanized antibodies may be derived from monoclonal antibodies capable of binding antigen, but any convenient mammal, including mice, rats, rabbits, or other vertebrates. Produced from animal sources.

The term “antibody” further encompasses antibody fragments. Examples of antibody fragments include Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , and Fv fragments; including but not limited to single chain Fv (scFv) molecules, without the associated heavy chain portion, A single-chain polypeptide containing only one light chain variable domain, or a fragment thereof containing three CDRs of the light chain variable domain, and (3) only one heavy chain variable region without an attached light chain portion A diabody having a primary structure of unbroken, contiguous contiguous amino acid residues, including a single-chain polypeptide containing or a fragment thereof containing three CDRs of the heavy chain variable region, or any of There are antibody fragments; as well as multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain (s) can include any constant domain sequence found in the non-Fc region of an intact antibody (eg, CH1 in an IgG isotype). And / or any hinge region sequence found in intact antibodies, and / or a leucine zipper sequence fused or placed into the heavy chain hinge region sequence or constant domain sequence. You can also. Suitable leucine zipper sequences include Kostelney et al., (1992)
Jun and fos leucine zippers taught by J. Immunol., 148: 1547-1553, and GCN4 leucine zippers described in US Pat. No. 6,468,532. Fab and F (ab ') ₂
Fragments lack Fc fragments of intact antibodies, typically by proteolysis with enzymes such as papain (for the production of Fab fragments) or pepsin (for the production of F (ab) ₂ fragments), Arise.

  If an antibody binds preferentially to an antigen relative to another antigen, the antibody will “specifically bind” to that antigen or to an epitope of that antigen. For example, an antibody may have only about 50%, 20%, 10%, 5%, 1% or 0.1% cross-reactivity for one or more other epitopes.

  The term “conservative substitution” refers to changes between amino acids with similar molecular properties in a broad sense. For example, exchanges among the aliphatic groups alanine, valine, leucine and isoleucine can be considered conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Other conservative exchanges include the aliphatic groups aspartic acid and glutamic acid; the amide group asparagine and glutamine; the hydroxyl group serine and threonine; the aromatic groups phenylalanine, tyrosine and tryptophan; the basic groups lysine, arginine and Between histidine; and between sulfur-containing groups methionine and cysteine. Sometimes substitutions within the methionine and leucine groups can also be considered conservative. Preferred conservative substituents are aspartic acid-glutamic acid; asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine-tryptophan; lysine-arginine;

  An “effective amount” is an amount sufficient to produce a beneficial or desired clinical result when treated. An effective amount can be administered to a patient in one or more doses. From a therapeutic point of view, an effective amount is an amount sufficient to reduce a patient's infection. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include the age, sex and weight of the patient, the condition being treated, the severity of the condition, the shape of the drug being administered and the effective concentration.

  The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody preferentially binds to a single specific epitope of a molecule that can be molecularly defined. An epitope of a protein will consist of a limited number of amino acid residues on the protein, such as 5-15 residues, in a linear or non-linear configuration.

  "Equivalent" when used in describing nucleic acid or nucleotide sequences refers to nucleotide sequences that encode functionally equivalent polypeptides. Equivalent nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and thus differ in sequence due to the degeneracy of the genetic code. Like. For example, nucleic acid variants may include those resulting from nucleotide substitutions, deletions, or additions. Such substitutions, deletions or additions may involve one or more nucleotides. Variants may have changes in the coding region, non-coding region, or both. Changes in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions.

“Homology” or alternatively “identity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology may be determined by comparing one position in each sequence that could be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matched or homologous positions common to these sequences. The term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity may be determined by comparing a single position in each sequence that could be aligned for comparison purposes. If an equivalent position in a compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position; the equivalent site is the same or similar amino acid residue (eg, steric and / or electronic) If the property is occupied by (such as similarity), the molecule is said to be homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at common positions in the compared sequences. A variety of alignment algorithms and / or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Wisconsin) and may be used with default settings. ENTREZ is available through Bethesda, Maryland, the National Institutes of Health, National Library of Medicine, and the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences is determined by the gap weight, such as weighting each amino acid gap as if it were a single amino acid or nucleotide mismatch between the two sequences. The determination may be made with the GCG program set to 1. Other techniques for alignment are Methods in Enzymology, vol. 266: Computer Methods for Macromolecular
Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of
Explained in Harcourt Brace & Co., San Diego, California, USA. Preferably, the columns are aligned using an alignment program that allows gaps in the sequence. Smith-Waterman is a type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Furthermore, the sequences may be aligned using a GAP program using the Needleman and Wansch alignment method. An alternative search strategy is to use MPSRCH software running on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach excels in the ability to pick out distant pairs and is particularly tolerant of small gaps and nucleotide sequence errors. The amino acid sequence encoded by the nucleic acid could be used to search both protein and DNA databases. Databases with individual sequences are described in Methods in Enzymology, ed. Doolittle above. Databases include Genbank, EMBL, and Japanese DNA database (DDBJ).

  The term “infection” as used herein is not clinically significant or may result in local cell damage due to competitive metabolism, toxins, intracellular replication or antigen-antibody responses. S. It refers to the invasion and proliferation of microorganisms such as Aureus into body tissues. If the body's defense mechanisms are effective, infection can be local, subclinical and transient. Local infections can persist or spread to become acute, subacute or chronic clinical infections or disease states. In addition, local infection may become systemic if microorganisms can reach the lymphatic or vascular system.

As used herein, the term “iron-regulated surface determinant system” or “Isd system” refers to a number of genes encoded by five different transcription units that collectively encode a putative heme uptake system. Including S. Says the Aureus Isd locus. The five transcription units are isdA, isdB, isdCDEFsrtBisdG, isdH, and isdI. Transcription of the isd gene is regulated by iron in the environment under the control of the Fur promoter. Four of the proteins encoded by the Isd locus, IsdA, IsdB, IsdC, and IsdH are covalently linked to the cell wall by an amide bond between the C-terminus of the polypeptide chain and the peptidoglycan. IsdA, IsdB, and IsdH are characterized as having a C-terminal sorting signal called the LPXTG motif (ie, the motif recognized by sortase A). IsdC has been characterized as having a C-terminal sorting signal called the NPQTN motif (ie, the motif recognized by sortase B in S. aureus). IsdA, IsdB, and IsdH are linked to the cell wall by a membrane-bound transpeptidase called sortase A (srtA) that cleaves a cell surface protein with the LPXTG motif and catalyzes the formation of an amide bond between the polypeptide and peptidoglycan. Yes. IsdC Sortase B on the cell wall
Other proteins encoded by the Isd locus linked by a transpeptidase similar to sortase A (srtB) include putative membrane translocation factors IsdD, IsdE, and IsdF, and iron from heme. There are IsdG and IsdI, cytoplasmic heme-iron binding proteins that may be involved in the extraction.

  As used herein, “IsdA polypeptide” refers to iron-regulated surface determinant A. The sequence of the IsdA polypeptide is as set forth in SEQ ID NO: 3 and is encoded by SEQ ID NO: 1. The term further encompasses any fragment, variant, analog, agonist, chemical derivative, functional derivative or functional fragment of an IsdA polypeptide. An “IsdA immunogen” is an IsdA polypeptide capable of eliciting an immune response in a subject.

  As used herein, “IsdB polypeptide” refers to iron-regulated surface determinant B. The sequence of the IsdB polypeptide is as set forth in SEQ ID NO: 6 and is encoded by SEQ ID NO: 4. The term further encompasses any fragment, variant, analog, agonist, chemical derivative, functional derivative or functional fragment of an IsdB polypeptide. An “IsdB immunogen” is an IsdB polypeptide that can elicit an immune response in a subject.

  As used herein, “IsdC polypeptide” refers to iron-regulated surface determinant C. The sequence of the IsdC polypeptide is as set forth in SEQ ID NO: 9, and is encoded by SEQ ID NO: 7. The term further encompasses any fragment, variant, analog, agonist, chemical derivative, functional derivative or functional fragment of an IsdC polypeptide. An “IsdC immunogen” is an IsdC polypeptide capable of eliciting an immune response in a subject.

  “Label” or “detectable label” includes, but is not limited to, radioisotopes, fluorophores, chemiluminescent components, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (eg A detectable molecule including biotin or hapten). “Fluorescent substance” refers to a substance or a part thereof capable of exhibiting a detectable range of fluorescence. Specific examples of suitable labels include fluorescein, rhodamine, dansyl, umbelliferone, Texas Red, luminol, NADPH, alpha- or beta-galactosidase and horseradish peroxidase.

  The term “mutant” as used herein with respect to a gene refers to a gene that encodes a mutant protein. The term “mutant” as used herein for a protein means a protein that does not play its normal or normal physiological role. S. Aureus polypeptide variants may result from amino acid substitutions, deletions or additions. Such substitutions, deletions or additions may involve one or more residues. Among these, S. Substitutions, additions and deletions that alter the properties or activity of the Aureus protein.

  The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to polymeric length nucleotides of any length, regardless of deoxyribonucleotides or ribonucleotides or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, one (or more) loci defined from linkage analysis, exons, introns, messenger RNA (mRNA) Transfer RNA, ribosomal RNA, ribozyme, cDNA, antisense nucleic acid, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, Nucleic acid probes and primers. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may have been added before assembly of the polymer or added after. There may be a non-nucleotide component in the middle of the nucleotide sequence. Furthermore, the polynucleotide may be modified after the formation of the polymer, such as by binding with a labeling component. The term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that does not occur in nature or that is linked to another polynucleotide in a non-natural organization. “Oligonucleotide” refers to a single-stranded polynucleotide having less than about 100 nucleotides, eg, less than about 75, 50, 25, or 10 nucleotides.

  The terms “polypeptide”, “peptide” and “protein” (if single chain) are used interchangeably herein to refer to a polymer of amino acids. The polymer may be linear or branched, may contain modified amino acids, and may have non-amino acids along the way. This term further encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. is there. The term “amino acid” as used herein refers to both natural and / or non-natural or synthetic amino acids, including both glycine and D- or L-form optical isomers, as well as amino acid analogs and peptidomimetics. Also say.

  The term “small molecule” refers to a compound having a molecular weight of less than about 5 kD, less than about 2.5 kD, less than about 1.5 kD, or less than about 0.9 kD. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, sugars, lipids or other organic (carbon containing) or inorganic molecules. Numerous pharmaceutical companies often have extensive libraries of chemical and / or biological mixtures of fungal, bacterial, or algae extracts that can be screened with any of the assays of the present invention. Can do. The term “small organic molecule” refers to small molecules that are often identified as organic or medical compounds and do not include molecules that are only nucleic acids, peptides or polypeptides.

  The term “specifically hybridizes” refers to detectable and specific nucleic acid binding. The polynucleotides, oligonucleotides and nucleic acids of the present invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that suppress appreciable amounts of detectable binding to non-specific nucleic acids. Stringent conditions as known in the art and discussed herein may be used to achieve selective hybridization conditions. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides and nucleic acids of the invention and the nucleic acid sequence of interest is at least 30%, 40%, 50%, 60%, 70%, 80% 85%, 90%, 95%, 98%, 99%, or more. In some cases, hybridization and washing conditions are performed according to conventional hybridization techniques and under stringent conditions as further described herein.

  The term “stringent conditions” or “stringent hybridization conditions” refers to conditions that promote specific hybridization between two complementary polynucleotide strands to form a duplex. Stringent conditions will be selected to be about 5 ° C. lower than the thermal melting point (Tm) of the polynucleotide duplex at a defined ionic strength and pH. The length of the complementary polynucleotide strands and their GC content will determine the Tm of the duplex and thus the hybridization conditions necessary to obtain the desired hybridization specificity. Tm is the temperature (under a defined ionic strength and pH) at which 50% of a polynucleotide sequence hybridizes to a perfectly complementary strand. In some cases, it may be preferable to increase the stringency of the hybridization conditions to be approximately equal to the Tm of a particular duplex.

Various techniques for estimating Tm are available. Typically, a GC base pair in a duplex is estimated to contribute to about 3 ° C. of its Tm, while an AT base pair is estimated to contribute to about 2 ° C., at which time the theoretical maximum The upper limit is about 80-100 ° C. However, more sophisticated forms of Tm are available that take into account GC stacking interactions, solvent effects, desired assay temperatures, and the like. For example, a probe having a dissociation temperature (Td) of approximately 60 ° C. is represented by the formula: Td
= ((((((3 x #GC) + (2 x #AT)) x 37)-562) / # bp) -5; (where #GC, #AT, and #bp are The number of guanine-cytosine base pairs, the number of adenine-thymine base pairs, and the number of total base pairs involved in chain formation).

  Hybridization may be performed in 5xSSC, 4xSSC, 3xSSC, 2xSSC, 1xSSC or 0.2xSSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24 hours. The temperature of hybridization may be increased to adjust the stringency of the reaction, for example from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C., or 65 ° C. Furthermore, the hybridization reaction may include other substances that affect the stringency. For example, when hybridization is performed in the presence of 50% formamide, the stringency of hybridization at a specified temperature increases. .

  The hybridization reaction may be followed by a single washing step, or two or more washing steps may be continued at the same or different salinity and temperature. For example, the stringency may be increased by increasing the washing temperature from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C. or higher. The washing step may be performed in the presence of a surfactant, such as 0.1 or 0.2% SDS. For example, two washing steps each at 65 ° C. after hybridization may be performed in 2 × SSC, 0.1% SDS for about 20 minutes, and two more washes at 65 ° C. each depending on the choice. The step may be performed in 0.2 × SSC, 0.1% SDS for about 20 minutes.

  Examples of stringent hybridization conditions include 50% formamide, 10 × denharts (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin) and 200 μg / ml denatured carrier DNA, such as sheared salmon sperm DNA. Overnight hybridization at 65 ° C. in a solution containing or consisting of these, followed by 2 washing steps of 2 × SSC, 0.1% SDS for about 20 minutes each at 65 ° C., and 0.2 × SSC, 0.1% There are two washing steps in SDS, each at 65 ° C. for about 20 minutes.

  Hybridization will include hybridizing two nucleic acids in solution or hybridizing one nucleic acid in solution to a nucleic acid attached to a solid support such as a filter. When one nucleic acid is on a solid support, a prehybridization step may be performed before the hybridization step. Prehybridization may be performed at the same solution and at the same temperature as the hybridization solution for at least about 1 hour, 3 hours, or 10 hours (without complementary polynucleotide strands).

Suitable stringency conditions are known to those skilled in the art or may be determined empirically by those skilled in the art. For example, Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1989), 6.3.1-12.3.6; Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; S.
Agrawal (ed.) Methods in Molecular Biology, volume 20; Tijssen (1993)
Laboratory Techniques in biochemistry and molecular biology-hybridization with
nucleic acid probes, eg, part I chapter 2 “Overview of principles of
hybridization and the strategy of nucleic acid probe assays ”, Elsevier, New York;
and Tibanyenda, N. et al., Eur. J.
Biochem. 139: 19 (1984) and Ebel, S. et al.,
See Biochem. 31: 12083 (1992).

  The term “substantially homologous” when used in reference to a nucleic acid or amino acid sequence is substantially identical or similar in sequence to each other, resulting in conformational homology and thus one or more organisms. A sequence that retains a useful degree of pharmacological (including immunological) activity. The term is not intended to mean the normal evolution of the sequence.

  “Subject” refers to a male or female mammal, including a human.

A “variant” of an Isd polypeptide refers to a molecule that is substantially similar to IsdA, IsdB, or IsdC. Variant peptides may be prepared covalently by direct chemical synthesis of variant peptides using methods known in the art. Furthermore, variants of Isd polypeptides may include, for example, residue deletions, insertions or substitutions within the amino acid sequence. In addition, using any combination of deletions, insertions, and substitutions will lead to a final construct if the final construct has the desired activity. These variants are encoded by performing site-directed mutagenesis of nucleotides in the DNA encoding the peptide molecule (as exemplified in Adelman et al., DNA 2: 183 (1983)). After the DNA is made, it can be prepared by expressing the DNA in a recombinant cell culture. The variant typically exhibits the same qualitative biological activity as the wild-type Isd polyepoxy degree. It is known in the art that all possible single amino acid substitutions of a given polypeptide could be synthesized (Geysen et al., Proc. Nat. Acad. Sci. (USA) 18 : 3998-4002 (1984)). The effect of different substitutions is not necessarily additive, but safely combines two preferred or neutral single substitutions at different residue positions in an Isd polypeptide without losing Isd protein activity It would be reasonable to predict that A method for preparing degenerate polypeptides is described in US Pat. No. 5,010,175 to Rutter; Haughter
et al., Proc.
Nat. Acad. Sci. (USA) 82: 5131-5135 (1985); Geysen et al., Proc. Nat. Acad. Sci. (USA) 18: 3998-4002 (1984); WO86 / 06487;
And as described in WO86 / 00991. In designing a substitution strategy, one skilled in the art will determine which residues to change and which amino acids or classes of amino acids are suitable substitutions. In addition, studies of sequence variations of family or naturally occurring homologous proteins may be considered. Some amino acid substitutions are often more tolerant than others, and these often correlate with similarities in size, charge, etc. between the original amino acid and the substitution. Amino acid insertions or deletions could also be made as described above. The substitution is preferably conservative. For example, both are incorporated here by reference in their entirety: Schulz et al., Principle of Protein Structure
(Springer-Verlag, New York (1978)); and Creighton, Proteins: Structure and
Molecular Properties (WH Freeman & Co.,
See San Francisco (1983)).

  A “chemical derivative” of an Isd polypeptide can include additional chemical moieties not normally part of the IsdA, IsdB, or IsdC amino acid sequence. Such chemical modifications are made to Isd polypeptides by reacting the targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with a selected side chain or terminal residue. Can be introduced. The amino terminal residue can be reacted with succinic acid or other carboxylic anhydrides. Other reagents suitable for derivatizing alpha-amino containing residues include amide-esters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzene sulfonic acid; O-methylisourea 2,4-pentanedione; and transminase-catalase that reacts with glyoxylate. Specific modification of tyrosyl residues themselves has been extensively studied, but of particular interest is the method of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Carboxyl side chains such as aspartyl or glutamyl include 1-cyclohexyl-3- [2-morpholinyl- (4-ethyl)] carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide It can be selectively modified by reaction with carbodiimide (R′N—C—N—R ′). Furthermore, aspartyl or glutamyl residues can be converted to asparaginyl and glutaminyl by reaction with ammonium ions.

  A “vector” is a self-replicating nucleic acid molecule that transports an inserted nucleic acid molecule into and / or between host cells. The term functions mainly for vectors that function for insertion of nucleic acid molecules into cells, for replication of vectors that primarily function for replication of nucleic acids, and for transcription and / or translation of DNA or RNA. An expression vector. Also included are vectors that provide more than one of the above functions. An “expression vector” as used herein is defined as a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. By “expression system” is meant a suitable host cell that usually consists of an expression vector that can function to yield a desired expression product.

3. Isd gene
The three genes, Isd locus, isdA, isdB, and isdC, are S. cerevisiae. It encodes a cell surface protein covalently linked to the Aureus cell wall. Figure 1-3 shows isdA (SEQ ID NO: 1), isdB (SEQ
ID NO: 4) and the nucleic acid sequence of isdC (SEQ ID NO: 7).

  Furthermore, the nucleic acids of the invention may comprise, consist of, or consist essentially of any of the isd nucleotide sequences described herein. Still other nucleic acids comprise, consist of, or consist of nucleotide sequences having at least about 70%, 80%, 90%, 95%, 98%, or 99% identity or homology to the isd gene It basically consists of an array. Substantially homologous sequences may be identified using stringent hybridization conditions.

  Isolated nucleic acids that differ from the nucleic acids of the invention due to the degeneracy of the genetic code are also within the scope of the invention. For example, many amino acids are designed by two or more triplets. Codons that specify the same amino acid, ie, synonyms (eg, CAU and CAC are synonymous for histidine) may result in “silent” mutations that do not affect the amino acid sequence of the protein. However, it is expected that there will be DNA sequence polymorphisms that do not lead to changes in the amino acid sequence of the polypeptides of the invention. One skilled in the art will recognize that one or more of the nucleic acids encoding a particular protein of the invention (from less than 1% of nucleotides up to about 3 at most) between certain species due to natural allelic differences. It will be understood that these differences may be in (or 5% or possibly more) nucleotides. Any such nucleotide differences and resulting amino acid polymorphisms are within the scope of the present invention.

  Nucleic acids encoding proteins having amino acid sequences that are evolutionarily related to the polypeptides disclosed herein are provided, where “evolutionally related” refers to (eg, allelic differences or differentials). It refers to a variant variant of the protein of the present invention obtained by a protein having a different amino acid sequence produced by natural splicing or by combinatorial mutagenesis.

  Also provided are fragments of the polynucleotides of the invention that encode biologically active portions of the polypeptides. As used herein, a fragment of a nucleic acid that encodes an active portion of a polypeptide disclosed herein is a nucleotide sequence that has fewer nucleotides than a nucleotide sequence that encodes the full-length amino acid sequence of a polypeptide of the invention. Retains at least a portion of the biological activity of a full-length Isd protein as defined herein, or, optionally, is functional as a modulator of the biological activity of the full-length protein. Say something that encodes a polypeptide. For example, such a fragment may mediate the interaction of the protein with another molecule (eg, polypeptide, DNA, RNA, etc.) among the original full-length protein from which the polypeptide was derived. Polypeptides containing the domain are included.

  The nucleic acids provided herein may further include linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant polypeptides.

  Nucleic acids encoding the Isd polypeptides provided herein may be obtained from mRNA or genomic DNA from any organism according to the protocols described herein or those known in the art. For example, a cDNA encoding the polypeptide of the present invention may be obtained by isolating total mRNA from organisms such as bacteria, viruses, mammals and the like. A double stranded cDNA can then be prepared from this total mRNA and then inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. The gene encoding the polypeptide of the present invention may be cloned using established polymerase chain reaction techniques according to the nucleotide sequence information provided by the present invention. In one aspect, a method of amplifying a nucleic acid of the invention or a fragment thereof provides (a) a pair of single stranded oligonucleotides each of which is complementary to the nucleic acid sequence of the invention and is at least 8 nucleotides in length. The partner sequence to which the oligonucleotide is complementary is at least 10 nucleotides away, and (b) the oligonucleotide comprises a nucleic acid comprising a nucleic acid of the invention Amplifying the nucleic acid by contacting the sample under conditions that allow amplification of the region located between the pair of oligonucleotides.

  Isd protein can be obtained from a recombinant vector, a host cell containing said recombinant vector, and the encoded S. coli protein. It may be expressed from a method of producing an Aureus polypeptide. Suitable vectors may be introduced into host cells using known techniques such as infection, transduction, transfection, transfection, electroporation and transformation. The vector may be, for example, a phagemid, a plasmid, a virus or a retrovirus vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementary host cells.

  The vector may contain a selection marker for growth in the host. In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it could be introduced into host cells after it has been packaged in vitro using a suitable packaging cell line.

  Suitable vectors are those that contain a cis-acting regulatory region for the polynucleotide of interest. A suitable trans-acting factor may be provided to the host, to a complementary vector, or to the vector itself upon introduction into the host cell.

  In some embodiments, the vector serves for specific expression that may be inducible and / or cell type specific. Particularly suitable among such vectors are those that can be induced by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

  Expression vectors useful in the present invention include chromosomal, episomal, and viral vectors, such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal factors, baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, fowlpox. Viruses, viruses such as pseudo-rabies virus and retroviruses, and vectors derived from combinations of these such as cosmids and phagemids.

  Such DNA inserts are operably linked to suitable promoters, such as the phage lambda PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and the retroviral LTR promoter, among others. It must be. Other suitable promoters will be known to those skilled in the art. The expression construct will further contain sites for transcription initiation and termination, and the transcription region will contain a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes a translation start site at the beginning of the polypeptide to be translated and a termination codon (UAA, UGA or UAG) appropriately positioned at the end. It would be nice to be.

  As indicated, the expression vector will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes in the case of eukaryotic cell lines and tetracycline, kanamycin or ampicillin resistance genes in the case of culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells: fungal cells such as yeast cells Insect cells such as Drosophila S2 and Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Suitable media and conditions for the above host cells are known in the art.

  Among the vectors suitable for use in bacteria are pQE70, pQE60 and pQE9, pQE10 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; There are vectors of the pET series available from Novagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

Known bacterial promoters suitable for use in the present invention include E. coli.
lacI and lacZ promoters, T3, T5 and T7 promoters, gpt promoter, lambda PR and PL promoters, trp promoter and xyI / tet
There is a chimeric promoter. Suitable eukaryotic promoters include CMV early promoters, HSV thymidine kinase promoters, early and late SV40 promoters, retroviral LTR promoters such as those of Rous sarcoma virus (RSV), and mouse metallothionein-I promoters. There is a metallothionein promoter.

Introduction of the construct into the host cell can be performed by calcium phosphate precipitation, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in numerous standard laboratory manuals (eg, Davis, et al., Basic Methods in Molecular Biology).
(1986)).

  Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotes will be enhanced by inserting an enhancer sequence into the vector. An enhancer is a cis-acting DNA factor, usually about 10 to 300 nucleotides, that acts to increase the transcriptional activity of the promoter in the host cell type. Examples of enhancers include the SV40 enhancer located from the nucleotide origin 100 to the 270 position behind the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the replication origin, and the adenovirus enhancer, There is.

  For secretion of the translated polypeptide into the endoplasmic reticulum lumen, into the periplasmic space, or into the extracellular environment, a suitable secretion signal may be incorporated into the expressed polypeptide, such as the amino acid sequence KDEL. Said signals may be endogenous to the polypeptide of interest or they may be heterologous signals.

The coding sequence of the polypeptide of interest may be incorporated as part of a fusion gene comprising a nucleotide sequence that encodes a different polypeptide. The invention includes a nucleic acid of the invention and at least one heterologous sequence linked in-frame to the nucleotide sequence of the nucleic acid of the invention so as to encode a fusion protein comprising the heterologous polypeptide. The isolated nucleic acid is considered. The heterologous polypeptide is fused to (a) the C-terminus of the polypeptide encoded by the nucleic acid of the present invention, (b) the N-terminus of the polypeptide, or (c) the C-terminus and N-terminus of the polypeptide. You may let them. In some cases, the heterologous sequence encodes a polypeptide that allows detection, isolation, solubilization and / or stabilization of the polypeptide to which it is fused. In yet another embodiment, the heterologous sequence is polyHis tag, myc, HA, GST, protein A, protein G, calmodulin binding peptide, thioredoxin, maltose binding protein, polyarginine, poly
It encodes a polypeptide selected from the group consisting of His-Asp, FLAG, a portion of an immunoglobulin protein, and a transcellular transport peptide.

If it is desired to produce immunogenic fragments of the polypeptides of the invention, a fusion expression system may be useful. For example, the rotavirus VP6 capsid protein may be used as an immunogenic carrier protein for a portion of a polypeptide, either in monomeric or viral particle form. A nucleic acid sequence corresponding to the polypeptide portion of the present invention for which an antibody to the antibody is to be raised is incorporated into a fusion gene construct comprising the coding sequence of the late vaccinia virus structural protein, and the fusion protein comprising the protein portion as part of the virion. A set of recombinant viruses expressing may be produced. In addition, hepatitis B surface antigen may be utilized for this role. Similarly, a chimeric construct encoding a fusion protein containing a portion of the polypeptide of the present invention and a poliovirus capsid protein may be produced to enhance immunogenicity (eg, EP Publication No: 0259149;
and Evans et al., (1989) Nature 339: 385; Huang et al., (1988) J. Virol. 62: 3855; and Schlienger et al., (1992) J. Virol.
66: 2).

The fusion protein will facilitate the expression and / or purification of the protein. For example, the polypeptide of the present invention may be prepared as a glutathione-S-transferase (GST) fusion protein. By using such a GST fusion protein, purification of the polypeptide of the present invention can be simplified by using, for example, a glutathione derivatized matrix (for example, Current Protocols in
Molecular Biology, eds.Ausubel et al., (NY: John Wiley & Sons,
(1991)). In another embodiment, if there is a fusion gene encoding a purified leader sequence, such as a poly- (His) / enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, Ni ²⁺ metal resin is used. It would be possible to purify the fusion protein after expression by affinity chromatography. This purified leader sequence can then be removed by treatment with enterokinase to provide a purified protein (eg, Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: See 8972).

Techniques for producing fusion genes are known. Basically, various DNA fragments encoding different polypeptides are joined using blunt ends or sticky ends for ligation, digested with restriction enzymes to suitable ends, filled with sticky ends as appropriate, and treated with alkaline phosphatase. Thus, it is performed according to a conventional method such as preventing unnecessary joining and performing ligation with an enzyme. In another embodiment, the fusion gene may be synthesized by conventional methods including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments using anchor primers can generate complementary overhangs between two consecutive gene fragments, which are then annealed to create a chimeric gene sequence. (For example,
Current Protocols in
See Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

  In other embodiments, the nucleic acids of the invention include plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubing, containers, capillaries, pads, slices, etc. It may be fixed on a solid surface. The nucleic acid of the present invention may be immobilized on a chip as part of an array. The array may comprise one or more polynucleotides of the invention as described herein. In certain embodiments, the chip includes one or more polynucleotides of the invention as part of an array of polynucleotide sequences.

  Another aspect relates to the use of the nucleic acids of the invention in “antisense therapy”. Antisense therapy as used herein refers to transcription and / or by specifically hybridizing or binding to intracellular mRNA and / or genomic DNA encoding one of the polypeptides of the invention under cellular conditions. Alternatively, it refers to administration or in situ production of oligonucleotide probes or their derivatives that inhibit the expression of the polypeptide, such as by inhibiting translation. The binding may be by conventional base pair complementarity, or by specific interaction with the major groove of the duplex, for example in the case of binding to a DNA duplex. In general, antisense therapy refers to a range of techniques commonly used in the art, including any therapy that relies on specific binding to an oligonucleotide sequence.

  The oligonucleotide may be bound to another molecule such as a peptide, a crosslinker transport agent induced by hybridization, a cleavage agent induced by hybridization, and the like. The antisense molecule may be a “peptide nucleic acid (PNA)”. PNA refers to an antisense molecule or anti-gene agent comprising an oligonucleotide comprising at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending with lysine. This terminal lysine provides solubility to the composition. PNA will bind preferentially to complementary single-stranded DNA or RNA to stop transcriptional elongation, and PEGylation will extend their lifetime in cells.

The antisense construct of the present invention may be delivered as an expression plasmid or the like that, when transcribed in a cell, yields RNA that is complementary to at least a unique portion of the mRNA encoding the polypeptide of the present invention. Alternatively, the antisense construct can be expressed ex vivo and hybridized to mRNA and / or genomic sequences encoding the polypeptide of the invention when introduced into a cell. It may be an oligonucleotide probe that causes inhibition. Such oligonucleotide probes may be modified oligonucleotides that are stable in vivo because they are resistant to endogenous nucleases such as exonucleases and / or endonucleases. Examples of nucleic acid molecules used as antisense oligonucleotides are the phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also US Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). In addition, general approaches to construct oligomers useful in antisense therapy are described, for example, by van der Krol et al.,
(1988) Biotechniques 6: 958-976; and
Stein et al., (1988) Cancer Res 48: 2659-2668.

In a further aspect, double-stranded small interfering RNA
(siRNA) and methods of administering the same RNA are provided. siRNA reduces or blocks gene expression. While not wishing to be bound by theory, it is widely believed that siRNAs inhibit gene expression by mediating sequence-specific mRNA degradation. RNA interference (RNAi) is a process of sequence-specific post-transcriptional gene silencing, particularly in animals and plants, initiated by double-stranded RNA (dsRNA) that is sequence homologous to the silenced gene
(Elbashir et al. Nature 2001;
411 (6836): 494-8). Therefore, it is considered that the expression of the nucleic acid of the present invention can be inhibited by using siRNA and long dsRNA having substantial sequence identity to all or part of the polynucleotide of the present invention.

  Alternatively, siRNAs that reduce or block the expression of the Isd polypeptides described herein may be determined by examining multiple siRNA constructs for the target gene. Such siRNAs for certain target genes may be chemically synthesized. The nucleotide sequence of an individual RNA strand is selected such that the strand has one complementary region for the target gene to be inhibited (ie, a complementary RNA strand is formed upon expression of the target gene). A complementary nucleotide sequence to a region of the mRNA transcript to be processed, or to a region of its processing product, or to a region of the (+) strand virus). The step of synthesizing the RNA strand may include solid phase synthesis, where individual nucleotides are joined end-to-end through the formation of 3'-5 'phosphodiester bonds between the nucleotides during successive synthesis cycles. Is done.

  Here, an siRNA comprising a nucleotide sequence consisting essentially of the sequence of an isd nucleic acid as described herein is provided. The siRNA molecule will comprise two strands, each strand comprising at least essentially complementary nucleotide sequences, one of which essentially corresponds to the sequence of the target gene. The sequence that essentially corresponds to the sequence of the target gene is referred to as the “sense target sequence” of the siRNA, and the sequence that is essentially complementary thereto is referred to as the “antisense target sequence”. The sense and antisense target sequences are about 15 to about 30 nucleotides in length; about 19 to about 25 nucleotides in length; about 19 to 23 nucleotides in length, or about 19, 20, 21, 22, or 23 nucleotides in length It may be. The length of the sense and antisense sequences is such that siRNAs with sense and antisense target sequences of that length can inhibit the expression of the target gene, preferably without significantly inducing the host interferon response. To be determined.

siRNA target sequence is web.mit.edu/mmcmanus/www/home1.2files/siRNAs
Predict using one of the algorithms provided by the mmcmanus World Wide Web with the extension.

  The sense target sequence may be fundamentally or substantially identical to the coding or non-coding portion of the target nucleic acid or a combination thereof. For example, the sense target sequence may be essentially complementary to the 5 'or 3' untranslated region, promoter, intron or exon of the target nucleic acid or its complementary sequence. It may also be essentially complementary to a region that includes the boundary between two such gene regions.

The nucleotide base composition of the sense target sequence may be about 50% adenine (As) and thymidine (Ts) and 50% cytidine (Cs) and guanosine (Gs). Alternatively, the base composition may be at least 50% Cs / Gs, such as about 60%, 70% or 80% Cs / Gs. Thus, the selection of the sense target sequence will be based on the nucleotide composition of the nucleotide. With regard to reachability of target nucleic acids by siRNA, such is the case with Lee et al. (2002) Nature
It can be determined as described in Biotech. 19: 500. This approach involves using an oligonucleotide complementary to the target nucleic acid as a probe to determine substrate accessibility, such as in cell extracts. After duplexing with this oligonucleotide probe, the substrate becomes sensitive to RNaseH. Therefore, the degree of RNaseH sensitivity to a probe, as judged by PCR, reflects the reachability of the selected site, and how good the corresponding siRNA inhibits transcription of this target gene. It will be a predictor of whether or not it will function. Algorithm specific primers may be used for polymerase chain reaction (PCR) assays or to identify antisense oligonucleotides for identifying first target sequences.

  The sense and antisense target sequences are preferably sufficiently complementary so that siRNA containing both sequences can inhibit the expression of the target gene, ie, can mediate RNA interference. For example, the sequences may be sufficiently complementary to allow hybridization under desired conditions, such as within a cell. Thus, the sense and antisense target sequences may be at least about 95%, 97%, 98%, 99%, or 100% identical, and at most 5, 4, 3, 2, 1, It may be different by one or zero nucleotides.

  It is also preferred that the sense and antisense target sequences are sequences that will not interact much with sequences other than the target nucleic acid or its complement. This can be confirmed, such as by comparing the selected sequence to other sequences in the genome of the target cell. The comparison of sequences can be performed according to methods known in the art, for example using the BLAST algorithm further described herein. Of course, small experiments can be performed to confirm that a particular first target sequence can specifically inhibit the expression of the target nucleic acid and that of other genes is essentially not. .

  Furthermore, siRNA may contain sequences in addition to sense and antisense sequences. For example, the siRNA may be an RNA duplex composed of two strands of RNA such that at least one strand has a 3 ′ overhang. The other strand may be blunt ended or have a protrusion. In embodiments where the RNA molecule is double stranded and both strands contain overhangs, the length of the overhangs may be the same for each strand or different. In certain specific embodiments, the siRNAs are each on a single RNA strand and consist of about 19 to 25 nucleotides in pairs, about 1 to about 3, especially about 2 nucleotides. Sense and antisense sequences, such that they have an overhang at the 3 ′ end of both RNAs. In order to further enhance the stability of the RNA of the present invention, the 3 ′ overhang can be stabilized against degradation. In certain embodiments, RNA is stabilized by the inclusion of purine-based nucleotides such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine-based nucleotides with modified analogs, for example, substitution of uridine 2 nucleotide 3 ′ overhangs with 2′-oxythymidine is allowed and does not affect RNAi efficiency. The lack of 2 'hydroxyl will also increase the nuclease resistance of the overhang, at least in tissue culture media. The RNA strand of siRNA may have a 5 'phosphate group and a 3' hydroxyl group.

  In one embodiment, the siRNA molecule comprises a double stranded RNA that forms a duplex. In another embodiment, the siRNA consists of a single RNA strand forming a hairpin loop to which the sense and antisense target sequences hybridize, and the sequence between the two target sequences is based on this hairpin structure loop. This is a spacer arrangement. The spacer sequence can be any combination of nucleotides and any length, provided that two complementary oligonucleotides linked by a spacer having this sequence can form a hairpin structure, At least a portion of the spacer must then form a loop at the closed end of the hairpin. For example, the spacer sequence can be about 3 to about 30 nucleotides, about 3 to about 20 nucleotides, about 5 to about 15 nucleotides, about 5 to about 10 nucleotides, or about 3 to about 9 nucleotides. The sequence may be any sequence that does not interfere with the formation of the hairpin structure. In particular, the spacer sequence is preferably not a sequence with significant homology to the first or second target sequence. Because it can interfere with the formation of the hairpin structure. It is also preferred that the spacer sequence is not similar to other sequences such as the genomic sequence of the cell into which the nucleic acid will be introduced. Because it may have undesirable effects in the cell.

  Those skilled in the art, when referring to nucleic acids such as RNA, that this RNA may consist of naturally occurring nucleotides or nucleotide derivatives that provide higher stability to the nucleic acids, etc. It will be understood. Any derivative is acceptable provided that the nucleic acid can function in the desired manner. For example, if an siRNA can inhibit the expression of a target gene, the siRNA may contain a nucleotide derivative.

  For example, siRNAs may contain one or more modified bases and / or modified backbones for stability or other reasons. For example, the phosphodiester bond of natural RNA may be modified to contain at least one of nitrogen or sulfur heteroatoms. Furthermore, for example, siRNA containing an unusual base such as inosine or a modified base such as a tritylated base can be used in the present invention. It will be appreciated that a variety of modifications can be made to the RNA that serve many useful purposes known to those of skill in the art. The term siRNA as used herein encompasses such chemically, enzymatically or metabolically modified forms of siRNA provided that it is derived from an endogenous template. .

There is no restriction | limiting in the aspect which synthesize | combines siRNA. Thus, using manual and / or automated methods,
Alternatively, it may be synthesized in vivo. In vitro synthesis can be performed chemically, or enzymatically using, for example, RNA polymerase (eg T3, T7, SP6) cloned for transcription of DNA (or cDNA) templates, or You may carry out by the mixing method of both. Furthermore, siRNA may be prepared by chemically synthesizing each of the two strands and hybridizing the two strands to form a duplex. In vivo, siRNA may be synthesized using recombinant techniques known in the art (eg, Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning,
Volumes I and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait
ed, 1984); Nucleic Acid Hybridisation (BD Hames & SJ Higgins eds.
1984); Transcription and Translation (BD Hames & SJ Higgins eds.
1984); Animal Cell Culture (RI Freshney ed. 1986); Immobilised Cells and Enzymes
(IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984);
Methods in Enzymology Series (Academic
Press, Inc.); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos eds. 1987,
Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (edited by Wu and Grossman, and Wu, respectively), Mayer and Walker, eds. (1987),
Immunochemical Methods in Cell and Molecular Biology (Academic Press, London),
Scopes, (1987), Protein Purification:
Principles and Practice, Second Edition (Springer-Verlag, NY), and Handbook of Experimental Immunology,
Volumes I-IV (see DM Weir and CC Blackwell eds 1986)). For example, bacterial cells can be transformed with an expression vector containing a DNA template from which siRNA is derived.

  The siRNA when synthesized outside the cell may be purified prior to introduction into the cell. Purification may be by extraction with a solvent (eg phenol / chloroform) or resin, precipitation (eg in ethanol), electrophoresis, chromatography, or a combination thereof. However, purification may result in loss of siRNA and therefore may be minimal or not performed at all. The siRNA may be dried for storage or may be dissolved in an aqueous solution that may contain buffering agents or salts to facilitate annealing and / or stabilization of the RNA strand.

  The double stranded structure may be formed on one self-complementary RNA strand or on two separate complementary RNA strands.

Since mammalian cells are known to be able to respond to extracellular siRNA, they are thought to have a dsRNA transport mechanism (Asher et al. (1969) Nature 223).
715-717). Accordingly, siRNA may be administered extracellularly in the mammalian body cavity, interstitial cavity, circulation, or introduced orally. Oral introduction methods include direct mixing of RNA with mammalian food, or manipulation of a species used as food to express the RNA, followed by feeding to the mammal to be introduced. There is a manipulated approach. For example, food bacteria such as Lactococcus lactis may be transformed to produce dsRNA (see WO93 / 17117, WO97 / 14806). Blood vessels or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are sites where RNA may be injected.

RNA may be introduced into cells intracellularly. In this respect, a physical method for introducing a nucleic acid may be used. siRNA, Zernicka-Goetz et al. (1997) Development
124, 1133-1137 and Wianny et al. (1998) Chromosoma 107, 430-439 may be used for administration.

  Other physical methods of introducing nucleic acids intracellularly include irradiation of particles coated with siRNA, such as gene gun technology where the siRNA is immobilized on a gold particle and shot directly into the wound site. Thus, the present invention proposes the use of siRNA for gene cancer in order to inhibit target gene expression. Furthermore, a composition suitable for gene cancer therapy comprising siRNA and gold particles is also provided. An alternative physical method is to electroporate cell membranes in the presence of siRNA. Large scale RNAi is possible with this method. Other methods known in the art for introducing nucleic acids into cells may be used, such as lipid mediated carrier transport, chemical mediated transport of calcium phosphate and the like. siRNA may be introduced together with a component that performs one or more of the following activities: enhances cellular RNA uptake, promotes duplex annealing, stabilizes the annealed strand, or Increase target gene inhibition.

  RNA can be administered using any known gene therapy method. A viral construct packaged in a viral particle will achieve both efficient introduction of the expression construct into the cell and transcription of the siRNA encoded by the expression construct. Thus, siRNA can also be generated in a cell. Vectors such as expression vectors containing nucleic acids encoding one or two strands of a given siRNA molecule may be used for this purpose. The nucleic acid will further include an antisense sequence that is essentially complementary to the sense target sequence. The nucleic acid may further include a spacer sequence between the sense and antisense target sequences. The nucleic acid will further include promoters that direct the expression of sense and antisense sequences in the cell, such as RNA polymerase II or III promoters and transcription termination signals. The array will be operably linked.

In certain embodiments, the nucleic acid comprises an RNA coding region (eg, a sense or antisense target sequence) operably linked to an RNA polymerase III promoter. Immediately following this RNA coding region is pol
There may be a pol III terminator sequence commanding the termination of RNA synthesis by III. A pol III terminator sequence generally has four or more consecutive thymidine ("T") residues. In a preferred embodiment, a collection of 5 consecutive T residues is used as a terminator, and this terminator stops the transcription of pol III at the second or third T of the DNA template. 3 uridines ("U")
Residues are only added to the 3 'end of the coding sequence. H1 of human or mouse origin, or from any other species
Various pol III promoters can be used in the present invention, including promoter fragments derived from RNA genes or U6 snRNA genes. In addition, the pol III promoter can be modified / engineered to incorporate other desired properties such as the ability to be induced by small chemical molecules in either a systemic or tissue specific manner. For example, in certain embodiments, the promoter may be activated by tetracycline. In another embodiment, the promoter may be activated by IPTG (lacI system).

  siRNA can be generated in a cell by transforming the cell with two nucleic acids such as a vector, in which each nucleic acid contains an expression cassette, each of which further comprises a promoter, an RNA coding sequence (one Is the sense target sequence and the other is the antisense target sequence) and a termination signal. Alternatively, a single nucleic acid may contain these two expression cassettes. In yet another embodiment, the nucleic acid encodes a single stranded RNA comprising a sense target sequence linked to a spacer linked to an antisense target sequence. The nucleic acid may be present in a vector, for example in an expression vector such as a eukaryotic expression vector that allows expression of sense and antisense target sequences in the introduced cells. Also good.

Vectors that generate siRNAs are, for example, Paul et al. (2002) Nature Biotechnology.
29: 505; Xia et al. (2002) Nature Biotechnology 20: 1006; Zeng et al. (2002) Mol. Cell 9: 1327; Thijn et
al. (2002) Science 296: 550; BMC
Biotechnol. 2002 Aug 28; 2 (1): 15; Lee et
al. (2002) Nature Biotechnology
19: 500; McManus et al. (2002) RNA 8: 842; Miyagishi et al. (2002) Nature Biotechnology 19: 497; Sui et al. (2002) PNAS
99: 5515; Yu et al. (2002) PNAS 99: 6047; Shi et al. (2003) Trends Genet.
19 (1): 9; Gaudilliere et al. (2002) J. Biol. Chem. 277 (48): 46442;
US2002 / 0182223; US 2003/0027783; WO 01/36646 and WO 03/006477. Further, commercially available vectors are also available. For example, pSilencer is available from Gene Therapy Systems and the pSUPER RNAi system is available from Oligoengine.

  Further provided herein are compositions comprising one or more siRNAs or one or more nucleic acids encoding an RNA coding region of a siRNA. The composition may be a pharmaceutical composition and may include a pharmaceutically acceptable carrier. Further, the composition may be provided in a device for administering the composition to cells or to a subject. For example, the composition may be in a syringe or on a stent. The composition may further comprise an agent that facilitates entry of the siRNA or nucleic acid into the cell.

In general, oligonucleotides are described by Caruthers et al., Methods in Enzymology (1992) 211: 3-19, which are incorporated herein by reference in their entirety.
Thompson et al., International PCT
Publication No. WO 99/54459; Wincott et
al., Nucl. Acids Res. (1995)
23: 2677-2684; Wincott et al., Methods
Mol. Bio., (1997) 74:59; Brennan et
al., Biotechnol. Bioeng. (1998)
61: 33-45; and Brennan, US Pat. No. 6,001,311 and the like, may be synthesized using protocols known in the art. In general, oligonucleotide synthesis uses conventional nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5 ′ end and phosphoramidite at the 3 ′ end. In one non-limiting example, small scale synthesis is performed on an Expedite 8909 RNA synthesizer sold by Applied Biosystems (Weiterstadt, Germany) on ChemGenes Corporation (01721 Massachusetts, USA, Homer Avenue, Ashland, USA). 200, ribonucleoside phosphoramidites sold by Ashland Technology Center). Alternatively, 96-well plate synthesizers, such as those manufactured by Protogene (Palo Alto, Calif., USA), or Usman et al., Each of which is incorporated herein by reference in its entirety. , J. Am. Chem. Soc. (1987) 109: 7845;
Scaringe et al., Nucl. Acids Res. (1990) 18: 5433; Wincott et al., Nucl. Acids Res.
(1990) 23: 2677-2684; and Wincott et al., Methods Mol. Bio. (1997) 74:59.

The nucleic acid molecules of the present invention may be synthesized separately and dsRNA
For example, ligation (Moore et al., Science (1992) 256: 9923; Draper et
al., International PCT Publication No. WO 93/23569; Shabarova et al., Nucl. Acids Res. (1991) 19: 4247; Bellon et al., Nucleosides & Nucleotides (1997)
16: 951; and Bellon et al., Bioconjugate Chem. (1997) 8: 204; or may be formed after synthesis, such as by hybridization after synthesis and / or deprotection. Nucleic acid molecules can be purified by conventional methods by gel electrophoresis, or alternatively by high performance liquid chromatography (HPLC; see Wincott et al., Supra), which is incorporated herein by reference in its entirety. Can be purified and resuspended in water.

In another embodiment, the level of a particular mRNA or polypeptide in the cell is reduced by introducing a ribozyme or a nucleic acid encoding such a ribozyme into the cell. Ribozyme molecules designed to cleave mRNA transcripts can be introduced into cells or expressed in cells to inhibit target gene expression (eg, Sarver et al., 1990). Science 247: 1222-1225 and US Pat. No. 5,093,246). One commonly used ribozyme motif is a hammerhead with low substrate sequence requirements. The design of the hammerhead ribozyme is disclosed in Usman et al., Current Opin. Struct. Biol. (1996) 6: 527-533. Usman also discusses the therapeutic use of ribozymes. Ribozymes are also described in Long et al., FASEB J. (1993) 7:25; Symons, Ann.
Rev. Biochem. (1992) 61: 641; Perrotta et al., Biochem. (1992)
31: 16-17; Ojwang et al., Proc. Natl. Acad. Sci. (USA) (1992)
89: 10802-10806; and US Pat. No. 5,254,678 can also be prepared and used. Cleavage of HIV-I RNA by ribozymes is described in US Pat. No. 5,144,019; methods of cleaving RNA using ribozymes are described in US Pat. No. 5,116,742; and methods for increasing ribozyme specificity are described in US Patent No. 5,225,337 and Koizumi et al., Nucleic Acid Res.
(1989) 17: 7059-7071. The preparation and use of ribozyme fragments in the hammerhead structure is also described in Koizumi et al., Nucleic Acids Res. (1989) 17: 7059-7071. Preparation and use of ribozyme fragments in hairpin structures
Burke, Nucleic Acids Res. (1992) 20: 2835. Ribozyme is also available from Daubendiek and Kool, Nat.
Biotechnol. (1997) 15 (3): 273-277 can also be prepared by rolling transcription.

Gene expression by targeting a deoxyribonucleotide sequence complementary to the regulatory region of the target gene (ie, gene promoter and / or enhancer) to form a triple helix structure that prevents transcription of the gene in the target cell in the body (Schematically Helene (1991) Anticancer Drug
Des., 6 (6): 569-84; Helene et al.
(1992) Ann. NY
Acad. Sci., 660: 27-36; and Maher (1992) Bioassays 14 (12): 807-15).

In further embodiments, RNA aptamers can be introduced into cells or expressed in cells. RNA aptamers are Tat and Rev RNA (Good et al.
(1997) Gene Therapy 4: 45-54) is a specific RNA ligand of such a protein that can specifically inhibit the translation of the protein.

4). Isd polypeptides S. Aureus polypeptides including IsdA (SEQ ID NO: 3), IsdB (SEQ ID NO: 6), and IsdC (SEQ ID NO: 9) (Figures 1-3) described herein include Naturally purified products, chemical synthesis products, and products produced by recombinant techniques from prokaryotic or eukaryotic host cells, including bacteria, yeast, higher plants, insects and mammalian cells, Is included. In some embodiments, the polypeptides disclosed herein are those that inhibit the function of an Isd polypeptide.

  A polypeptide may further comprise, consist of, or consist essentially of any of the amino acid sequences disclosed herein. Yet another polypeptide consists of an amino acid sequence comprising an amino acid sequence having at least about 70%, 80%, 90%, 95%, 98% or 99% identity or homology to the Isd polypeptide, Alternatively, it basically consists of an amino acid sequence. For example, a polypeptide that differs from the sequence of the native Isd protein by about 1, 2, 3, 4, 5, or more amino acids is also conceivable. This difference may be a substitution, deletion or addition, such as a conservative substitution. This difference is preferably in a region that is not largely conserved between different species. Such a region could be identified by aligning the amino acid sequences of Isd proteins from various species. These amino acids can be substituted, for example, with those found in other species. Other amino acids that may be substituted, inserted or deleted at these or other positions can be identified by combining mutagenesis studies with biological assays.

  Furthermore, the protein may contain one or more unnatural amino acids. For example, non-traditional amino acids or chemical amino acid analogs can be introduced as a substitution or addition to the protein. Non-traditional amino acids include, but are not limited to, D-form isomers of normal amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, Epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butyl There are designer amino acids such as alanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acid, beta-methyl amino acid, Calpha-methyl amino acid, nalpha-methyl amino acid, and general amino acid analogs. Furthermore, the amino acid may be D-type (dextrorotatory) or L-type (left-handed).

  Still other proteins encompassed herein are those containing modified amino acids. Examples of proteins may be modified by glycosylation, PEGylation, phosphorylation, or any similar process that leaves at least one biological function of the derived protein of the group. Derivative protein.

  The protein may be used as a substantially pure formulation, such as in this case, at least about 90% of the protein in the formulation is the desired protein. Compositions containing at least about 50%, 60%, 70%, or 80% of the desired protein may also be used. S. Aureus polypeptides can be obtained from recombinant cell cultures by ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography. And can be recovered and purified by known methods, including high performance liquid chromatography ("HPLC") used for lectin chromatography and purification. The protein may be used as a substantially pure formulation, such that at least about 90% of the protein in the formulation is the desired protein. Compositions containing at least about 50%, 60%, 70%, or 80% of the desired protein may also be used.

  The protein may be denatured or remain undenatured, and as a result may be aggregated or unaggregated. The protein can be denatured according to methods known in the art.

  In some embodiments, the Isd polypeptide described herein comprises a domain-containing fusion that increases its solubility and / or facilitates its purification, identification, detection, and / or structural characterization. It may be a protein. Examples of domains include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp, or FLAG fusion protein. And there are tags. Further examples of domains include domains that alter the in vivo localization of proteins, such as signal peptides, type III secretion system targeting peptides, transcellular transport domains, nuclear localization signals, and the like. In various embodiments, the polypeptides of the present invention may include one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain, or may contain fusions to two or more different domains. The fusion may be at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N and C-terminus of the polypeptide. In addition, a linker sequence may be included between the polypeptide of the present invention and the fusion domain to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. Are within the scope of the present invention. In another embodiment, the polypeptide may be constructed to include a protease cleavage site between the fusion polypeptide and the polypeptide of the invention for tag removal after or after protein expression. Examples of suitable endoproteases are, for example, factor Xa and TEV protease. The protein may be fused to the signal sequence. For example, when prepared by recombination, a nucleic acid encoding the peptide may be linked to a signal sequence at its 5 ′ end so that the protein is secreted from the cell.

In some embodiments, the polypeptides of the invention may be chemically synthesized, synthesized by ribosomes in a cell-free system, or synthesized by ribosomes in cells. Chemical synthesis of the polypeptides of the invention includes stepwise solid phase synthesis, semi-synthesis through conformation-assisted peptide fragment religation, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Can be performed using a variety of methods known in the art. Natural chemical ligation utilizes a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. This transient thioester-linked intermediate is then spontaneously rearranged to provide a full-length ligation product with a natural peptide bond at the ligation site. The full length ligation product is chemically identical to the protein produced by cell-free synthesis. The full length ligation product may be folded modified and / or oxidized if possible to form natural disulfide-containing protein molecules (eg, US Pat. Nos. 6,184,344 and 6,174,530; and Muir et al.
al., Curr. Opin. Biotech. (1993):
vol. 4, p 420; Miller et al., Science
(1989): vol. 246, p 1149; Wlodawer et al.,
Science (1989): vol. 245, p 616;
Huang et al., Biochemistry (1991): vol. 30, p 7402; Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; Rajarathnam et al. , Science (1994): vol. 264, p 90; RE Offord, “Chemical Approaches
to Protein Engineering ”, in Protein Design and the Development of New
therapeutics and Vaccines, JB Hook, G. Poste, Eds., (Plenum Press, New York,
1990) pp. 253-282; Wallace et al., J. Biol. Chem. (1992): vol. 267, p 3852;
Abrahmsen et al., Biochemistry
(1991): vol. 30, p 4151; Chang, et al.,
Proc. Natl. Acad. Sci. USA (1994) 91:
12544-12548; Schnlzer et al., Science (1992): vol., 3256, p 221; and Akaji et
al., Chem. Pharm. Bull. (Tokyo)
(1985) 33: 184).

    In some embodiments, it may also be advantageous to provide native or experimentally derived homologues of the polypeptides of the invention. Such homologues will function with limited ability as modulators that promote or inhibit some of the biological activity of the native polypeptide. Thus, certain biological effects can be reduced by treatment with homologs of limited function compared to treatment with agonists or antagonists that are directed to all of the biological activity of the polypeptides of the invention. Can be triggered by side effects. For example, it interferes with the ability of the wild-type polypeptide of the present invention to bind to a specific protein, but does not substantially interfere with complex formation between the natural polypeptide and other intracellular proteins. A new antagonistic homologue could be made.

  The polypeptide may be derived from the full-length polypeptide of the present invention. An isolated peptidyl portion of such a polypeptide will be obtained by screening a recombinantly produced polypeptide from the corresponding fragment of the nucleic acid encoding such a polypeptide. In addition, fragments may be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, the protein may be arbitrarily divided into fragments having a desired length without overlapping the fragments, or may be divided into overlapping fragments having a desired length. Fragments may be made (by recombinant or chemical synthesis) and examined to identify peptidyl fragments having the desired properties, such as the ability to function as a modulator of the polypeptides of the invention. In one exemplary embodiment, the peptidyl portion of the protein of the present invention can be tested for binding activity or inhibitory activity by expressing the peptidyl portion of the protein of the present invention as a thioredoxin fusion protein each containing a discrete portion of the protein of the present invention. (See, eg, US Pat. Nos. 5,270,181 and 5,292,646; and PCT Publication WO94 / 02502).

  In another embodiment, truncated polypeptides may be prepared. A truncated polypeptide is one having 1 to 20 or more amino acid residues taken from either or both of the N-terminus and C-terminus. Such truncated polypeptides may be easier to express, purify or characterize than full-length polypeptides. For example, truncated polypeptides are more likely to crystallize and produce higher quality diffractive crystals than full-length polypeptides, or HSQC spectra that have high luminance peaks and minimal overlapping peaks. it is conceivable that. In addition, the use of truncated polypeptides may identify stable and active domains that are easier to characterize among full-length polypeptides.

  Furthermore, the structure of the polypeptide of the present invention is directed to the purpose of improving therapeutic or prophylactic efficacy, or stability (eg, ex vivo shelf life, in vivo proteolytic resistance, etc.). It is also possible to modify. Such a modified polypeptide is considered a “functional equivalent” of a polypeptide as further detailed herein, if it is designed to retain at least one activity of the native protein. Such modified polypeptides can be made, for example, by amino acid substitutions, deletions, or additions, and substitutions in this case will also consist entirely or partially of conservative amino acid substitutions.

  For example, conservative amino acid substitutions alone, such as substituting leucine for isoleucine or valine, aspartic acid for glutamic acid, and threonine for serine, will not have a significant effect on the biological activity of the resulting molecule. It is reasonable to predict. Whether a functional homolog can be produced as a result of a change in the amino acid sequence of a polypeptide can be easily determined by evaluating the ability of the variant polypeptide to produce a response similar to that of the wild-type protein. . Polypeptides with two or more substitutions will be readily tested in the same manner.

  Methods are provided for generating a set of combinatorial and truncation variants of the polypeptides of the invention and are particularly useful for identifying potential variant sequences (eg, homologs). The purpose of screening such a combinatorial library is to create a homolog or the like that seems to regulate the activity of the polypeptide of the present invention, or, optionally, to create a homolog with completely novel activity. It is. Combinatorial-derived homologues having selective efficacy compared to natural proteins may be produced. Such homologues could be used in the development of therapeutics.

  Similarly, mutagenesis may result in homologs with intracellular half-lives that are dramatically different from the corresponding wild-type protein. For example, the altered protein could be more stable or unstable to other cellular processes that cause proteolysis or cause destruction or inactivation of the protein. Such homologues and the genes that encode them may be used to alter protein expression by regulating the half-life of the protein. As mentioned above, such proteins could be used in the development of therapeutics or therapies.

  In a similar manner, a protein homologue that acts as an antagonist may be generated by the combinatorial method described above, since it can interfere with the activity of the corresponding wild-type protein.

  In an exemplary embodiment of this method, the amino acid sequences of a population of protein homologues are preferably aligned for the highest homology possible. Such a population of variants may include, for example, homologs from one or more species, or homologs from the same species but different due to mutation. An amino acid at each position of the sequence after alignment is selected to produce a degenerate set of combinatorial sequences. In some embodiments, the combinatorial library is created utilizing a degenerate library of genes that encode a library of polypeptides each containing at least a portion of a potential protein sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence, and the degenerate set of potential polynucleotide sequences as individual polypeptides or, optionally, a set of larger (such as for phage display) It may be expressed as a fusion protein.

There are numerous ways in which a library of potential homologs could be generated from a degenerate oligonucleotide sequence. Chemical synthesis of degenerate gene sequences may be performed with an automated DNA synthesizer, and then the synthetic gene may be ligated into a vector suitable for expression. One purpose of the degenerate set of genes is to provide all of the sequences that encode the desired set of potential protein sequences in a single mixture. The synthesis of degenerate oligonucleotides is known in the art (eg Narang (1983) Tetrahedron
39: 3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos.
Macromolecules, ed. AG Walton, Amsterdam:
Elsevier pp. 273-289; Itakura et al.
(1984) Annu. Rev. Biochem. 53: 323;
Itakura et al. (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477). Such techniques have been used for directed development of other proteins (eg Scott et al. (1990) Science 249: 386-390; Roberts et al. (1992) PNAS USA 89: 2429-2433; Devlin et
al. (1990) Science 249: 404-406;
Cwirla et al. (1990) PNAS USA 87: 6378-6382; and US Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis may be used to create the combinatorial library. For example, alanine scanning mutagenesis etc. (Ruf et al. (1994) Biochemistry 33: 1565-1572; Wang et al. (1994) J. Biol. Chem. 269: 3095-3099; Balint et al. (1993 ) Gene
137: 109-118; Grodberg et al. (1993) Eur. J. Biochem. 218: 597-601; Nagashima et al. (1993) J. Biol. Chem. 268: 2888-2892; Lowman et al. (1991 ) Biochemistry
30: 10832-10838; and Cunningham et al., (1989) Science 244: 1081-1085), linker scanning mutagenesis (Gustin et al. (1993) Virology 193: 653-660; Brown et al. 1992) Mol. Cell Biol. 12: 2644-2652; McKnight et al. (1982) Science
232: 316); saturation mutagenesis (Meyers et al. (1986) Science 232: 613); PCR mutagenesis (Leung et al. (1989) Method Cell Mol Biol 1: 11-19); or random mutagenesis (Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press,
Cold Spring Harbor, NY; and Greener et
al. (1994) Strategies in Mol Biol
7: 32-34), protein homologs (both agonist and antagonist forms) could be made and isolated from the library. Linker scanning mutagenesis is an attractive method for identifying truncated forms of bioactive proteins, particularly in combinatorial environments.

  A wide variety of techniques are known in the art for screening gene products of combinatorial libraries created by point mutations and truncations, and for screening cDNA libraries by searching for gene products having specific properties. Such techniques will generally be adapted to suit rapid screening of protein homolog gene libraries generated by combinatorial mutagenesis. The most widely used techniques for screening large gene libraries typically involve cloning a gene library into a replicable expression vector and transforming the appropriate cells with the resulting vector library. Converting and expressing the combinatorial gene under conditions such that upon detection of the desired activity, it is relatively easy to isolate the vector encoding the gene from which the product was detected. .

In one exemplary screening assay embodiment, candidate combinatorial gene products are displayed on the cell surface, and the ability of specific cells or virus particles to bind to the combinatorial gene product is detected by a “panning assay”. For example, cloning a gene library into the surface membrane protein gene of a bacterial cell (Ladner et al., WO
88/06630; Fuchs et al., (1991) Bio / Technology 9: 1370-1371; and Goward et
al., (1992) TIBS 18: 136-140) and the resulting fusion protein is detected by panning, such as using a fluorescently labeled molecule that binds to this cell surface protein, such as FITC-substrate. May be scored for functionally homologues. Cells may be visually inspected and separated under a fluorescence microscope, or may be separated with a fluorescence-labeled cell sorter if possible from the cell morphology. Using this method, it will be possible to identify substrates or other polypeptides that can interact with the polypeptides of the invention.

In a similar manner, the gene library may be expressed on the surface of the viral particle as a fusion protein. For example, in filamentous phage systems, foreign peptide sequences may be expressed on the surface of infectious phage to provide two advantages. First, since these phage are believed to attach to the affinity matrix at a very high density, a large number of phage could be screened at once. Second, because each infectious phage presents a combinatorial gene product on its surface, once a particular phage is recovered from the affinity matrix in low yield, the phage may be further infected and amplified. . Almost identical E. coli filamentous phages M13, fd, and f1 groups are most frequently used in phage display libraries, but it is not possible to use the phage gIII or gVIII membrane proteins for the final virion. This is because a fusion protein can be produced without destroying proper packaging (Ladner et al., PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690;
Marks et al., (1992) J. Biol. Chem. 267: 16007-16010;
Griffiths et al., (1993) EMBO J. 12: 725-734; Clackson et al., (1991) Nature 352: 624-628; and Barbas et al., (1992) PNAS USA 89: 4457-4461). Other phage membrane proteins may be used as appropriate.

  The polypeptides disclosed herein may be reduced to create mimetics such as peptides or non-peptide substances that can mimic the binding of a reference protein to another intracellular partner. Mutational techniques such as those described above and the thioredoxin system are also particularly useful for mapping determinants that participate in protein-protein interactions with other proteins in one protein. For example, in a given protein, an important residue involved in molecular recognition of the substrate protein is determined, and this residue is used to create a peptide mimetic that will bind to this substrate protein. Also good. The peptide mimetic is then bound to the substrate, covering the key residues necessary for interaction with the wild-type protein, and used as an inhibitor of the wild-type protein, between the protein and the substrate. Will interfere with the interaction. For example, by mapping the amino acid residue involved in the binding of the substrate polypeptide in a certain protein, such as by using scanning mutagenesis, a peptide mimetic compound that mimics this residue when bound to the substrate is created. I can do it.

  For example, the Isd protein derivatives described herein may be chemically modified peptides and peptide mimetics. Peptide mimetics are compounds based on or derived from peptides and proteins. Peptide mimetics can be obtained by structural modification of known peptide sequences using unnatural amino acids, conformational constraints, isosteric substitution, and the like. Such peptide mimetics constitute a continuum of structural space between peptides and non-peptide synthetic structures. Thus, peptide mimetics may be useful in delineating pharmacophores and translating peptides into non-peptide compounds with parent peptide activity.

Furthermore, mimetope of the peptide can also be provided. Such peptide mimetics are non-hydrolyzable (eg, highly stable to proteases or other physiological conditions that degrade the corresponding peptide), have high specificity, and / or cell differentiation. It is possible to have attributes such as high potency for stimulating. For purposes of illustration, such non-hydrolyzable peptide analogs of such residues are benzodiazepines (eg, Freidinger et al., In peptides: Chemistry and Biology, GR
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)), azepines (eg Huffman et al., In Peptides:
Chemistry and Biology, GR Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), substituted gamma lactam rings (Garvey et al., In Peptides: Chemistry and Biology, GR
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptide (Ewenson et al., (1986) J. Med. Chem. 29: 295; and Ewenson et
al., in Peptides: Structure and
Function (Proceedings of the 9th American Peptide Symposium) Pierce
Chemical Co. Rockland, IL, 1985), β-turn dipeptide core (Nagai et al., (1985) Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 1231), and β-aminoalcohol (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et
al. (1986) Biochem Biophys Res Commun
134: 71).

  In addition to the wide variety of side chain substitutions that can be made to create peptide mimetics, the present specification specifically discusses the use of conformationally constrained mimics of peptide secondary structures. A number of surrogates have been developed in place of peptide amide bonds. Surrogates commonly used for amide bonds include the following groups: (i) trans-olefins, (ii) fluoroalkenes, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.

Surrogate example:

  In addition, peptide mimetics based on more substantial modifications of the peptide backbone can be used. Peptide mimetics that fall into this category include (i) retro-inverso analogs, and (ii) N-alkylglycine analogs (so-called peptoids).

Examples of analogs:

  In addition, combinatorial chemical methods are attracting attention in the development of new peptide mimetics. For example, the so-called “peptide morphing” strategy of one embodiment focuses on the random generation of a library of peptide analogs containing a wide range of peptide bond substitutions.

  In certain embodiments, peptide mimetics can be obtained as retro-inverso analogs of the peptide. Such retro-inverso analogs can be made according to methods known in the art, such as those described in US Pat. No. 4,522,752 to Sisto et al. Certain retro-inverso analogs can be made, for example, as described in WO 00/01720. It will be appreciated that mixed peptides may be made, eg, containing some normal peptide bonds. As a general guideline, it is common to change the site most prone to proteolysis, and an amide bond that is less prone to change is optimal for mimetic switching. The final product or its intermediate can be purified by HPLC.

  The peptide may comprise at least one amino acid or all amino acids that are D-type stereoisomers. Other peptides may include at least one inverted amino acid. The inverted amino acid may be a D-type isomer. All amino acids of a peptide may be inverted and / or all amino acids may be D stereoisomers.

  In another exemplary embodiment, the peptide mimetic can be obtained as a retro-enantio analog of a peptide. Such retro-enantio analogs are synthesized with commercially available D-amino acids (or analogs thereof) and standard solid phase or liquid phase peptide synthesis techniques as described, for example, in WO 00/01720. be able to. The final product will be purified by HPLC to give a pure retro-enantio analog.

In yet another exemplary embodiment, trans-olefin derivatives can be made in place of the peptides. The trans-olefin analog is YK Shue et al. (1987) Tetrahedron.
It can be synthesized as described in Letters 28: 3225 or WO 00/01720. Furthermore, pseudodipeptides synthesized by the above method can be linked to other pseudodipeptides to create peptide analogs with several olefin functional groups instead of amide functional groups.

Yet another class of peptide mimetic derivatives are phosphonic acid derivatives. The synthesis of such phosphonic acid derivatives can be adapted from known synthetic schemes. For example, Loots et al. In Peptides: Chemistry and Biology, (Escom Science
Publishers, Leiden, 1988, p. 118); Petrillo et
al. in Peptides: Structure and
Function (Proceedings of the 9th American Peptide Symposium, Pierce
See Chemical Co. Rockland, IL, 1985).

Numerous other peptide mimetic structures are known in the art and can be readily adapted for use in such peptide mimetic. For example, peptide mimetics include 1-azabicyclo [4.3.0] nonane surrogate (see Kim et al. (1997) J. Org. Chem. 62: 2847) or N-acyl piperazine acid. (Xi et al. (1998) J. Am. Chem.
Soc. 120: 80), or a 2-substituted piperazine moiety with a constrained amino acid analog (Williams et al. (1996) J. Am.
Med. Chem. 39: 1345-1348). In still other embodiments, some amino acid residues are replaced with, for example, a monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a diaromatic, aromatic-heteroaromatic, or dihetero. It can be substituted with an aryl or diaryl moiety such as an aromatic nucleus.

  Such peptide mimetics can be optimized, for example, by combinatorial synthesis techniques combined with high throughput screening.

  In addition, other examples of mimetope include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotype antibodies And / or catalytic antibodies, or fragments thereof. A mimetope can be obtained, for example, by screening a library of natural and synthetic compounds looking for compounds that can inhibit cell survival and / or tumor growth. Furthermore, mimetopes can be obtained, for example, from libraries of natural and synthetic compounds, specifically chemical or combinatorial libraries (ie, libraries of compounds that differ in sequence or size but have the same building blocks). Furthermore, mimetope can be obtained by rational drug design. In a rational drug design method, the three-dimensional structure of a compound of the present invention can be analyzed by nuclear magnetic resonance (NMR) or X-ray crystallography. Thus, if this three-dimensional structure is used, a potential mimetope structure can be predicted by computer modeling or the like. The predicted mimetope structure can then be generated, for example, by chemical synthesis, recombinant DNA techniques, or by isolating the mimetope from natural sources (eg, plants, animals, bacteria and fungi). it can.

  “Peptides, variants and derivatives thereof” or “peptides and analogs thereof” are included in “peptide therapeutics” and are intended to include any of the peptides or modified forms thereof, such as the peptide mimetics described herein, for example. Has been. Suitable peptide therapeutics are those that reduce cell viability or increase apoptosis. For example, by a factor of at least about 2, 5, 10, 30 or 100, as judged by the assays described herein, these will decrease cell viability or increase apoptosis. The activity of the Isd protein, fragment or variant thereof may be assayed using a suitable substrate or binding partner, or other reagent suitable for testing for the predicted activity as described below.

  In another embodiment, the activity of the polypeptide may be determined by assaying for the expression level of the RNA and / or protein molecule. Transcription levels can be determined by using, for example, Northern blots, hybridization to oligonucleotide arrays, or assaying the level of the resulting protein product. The level of translation can be determined, for example, by using Western blotting, or by identifying a detectable signal (eg, fluorescence, luminescence, enzyme activity, etc.) that produces the protein product. Depending on the particular situation, it may be preferable to detect the level of transcription and / or translation of a single gene or multiple genes.

  Alternatively, it may be preferable to measure the overall rate of DNA replication, transcription and / or translation within the cell. Generally this will be done by growing the cells in the presence of detectable metabolites that are incorporated into the resulting DNA, RNA, or protein product. For example, the rate of DNA synthesis can be determined by growing cells in the presence of BrdU that is incorporated into newly synthesized DNA. Thus, the amount of BrdU could be determined histochemically using anti-BrdU antibodies.

  In other embodiments, the polypeptides of the invention include plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubing, containers, capillaries, pads, slices, and the like. It may be fixed to a solid surface. The polypeptides of the present invention may be immobilized on a “chip” as part of an array. An array having a plurality of addresses may contain one or more polypeptides of the invention at one or more of these addresses. In one embodiment, the chip contains one or more polypeptides of the invention as part of an array.

5. Isd vaccine
IsdA, IsdB, and IsdC polypeptides can be obtained from S. cerevisiae. A cell surface protein expressed by Aureus and essential for complete bacterial power in vivo (shown using a mouse model of renal infection). Furthermore, IsdA is immunodominant so that anti-IsdA is detected in human serum where the antibody is covalescent. Thus, IsdA, IsdB, and / or IsdC polypeptides may be obtained from S. cerevisiae. It may be used as a vaccine therapy to treat Aureus infection.

  IsdA, IsdB, and / or IsdC polypeptides or polynucleotides may be formulated into a vaccine and to induce an immune response (eg, cellular or humoral) against IsdA, IsdB, and / or IsdC in a subject. May be administered to the subject.

An example of an IsdA protein for inclusion in a vaccine is a full-length IsdA polypeptide or an IsdA peptide. In some embodiments, recombinant IsdA protein will be used in the vaccine. In an alternative embodiment, the IsdB or IsdC protein used as a vaccine is a full length IsdB or IsdC, even a peptide fragment of IsdB or IsdC,
Alternatively, it may be a recombinant IsdB or IsdC protein.

Isd is antigenic and used as a vaccine
Peptides can be distinguished using a variety of methods. In one approach, peptides containing antigenic sequences may be selected based on widely accepted potential antigenicity and / or exposure criteria. Such criteria include affinity and hypothetical antigenic index as determined by protein surface exposure analysis. The determination of suitable criteria is known to those skilled in the art, for example Hopp et al., Proc Natl Acad Sci USA 1981; 78: 3824-8;
Kyte et al., J Mol Biol 1982; 157: 105-32; Emini, J Virol
1985; 55: 836-9; Jameson et al., CA BIOS 1988; 4: 181-6;
and Karplus et al., Naturwissenschaften 1985; 72: 212-3. Amino acid domains predicted to be exposed on the surface by these criteria will be selected preferentially over domains predicted to be more hydrophobic.

The IsdA, IsdB and / or IsdC portion determined to be antigenic could be chemically synthesized from individual amino acids by methods known in the art. A suitable method to synthesize protein fragments is Stuart and Young's “Solid Phase Peptide Synthesis,” Second Edition, Pierce.
Described in Chemical Company (1984).

  Alternatively, IsdA, IsdB or IsdC antigenic linear epitopes could be identified by mimetope analysis with the corresponding Isd antibody. Briefly, for mimetope analysis, the polypeptide will be divided into smaller fragments of overlapping fragments. For example, peptides with 15 overlapping amino acids are synthesized to cover the full length polypeptide. Each 15 amino acid peptide overlaps by 3 amino acids. Alternatively, peptide fragments of 15 amino acids may be designed in tandem order to cover the entire linear amino acid sequence. Each peptide may then be biotinylated to allow binding to streptavidin-coated wells in a 96 well plate. The reactivity of various antisera may be detected by enzyme-linked immunosorbent assay (ELISA). After blocking non-specific binding, anti-Isd antibody may be added to each well followed by peroxidase-conjugated secondary antibody and peroxidase substrate. The anti-Isd antibody may be an affinity-purified anti-full-length recombinant Isd or an affinity-purified anti-IsdA peptide. Alternatively, the anti-Isd antibody may be directed against IsdB or IsdC. The optical density of each well may be read at 450 nm and the double or triple wells may be averaged. The mean value obtained from a control ELISA (ie pre-immune serum) from a similar ELISA is subtracted from this test immunoglobulin value and the resulting value is tabulated to determine which linear epitope is recognized by the immunoglobulin. You may judge.

  In addition, antigenic fragments may be determined using competitive binding assays using synthetic peptides representing linear epitopes. In some embodiments, the antigenic fragment will inhibit the uptake of labeled iron.

  Further provided herein are DNA vaccines comprising nucleotide sequences encoding IsdA, IsdB, and / or IsdC peptides. Examples of DNA vaccines are those that encode two or more IsdA peptides. Alternative DNA vaccines would encode two or more IsdB or IsdC peptides, or any combination of two or more IsdA, IsdB, or IsdC peptides. The efficacy of a candidate vaccine (peptide or DNA) could be tested in a suitable animal model such as rat, mouse, guinea pig, monkey and baboon. The protective or positive effect of the vaccine should be reflected in the fertility decline of experimental animals.

Nucleic acids encoding IsdA, IsdB, or IsdC immunogens are obtained by amplification by polymerase chain reaction (PCR) of genomic DNA, cDNA, RNA (eg, by RT-PCR) or gene fragments derived from the cloned sequence. I will be. PCR primers are selected from the gene or cDNA based on known sequences that cause amplification of relatively non-repetitive fragments. Computer programs may be used to design primers with the required specificity and optimal amplification objectives. For example Oligo version 5.0
See (National Biosciences). Factors compatible with the design and selection of amplification primers can be found, for example, in Rylchik, W.
(1993) “Selection of Primers for Polymerase Chain Reaction.” In Methods in Molecular
Biology, vol. 15, White B. ed., Humana Press, Totowa, NJ. The sequence may be obtained from GenBank or other public sources. Alternatively, the nucleic acids of the present invention can be obtained by standard methods known in the art, such as an automated DNA synthesizer (such a synthesizer can be
It may be synthesized using a commercially available product such as Biosystems. A cloning vector suitable for an Isd polypeptide expressed in a host or cell could be constructed according to standard techniques described above.

  Alternatively, the Isd immunogen may be prepared from enzymatic cleavage of an intact Isd polypeptide. Examples of proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin. Intact polypeptides can be incubated with one or more proteinases, either simultaneously or sequentially. Alternatively, or in addition, intact Isd polypeptide can be treated with a disulfide reducing agent. The peptides may then be separated from each other by techniques known in the art including, but not limited to, gel filtration chromatography, gel electrophoresis, and reverse phase HPLC.

6). Isd antibody and use thereof
To generate antibodies against IsdA, IsdB, and / or IsdC, the host animal may be injected with a full-length Isd polypeptide or Isd peptide. The host may be injected with different length peptides containing the desired sequence. For example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 , 125, 130, 135, 140, 145 or 150 amino acids may be used. Alternatively, if a portion of a protein defines an epitope but is too short to be antigenic, it may be conjugated to a carrier molecule to generate an antibody. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequence, TrpE, and human or bovine serum albumin. Binding can be done by methods known in the art. One such method is to combine the cysteine residues of the fragment with the cysteine residues of the carrier molecule.

In addition, antibodies against three-dimensional epitopes, ie non-linear epitopes, may be prepared, for example, based on protein crystal data. The antibody obtained by the injection may be screened against a short antigen of IsdA, IsdB, or IsdC. Antibodies prepared against the Isd peptide will be tested for activity against the peptide and activity against the full-length Isd protein. The antibody has an affinity of at least about 10 ⁻⁶ M, 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M or 10 ⁻¹² M, Isd peptide and / or complete May have for long Isd protein.

Cells suitable for the DNA sequence of interest, and host cells for antibody expression and secretion, can be found in the American Type Culture Collection (“Catalogue of Cell Lines and Hybridomas”).
^{5 th edition (1985) Rockville,} Md., USA) , including, can be obtained from a number of sources.

Polyclonal and monoclonal antibodies may be made by methods known in the art. Monoclonal antibodies can be obtained from Kohler and Milstein, Nature
1975; 256: 495-7; and Campbell's “Monoclonal Antibody Technology, The Production and Characterization
of Rodent and Human Hybridomas ”in Burdon et
al., Eds. Laboratory Techniques in Biochemistry and Molecular Biology,
Volume 13, Elsevier Science Publishers, Amsterdam (1985) and immunological methods described by Huse et al, Science (1989) 246: 1275-81 May be produced by a hybridoma produced.

Antibody purification methods are known in the art. For example, Harlow and Lane (1988) Antibodies: A Laboratory Manual,
See Cold Spring Harbor Laboratory, NY. Purification methods include salting out precipitation (eg, with ammonium sulfate), ion exchange chromatography (eg, running on a cation or anion exchange column at neutral pH and eluting with a step gradient with increasing ionic strength), Gel filtration chromatography (including gel filtration HPLC) and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-antibodies. Antibodies can also be purified on affinity columns according to methods known in the art.

Other embodiments include functional equivalents of antibodies, including, for example, chimerized, humanized, and single chain antibodies and fragments thereof. PCT application WO 93/21319; European patent application No. 239,400; PCT application WO 89/09622; European patent application
388,745; and European Patent Application EP 332,424.

Functional equivalents include polypeptides having substantially the same amino acid sequence as the variable or hypervariable region of the antibodies of the invention. A "substantially the same" amino acid sequence, where Pearson and Lipman, (1988) Proc
Natl Acd Sci USA 85:
Defined as a sequence having at least 70%, preferably at least about 80%, and more preferably at least 90% homology to another amino acid sequence as determined by FASTA search according to 2444-8 deep.

  The chimerized antibody includes a constant region derived substantially or exclusively from a human antibody constant region, and a variable region derived substantially or exclusively from a variable region sequence derived from a mammal other than a human. Will. Humanized antibodies are derived substantially or exclusively from constant and variable regions other than complementarity determining regions (CDRs) derived from the corresponding human antibody region substantially or exclusively, and from non-human mammals. Would include the CDR.

  Suitable mammals other than humans will include any mammal that would be the source of monoclonal antibodies. Suitable examples of mammals other than humans may include, for example, rabbits, rats, mice, horses, goats, or primates.

  Antibodies against IsdA, IsdB or IsdC prepared as described above may also be used as anti-infective agents. In other embodiments, antibodies that recognize functional Isd fragments may be used in random peptide phage display technology (Eidne et al., Biol Reprod. 63 (5): 1396-402 (2000)). Briefly, a 15- or 12-mer random peptide phage display library can be used to determine peptides that are likely to interact with functional Isd peptides by competitive displacement of Fab fragments of Isd antibodies. . For this purpose, a fixed S.P. After aureus cells have been deposited in the wells of a multiwell plate, immunostaining with IsdA, IsdB or IsdC is evaluated in the absence and presence of native and random peptides expressed by the phage library. Also good. Once competitive peptides are identified by amino acid sequence analysis, larger amounts of peptides can be synthesized and used as alternative molecular antagonists for antibodies targeted at functional fragments. Another alternative is to screen small molecule libraries for the ability to competitively displace Fab fragments into functional IsdA, IsdB, or IsdC fragments. The molecular antagonists identified in this manner could be used to neutralize the effects of antibodies raised in an immune response against an Isd polypeptide or polynucleotide vaccine.

  In a further embodiment, an antibody against IsdA, IsdB, or IsdC (whole antibody or antibody fragment) is conjugated to a biocompatible substance such as a polyethylene glycol molecule (PEG) according to methods known to those skilled in the art to The half-life may be increased. See for example US Pat. No. 6,468,532. Functionalized PEG polymers are available, for example, from Nektar Therapeutics. Commercially available PEG derivatives include, but are not limited to, amino-PEG, PEG amino acid ester, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG amino acid, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl ester of amino acid PEG, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinyl sulfone, PEG-maleimide, PEG-orthopyridyl-disulfide, heterofunctional PEG, PEG vinyl derivatives, PEG silane, and PEG phosphoride, There is. The reaction conditions for conjugating these PEG derivatives will vary depending on the polypeptide, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the selection of PEG derivatives include the desired point of attachment (eg, lysine or cysteine R group), hydrolytic stability and reactivity of the derivative, binding stability, toxicity and antigenicity, for analysis There is aptitude etc.

6). Pharmaceutical Compositions Purified IsdA, IsdB, or IsdC polypeptides and nucleic acids can be formulated, orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intranasally, intravaginally, and disrupted (ie, branched). Scratch the top layer of the skin using a needle etc.)
Or it could be introduced as a vaccine through any other standard immunization route. Furthermore, the Isd polypeptide may be orally delivered as a vaccine through an enteric capsule that will be dissolved in the intestine and taken up by Peyer's patch antigen-presenting cells. Oral delivery of the Isd polypeptide may be supplemented with injection of the Isd polypeptide.

  Furthermore, as described herein, S.P. Aureus anti-Isd antibodies, isd antisense nucleic acids and siRNA may be administered by a variety of means, as is known in the art, depending on their intended use. For example, such S.I. When aureus antagonist compositions are administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Alternatively, the formulations of the invention may be administered parenterally as injections (intravenous, intramuscular or subcutaneous), infusion formulations or suppositories. For application by the ocular mucosal route, the compositions of the invention may be formulated as eye drops or ophthalmic ointments. These formulations may be prepared by conventional means, but if necessary, the composition may be prepared, for example, as a pharmaceutical additive, binder, disintegrant, lubricant, corrective, solubilizer, suspending agent. , Any conventional additives such as emulsifiers or coating agents.

  In the formulations of the present invention, wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants. May be present in the formulated drug.

  Such compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulation may be provided in unit dosage form as appropriate and may be prepared by any method known in the pharmaceutical industry. The amount of composition that may be combined with a carrier material to produce a single dose will vary depending upon the subject being treated, and the particular mode of administration.

  The methods for preparing these formulations include the step of bringing the composition of the present invention into association with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are obtained by uniformly and intimately associating the drug with a liquid carrier, or a finely divided solid carrier, or both, and then shaping the product as necessary. Prepared.

  Formulations suitable for oral administration include capsules, cachets, pills, tablets, lozenges (flavored bases, usually sucrose and acacia gum or tragacanth, each containing the prescribed amount of the composition as an active ingredient ), In the form of powder, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or incense It may be a tablet (using an inert base such as gelatin and glycerin, or sucrose and acacia gum). The composition of the present invention may be administered as a bolus, electuary or paste.

  For solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the composition may be one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, And / or the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or (3) Wetting agents such as glycerol; (4) Disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate; (5) Paraffin, etc. (6) Absorption accelerators such as quaternary ammonium compounds; (7) Acetyl alcohol and monostearic acid (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; 10) Mix with any of the colorants. In the case of capsules, tablets and pills, the composition may further comprise a buffer. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules using pharmaceutical additives such as lactose or lactose or high molecular weight polyethylene glycols.

  A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets use binders (eg gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg sodium starch glycolate or crosslinked sodium carboxymethylcellulose), surfactants or dispersants. Can be prepared. Molded tablets may be made by molding in a suitable machine a mixture of the composition moistened with an inert liquid diluent. Tablets and other solid dosage forms such as sugar-coated tablets, capsules, pills and granules can be incised or coated and shelled, such as enteric coatings and other known in the pharmaceutical industry, depending on the choice It may be prepared with a coating.

  Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the composition, the liquid dosage form comprises an inert diluent commonly used in the art such as water or other solvents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof Solubilizers and emulsifiers will be included.

  Suspensions, in addition to this composition, suspend eg ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. An agent may be included.

  Formulations for rectal or vaginal administration may be provided as suppositories, the suppository comprising, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate and the like, which is solid at room temperature. It may be prepared by mixing with one or more suitable non-irritating pharmaceutical additives or carriers that will melt in the body cavity and release the active substance to become liquid at body temperature. Formulations suitable for vaginal administration further include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing carriers known to be suitable in the art.

  Dosage forms for transdermal administration of the composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as may be required.

  Ointments, pastes, creams and gels include, in addition to the composition, animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, Or pharmaceutical additives such as mixtures thereof may be included.

  Powders and sprays may contain pharmaceutical additives such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances, in addition to the composition. The spray may additionally contain customary propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

  Depending on the choice, the compositions of the invention may be administered in an aerosol. This is accomplished by preparing an aqueous aerosol, liposomal formulation, or solid particles containing the compound. A non-aqueous (eg, fluorocarbon propellant) suspension could be used. A sonic nebulizer may be used. This is because they reduce exposure of the agent to shear forces that can cause degradation of the compound contained in the composition.

  Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the composition together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of the particular composition in question, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), serum There are innocuous proteins such as albumin, amino acids such as sorbitan esters, oleic acid, lecithin, glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

In addition, Isd-based vaccines may be administered parenterally as injection solutions (intravenous, intramuscular or subcutaneous). One or more adjuvants may optionally be included in the vaccine composition of the present invention. Any suitable adjuvant may be used, such as aluminum hydroxide, aluminum phosphate, vegetable and animal oils, but the amount of adjuvant will depend on the nature of the particular adjuvant used. Further, the anti-infectious vaccine composition includes at least saccharides such as sorbitol, mannitol, starch, sucrose, dextrin, glucose, proteins such as albumin or casein, and buffers such as alkali metal phosphates. A kind of stabilizer may be contained.
A suitable adjuvant is the SynerVax® adjuvant.

  A pharmaceutical composition of the present invention suitable for parenteral administration comprises the composition as one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or In combination with sterile powders that can be reconstituted just before use to form sterile injectable solutions or dispersions, including antioxidants, buffers, bacteriostats, and recipes intended for such formulations Solutes that are isotonic with the blood of the ent, or suspensions or thickeners may be included.

  Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, olive oil, and the like Vegetable oils, as well as injectable organic esters such as ethyl oleate. The proper fluidity may be maintained, for example, by using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

  Furthermore, the Isd immunogen or Isd antibody of the present invention may be encapsulated in liposomes and administered by injection. A commercially available liposome delivery system is obtained from Novavax, Rockville, Maryland, under the name Novasomes®. These liposomes are specifically formulated for immunogen or antibody delivery. In certain embodiments of the invention, Novasomes® containing Isd peptides or antibody molecules bound to the surface of these non-phospholipid positively charged liposomes may be used.

  Using the pharmaceutical compositions described herein, S. cerevisiae including but not limited to Frunkel, chronic Frunkel disease, impetigo, acute osteomyelitis, pneumonia, endocarditis, burn-like skin syndrome, toxic shock syndrome, and food poisoning. It would be possible to prevent or treat a condition or disease caused by an aureus infection.

8). Examples of screening assays for inhibitors of Isd In general, agents or compounds that can reduce pathogenic bacterial power by interfering with iron-regulated surface determinants (Isd) can be obtained using the disclosed assays. Screening large libraries, both natural products or synthetic (or semi-synthetic) extracts or chemical libraries, can be identified. One skilled in the art of drug discovery and development will appreciate that the precise source of an agent (eg, test extract or compound) is not critical to the screening methods of the present invention. Thus, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such agents, extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, synthetic compounds, and existing compounds. There is a modified type. To generate random or directed synthesis (eg, semi-synthetic or total synthesis) of any number of compounds, including but not limited to carbohydrate-, lipid-, peptide-, and nucleic acid-based compounds Numerous methods are available. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Biotics (Sussex, UK), Xenova (Slow, UK), Harbor
Commercial sources are available from a number of sources including the Branch Oceangraphics Institute (Fountain Peace, Florida) and PharmnaMar, USA (Cambridge, Mass.). In addition, natural and synthetically generated libraries are generated according to methods known in the art, for example, standard extraction and fractionation methods, as required. Furthermore, if necessary, any library compound is readily modified using standard chemical, physical, or biochemical methods.

  In addition, one of ordinary skill in the field of drug discovery and development can replicate or replicate sequences of substances whose anti-pathogenic activity is known (eg taxonomic de-replication, biological de-replication, and chemistry). It is easy to understand that a de-replication method or a deletion method) should be used if possible.

  Once the crude extract is found to have anti-pathogenic or antibacterial activity or binding activity, further fractionation of positive lead compounds is performed to isolate the chemical components responsible for the observed effects It is necessary. Thus, the purpose of the extraction, fractionation, and purification process is the careful characterization and identification of chemical entities in crude extracts with anti-pathogenic activity. Methods for fractionating and purifying such heterogeneous extracts are known in the art. If necessary, compounds that have been shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art.

  Potential inhibitors or antagonists of Isd-encoded polypeptides include organic molecules, peptides, peptide mimetics that bind to the nucleic acid sequences or polypeptides of the invention to inhibit or extinguish their activity. , Polypeptides, and antibodies. Potential antagonists further include small molecules that bind to the binding site of the polypeptide and occupy it, preventing binding to cell binding molecules and thus normal biological activity. Other potential antagonists include antisense molecules.

  In addition, S. cerevisiae identified by the screening assay described herein. Aureus anti-Isd antagonists may be administered by a variety of means depending on their intended use, as described above.

8.1 Interaction assays Assays using purified and recombinant IsdA, IsdB, and IsdC polypeptides to search for agents that bind to Isd gene products and disrupt protein-to-protein interactions. May be developed. Potential inhibitors or antagonists of IsdA, IsdB, or IsdC include small organic molecules, peptides, polypeptides, peptide mimetics that reduce or eliminate their activity by binding to either IsdA, IsdB, or IsdC, and There will be antibodies.

  In some embodiments, an agent that binds to an Isd polypeptide and inhibits iron uptake may be identified, wherein the method comprises (i) an action on the Isd polypeptide with a suitable interacting molecule. Contacting in the presence of a substance under conditions that allow interaction between the Isd polypeptide and the interacting molecule in the absence of the agent; (ii) interacting with the Isd polypeptide Determining the level of interaction between the active molecule and the level of interaction between the Isd polypeptide and the interactive molecule in the presence of the agent in the absence of the agent. What is different compared to is that the agent is an indicator that the interaction between the Isd polypeptide and the interacting molecule is inhibited.

  In another embodiment, agents that disrupt the interaction between the Isd polypeptide and the interacting molecule may be identified. In some binding assays, a reaction mixture can be made to include at least one biologically active portion of either IsdA, IsdB, or IsdC, the agent of interest, and a suitable interacting molecule. Good. Examples of interacting molecules would be hemoprotein, hemin, transferrin, fibrinogen or fibronectin. In certain exemplary embodiments, the agent of interest is an antibody against a particular Isd polypeptide. When an antibody binds to an Isd polypeptide, the binding function of the Isd polypeptide to heme or hemoprotein will be inhibited. Detection and quantification of an interaction between a particular Isd polypeptide and a suitable interacting molecule provides a means of determining the efficacy of an agent in inhibiting the interaction. The efficacy of the agent can be assessed by creating dose response curves taken from data obtained using various concentrations of the agent to be tested. In addition, a control assay can also be performed to provide a baseline for comparison. In this control assay, the interaction of a particular Isd polypeptide with a suitable interacting molecule may be quantified in the absence of the agent being tested.

  The interaction between a particular Isd polypeptide and a suitable interacting molecule could be detected by a variety of techniques. The modulation of complex formation can be quantified by immunoassay or by chromatographic detection using, for example, a detectably labeled protein such as a radioactive label, a fluorescent label, or an enzyme labeled polypeptide.

  Measurement of the interaction of a particular Isd protein with a suitable interacting molecule may be observed directly using surface plasmon resonance techniques with an optical biosensor device. This method is particularly useful for measuring the interaction of large polypeptides (greater than 5 kDa) and can be adapted for screening for inhibitors of protein-protein interactions.

Alternatively, immobilization of a specific Isd polypeptide or a suitable interacting molecule can be used to separate the complex from the uncomplexed form of one or both proteins, or to adapt the assay to automation. It would be preferable. Binding of a particular Isd protein to an interacting molecule, for example in the presence and absence of a candidate agent, can be performed in any container suitable for containing the reactants. Examples include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows the protein to bind to a matrix. For example, glutathione-S-transferase / IsdA (GST / IsdA) fusion protein is adsorbed to glutathione sepharose beads (St. Louis, Mo., Sigma) or glutathione derivatized microtiter plates and then, for example, ³⁵ S-labeled reciprocating plates. After blending with the active molecule and the agent to be tested, the mixture can be incubated under conditions that induce complex formation, such as physiological conditions of salt and pH, although more stringent conditions are preferred. Will. After incubation, wash the beads to remove unbound label, immobilize the matrix, determine the radioactive label directly (eg, place the beads in scintillant), or after dissociating the complex Judgment can be made during Qing. Alternatively, the complex can be dissociated from the matrix, separated by SDS-PAGE, and the level of interacting molecules found in the bead fraction can be quantified from the gel using standard electrophoresis techniques. it can.

  Other techniques for immobilizing proteins and other molecules on the matrix are available for use in this assay. For example, either a specific Isd protein or a suitable interacting molecule can be immobilized utilizing the binding of biotin and streptavidin. For example, biotinylated IsdA, IsdB, or IsdC is prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, Ill.) It can be fixed to wells of a 96-well plate (Pierce Chemical) coated with streptavidin. Alternatively, antibodies that are reactive with either IsdA, IsdB, or IsdC, but that do not interfere with the interaction with the interacting molecule of the polypeptide may be derivatized to the wells of the plate. IsdA, IsdB, or IsdC may be captured in this well by antibody binding. As noted above, the interactive molecule and test compound formulations may be incubated in a well presenting the polypeptide on the plate, and the amount of compound captured in the well determined by the presence of the agent being tested. It can be quantified in the absence or absence. Examples of methods for detecting such complexes include those described above for GST-immobilized complexes, as well as immunodetection methods for complexes using interacting molecules and reactive antibodies, or interactive There are enzyme binding assays that rely on the detection of enzyme activity associated with a molecule.

For example, the enzyme can be chemically bound or provided as a fusion protein with an interacting molecule. Illustratively, the interacting molecule is chemically cross-linked or gene fused to horseradish peroxidase and the amount of polypeptide captured in this complex is determined by the chromogenic substrate of the enzyme, e.g., 3,3. '-Diamino-benzazine tetrahydrochloride or 4-chloro-1-naphthol can be evaluated. Similarly, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation can be quantified using 1-chloro-2,4-dinitrobenzene for its GST activity. (Habig et al. (1974) J.
Biol. Chem. 249: 7130).

8.2 Expression Assays In further embodiments, iron uptake antagonists may affect the expression of isdA, isdB, and isdC nucleic acids or proteins. In this screening, S. After treating Aureus cells with the compound of interest,
The effect of this compound on isdA, isdB, and isdC nucleic acid or protein expression may be assayed.

  In some embodiments, an agent that inhibits the expression of an Isd polypeptide in Staphylococcus aureus may be identified, wherein the method comprises (i) wild-type in the presence or absence of the agent. Culturing a Staphylococcus aureus strain and (ii) comparing the expression of the Isd polypeptide, wherein the decrease in the expression of the Isd polypeptide in cells treated with the agent is greater The agent is an indicator of inhibiting the expression of the Isd polypeptide in Staphylococcus aureus.

  In an alternative embodiment, an agent that inhibits the expression of isd nucleic acid in Staphylococcus aureus may be identified, wherein the method comprises (i) wild-type in the presence or absence of said agent. Culturing a Staphylococcus aureus strain and (ii) comparing the expression of the isd nucleic acid, wherein the decrease in the expression of the isd nucleic acid in cells treated with the agent is greater than the agent Is an indicator of inhibiting the expression of isd nucleic acids in Staphylococcus aureus.

  For example, S. cerevisiae cultured in the presence or absence of the agent to be tested. From Aureus cells, total RNA was obtained using any suitable technique such as the one-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski et al. (1987) Anal. Biochem. 162: 156-159, It can be isolated. Next, reversal of isdA, isdB, or isdC expression in combination with Northern blot analysis, polymerase chain reaction (PCR), reverse transcription combined with polymerase chain reaction (RT-PCR), and ligase chain reaction. You may test by suitable methods, such as copying (RT-LCR).

Northern blot analysis can be performed as described in Harada et al. (1990) Cell 63: 303-312. Briefly, S. cerevisiae cultured in the presence of the agent to be examined. Prepare total RNA from Aureus cells. For Northern blotting, this RNA is denatured in a suitable buffer (eg glyoxal / dimethyl sulfoxide / sodium phosphate buffer), subjected to agarose gel electrophoresis and copied to a nitrocellulose filter. Once the RNA is bound to the filter by the UV linker, the filter is prehybridized in a solution containing formamide, SSC, Denharz solution, denatured salmon sperm, SDS, and sodium phosphate buffer. S. Aureus isdA, isdB, or isdC DNA sequences may be labeled by any suitable method (eg, ³² P-multiple addition DNA labeling system (Amersham)) and used as a probe. After hybridizing overnight, the filter is washed and exposed to X-ray film. In addition, controls can be performed to provide a baseline for comparison. In the control, S. Expression of isdA, isdB, or isdC in Aureus may be quantified in the absence of the agent being tested.

Alternatively, the level of mRNA encoding isdA, isdB, or isd polypeptide may be assayed, such as by using the RT-PCR method described in Makino et al. (1990) Technique 2: 295-301. Also good. Briefly, this method involves S. cerevisiae cultured in the presence of the agent to be tested. Adding total RNA isolated from Aureus cells to a reaction mixture containing RT primers and a suitable buffer. After incubation to anneal the primer, RT buffer, dNTP, DTT, RNase inhibitor and reverse transcriptase can be added to the mixture. Incubate to reverse-transcribe the RNA, and then PCR the RT product using a labeled primer. Alternatively, instead of labeling the primer, labeled dNTPs can be included in the PCR reaction mixture. PCR amplification can be performed with a DNA thermocycler according to conventional methods. After a suitable number of amplifications, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of a suitable band may be quantified using an image analyzer. RT and PCR reaction components and conditions, reagent and gel concentrations, and labeling methods are known in the art. Variations on the RT-PCR method will be apparent to those skilled in the art. Another PCR method capable of detecting the nucleic acid of the present invention is the PCR Primer: A Laboratory Manual (Dieffenbach et al. Eds., Cold
Spring harbor
Lab Press, 1995). In addition, controls can be provided to provide a baseline for comparison. In this control, S.P. Expression of isdA, isdB, or isdC polypeptide in Aureus may be quantified in the absence of the agent being tested.

  Alternatively, expression of isdA, isdB, and isdC polypeptides may be determined by S. cerevisiae. Aureus cells may be quantified using an antibody-based method such as an immunoassay after treatment with the agent to be examined. Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement, but not limited to Any suitable immunoassay can be used, including competitive and non-competitive assay systems using techniques such as body-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.

  For example, an isdA, isdB, or isdC polypeptide is a S. cerevisiae that has been treated with the agent being tested. It can be detected in a sample obtained from Aureus cells, for example, by a two-step sandwich assay. In the first step, a specific polypeptide is captured using a capture reagent (eg, either an IsdA, IsdB, or IsdC antibody). Depending on the selection, this capture reagent can be immobilized on a solid phase. In the second step, the captured marker is detected using a detection reagent labeled directly or indirectly. In one embodiment, the detection reagent is an antibody. S. cerevisiae treated with the agent to be examined. The amount of IsdA, IsdB, or IsdC polypeptide present in Aureus cells is determined using untreated S. cerevisiae. It can be calculated with reference to the amount present in the Aureus cells.

Suitable enzyme labels include, for example, those of the oxidase group that catalyze the production of hydrogen peroxide by reacting with a substrate. Glucose oxidase is particularly suitable because it has good stability and its substrate (glucose) can be easily used. The activity of a certain oxidase label could be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme labeled antibody / substrate reaction. Other suitable labels except enzyme, iodine ⁽¹²⁵ I, ¹²¹ I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), there is a radioactive isotope such as tritium ⁽³ H).

  Examples of suitable fluorescent labels include fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, o-phthaldehyde labels, and fluorescamine labels.

  Examples of suitable enzyme labels include maleate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase , Beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels.

Examples The invention generally described above will be more readily understood with reference to the following examples. However, the following examples are included solely for the purpose of depicting specific aspects and embodiments of the invention and are not intended to limit the invention in any manner.

Example 1: Expression of IsdA, IsdB and IsdC proteins
IsdA, IsdB, and IsdC proteins or fragments thereof are expressed under iron restriction (S. aureus-Fe) as shown in FIG. The SDS-PAGE gel shown in FIG. 4 shows IsdA, IsdB, and IsdC proteins as S.P. It shows that it is three of the most important iron-regulated proteins expressed by Aureus. These proteins are S. cerevisiae. Since it is expressed when Aureus cells are cultured in an iron-rich medium (S. aureus-Fe), it is inferred that they are probably highly expressed in vivo.

  When IsdE is overexpressed, such as IsdA, IsdB, IsdC, and fusions in E. coli, the lysate is colored deeply. Using absorbance and magnetic circular dichroism spectroscopy, confirm that this coloration is due to the ability of these proteins to collect various forms of protoporporin and heme from within the E. coli cytoplasm. , Confirmed their role in heme binding.

Example 2: Production of isd gene knockout mutant Further, the coding regions of isdA, isdB, and isdC or their portions are individually interrupted to produce a strain containing a single mutation in each of the isd genes. be able to. A cassette encoding resistance to tetracycline was inserted into the isdA coding region and interrupted. The isdB coding region was interrupted by inserting a cassette encoding resistance to erythromycin. The isdC coding region was interrupted by inserting a cassette encoding resistance to kanamycin. Next, each mutation was moved to the same genetic background using phage transduction, and Sebulsky et al., (2001) J.
Bacteriol. 183: 4994-5000 was selected using the appropriate resistance as described. Furthermore, a strain containing a mutation in which two or more of the isd genes are knocked out (for example, a strain mutated with isdA, isdB, and isdC) may be prepared.

Example 3 Survival Study in a Mouse Model of Renal Infection Female Swiss-Webster mice weighing 25 g were purchased from Charles River Laboratories, Canada and placed in a microisolator cage. Tryptic Soy bacteria
Grown overnight in Broth (TSB), collected and washed 3 times in sterile saline. In pilot experiments, S. Aureus Newman is well established in this model better than RN6390 and is injected into the tail vein to obtain acute but non-fatal renal infection. The optimal amount of Aureus Newman has been demonstrated to be 1 × 10 ⁷ CFU. Bacteria suspended in sterile saline were administered intravenously through the tail vein. The number of viable bacteria injected was confirmed by plating serial dilutions of inoculum on TSB. On day 6 after injection, the mice were killed and the kidneys were removed aseptically. The kidneys were homogenized using a PowerGen 700 homogenizer for 45 seconds in sterile PBS containing 0.1% Triton X-100, and the homogenate dilutions were plated on TSB agar to count viable bacteria. Data presented is log CFU collected per mouse.

  The results show that S. cerevisiae is either in a strain that has a mutation in IsdA alone or in all of IsdA, IsdB, and IsdC. S. It was demonstrated using a mouse renal abscess model of Aureus infection. Interestingly, at 6 days after injection, the recovered mutant bacteria were reduced by 90% of the number recovered from the wild type, and when these proteins were expressed on the bacterial cell surface, It shows an essential role in bacterial health. In addition, inhibition of these proteins in vivo prevents infection of bacteria that express Isd (ie in the case of Isd-based vaccines), or Isd expression once infection has begun. It also shows that it may lead to the removal of bacteria.

Example 4: S. cerevisiae under increasing hydrogen peroxide concentration. Survival of Aureus Isd protein bound to heme is It appears to act as an oxidative buffer that protects Aureus cells from the deleterious effects of free radicals. When a Newman strain incubated in the presence of heme is directly compared to a Newman strain that lacks IsdA, IsdB, and IsdC and is incubated in the presence of heme, the mutant cells cannot survive with higher concentrations of hydrogen peroxide. It is shown that there was not (FIG. 6). Thus, mutants lacking the expression of multiple Isd proteins are highly sensitive to hydrogen peroxide stimulation.

FIG. 7 shows wild-type S. cerevisiae migrated on SDS-PAGE stained with (A) Coomassie and (B) TMBZ (tetramethylbenzidine). Aureus and S. Expression of IsdA in both Aureus isdA :: km ^C is shown at the time of plus or minus of heme. Catalase activity associated with heme-bound IsdA cleaves the TMBZ compound to give a colored reaction product. Thus, heme-bound IsdA has catalase activity that would help resist oxidative lethality by phagocytes.

Example 5: Isd Vaccine A vaccine comprising a recombinant IsdA polypeptide is a systemic and local Protective immunity can be established in mice against Aureus infection. Recombinant IsdA protein may be prepared using standard techniques. Groups of 12-15 Swiss-Webster mice (25 g) can be used for all immunization experiments and injected intraperitoneally (IP). The mice can then be boosted by injection at various different times. Serum can be observed for anti-IsdA antibody titer over the course of the experiment. At approximately 30 days, mice received 1 × 10 ⁷ S. cerevisiae intravenously. Aureus can be loaded and observed for an additional 7 days. We have previously shown that injecting this number of organisms results in a non-fatal kidney infection. At various times after infection, mice can be killed and the number of organisms infected with kidney tissue can be observed. In addition, passive immunization experiments can be performed using serum collected from previously immunized mice to examine their effects in preventing infection in other groups of mice. Similar immunization experiments can be performed with IsdB and IsdC polypeptides.

Example 6: IsdA, IsdB, and IsdC antibodies Production of monoclonal antibodies against full-length Isd protein.
BALB / c mice can first be immunized with an intraperitoneal injection of full-length recombinant IsdA, IsdB, or IsdC and then about 6 weeks later boosted with native IsdA, IsdB, or IsdC as well. Mice can be immunized with a suitable adjuvant. Serum is obtained from mice approximately 10 days after the second injection and can then be tested for anti-HRP activity by ELISA. Mice whose serum exhibits high levels of anti-HRP activity can be selected for cell fusion. The spleen can be collected from these mice and perfused with Dulbecco's modified Eagle's medium (DMEM) to prepare a cell suspension.

Splenocyte suspensions containing B lymphocytes and macrophages can be prepared by spleen perfusion. This cell suspension can be washed and collected by centrifugation. Myeloma cells can also be washed in this manner. Viable cells can be counted and placed in a 37 ° C. water bath. 1 mL of 50% polyethylene glycol (PEG) can be added to DMEM. Balb / c
Splenocytes can be fused to SP 2 / 0-Ag 14 mouse myeloma cells with PEG, and the resulting hybridomas are selected from tissue cultures selected from hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT). It can be grown in medium supplemented with 20% fetal calf serum. Living cells can be grown to confluence. Spent media can be checked for antibody titer, specificity, and affinity. After incubating the cells in PEG for 1-1.5 minutes at 37 ° C., PEG was diluted by slowly adding DMEM medium. Cells may be pelleted and 35-40 mL DMEM containing 10% fetal bovine formation may be added. Cells can then be aliquoted into tissue culture plates and incubated in an incubator humidified at 37 ° C., 5% CO ₂ .

The next day, DMEM-FCS containing hypoxanthine (H), aminopterin (A) and thymidine (T) medium (HAT medium) can be added to each well. The concentration of HAT in the medium to be added can be double the required final concentration, i.e., H _final = 1 x 10 ^-4 M; A _final = 4 x 10 ^-7 M; and T _final = 1.6 × 10 ^-5 M.

  The plates can then be incubated with HAT medium every 2 to 3 days for 2 weeks. Subsequently, the fused cells can be cultured in DMEM-FCS containing HAT medium. When the fused cells are confluent from 1/2 to 3/4 on the bottom of the well, the supernatant tissue culture fluid can be removed and tested for IsdA, IsdB, or IsdC specific antibodies by ELISA. Positive wells can be cloned by limiting dilution on macrophage or thymocyte feeder plates and cultured in DMEM-FCS. The cloned wells can be examined and recloned three times before obtaining a statistically significant monoclonal antibody. Spent media can be tested for antibody producing clones.

B. Production of polyclonal antibodies against full-length Isd protein Two rabbits can be immunized with unbound purified recombinant IsdA, IsdB and / or IsdC as antigen. Briefly, 1 mg of recombinant IsdA, IsdB, or IsdC is resuspended in 1 ml phosphate buffered saline, emulsified with an equal volume of complete Freund's adjuvant, and approximately 1 ml (approximately half of the total volume). ) Can be injected intraperitoneally into each rabbit. Second and third immunizations can be performed after 2 and 3 weeks using incomplete Freund's adjuvant. Serum may be examined using an enzyme linked immunosorbent assay (ELISA) to determine recombinant IsdA, IsdB or IsdC specific antibody titers. Anti-recombinant IsdA, IsdB, and / or IsdC-containing sera showing high antibody titers in ELISA results can be purified by affinity chromatography on a Sepharose column conjugated with the corresponding Isd polypeptide. Anti-recombinant IsdA, IsdB, and IsdC immunoglobulins are tested for S. cerevisiae. The ability to attenuate the bacterial power of Aureus infection can be examined.

Example 7: Expression assay An assay to screen for agents that disrupt IsdA expression in Aureus can be performed as follows. Wild type S. Aureus cells can be cultured overnight in trypsin soy broth (TSB) (Difco) in the presence or absence of a test agent. After culturing for 24 hours, the cells were washed in 1 × PBS (phosphate buffered saline) and then 10 μg of lysostaphin STE solution (0.1 M NaCl, 10 mM Tris-HCl).
[pH 8.0], 1 mM EDTA [pH 8.0]) and can be dissolved at 37 ° C. The cell lysate can then be transferred to a plate pre-coated with anti-IsdA antibody and incubated at room temperature for 45-60 minutes. As a control, untreated S.P. Cell lysates from Aureus cells can be used. After washing three times with water, a secondary antibody conjugated to either alkaline phosphatase (A) or horseradish peroxidase (HRP) can be added and incubated for 1 hour. The individual plates can then be washed to separate the bound from the free antibody complex. The bound antibody can be detected using a chemiluminescent substrate (alkaline phosphatase or horseradish Super Signal luminol solution for horseradish peroxidase). A chemiluminescent signal can be detected using a microplate luminometer. The absence of a signal in a cell lysate sample obtained from cells treated with the agent to be tested would be an indication that the agent to be tested inhibits IsdA expression. Similar expression assays may be performed for IsdB and IsdC.

In practicing the present invention, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology are within the skill of the art. Would be used. Such techniques are described in the literature. For example, Molecular Cloning: A
Laboratory Manual, 2 ^nd Ed., Ed.by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DN
Glover ed., 1985); Oligonucleotide
Synthesis (MJ Gait ed., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic
Acid Hybridization (BD Hames & SJ Higgins
eds. 1984); Transcription And Translation
(BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987);
Immobilized Cells And Enzymes (IRL
Press, 1986); B. Perbal, A Practical Guide
To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells
(JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155
(Wu et al. Eds.), Immunochemical Methods In Cell And Molecular
Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology,
Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); Antibodies: A
Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)), Manipulating the Mouse Embryo, (Cold
See Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

Incorporation by reference All publications and patents referred to herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. It shall be. In case of conflict, the present application, including any definitions here, will be superior.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

FIG. 1 shows (A) the nucleic acid sequence encoding IsdA (SEQ ID NO: 1), (B) the reverse complement of SEQ ID NO: 1 (SEQ ID NO: 2), and (C) the corresponding amino acid of IsdA. The sequence (SEQ ID NO: 3) is shown. FIG. 2 shows (A) the nucleic acid sequence encoding IsdB (SEQ ID NO: 4), (B) the reverse complement of SEQ ID NO: 4 (SEQ ID NO: 5), and (C) the corresponding amino acid of IsdB. The sequence (SEQ ID NO: 6) is shown. FIG. 3 shows (A) the nucleic acid sequence encoding IsdC (SEQ ID NO: 7), (B) the reverse complement of SEQ ID NO: 7 (SEQ ID NO: 8), and (C) the corresponding amino acid of IsdC. The sequence (SEQ ID NO: 9) is shown. FIG. 4 shows S. cerevisiae grown in iron-rich and iron-depleted media. It is an SDS-PAGE gel showing a whole cell lysate of Aureus. FIG. 5 shows S. cerevisiae collected from the kidneys of mice 6 days after injection of 10 ⁷ bacteria into the tail vein. It is a graph which shows an Aureus number. FIG. 6 shows wild-type S. cerevisiae conjugated to heme. Aureus Isd protein knocks out isdA, isdB, and isdC. High hydrogen peroxide compared to S. aureus is a table showing that can survive in the (H ₂ O ₂₎ conditions (P indicates that bacterial viability over 90%) 7A and 7B show (A) Coomassie (for total protein) and (B) TMBZ (tetramethylbenzidine), wild type and isdA knockout S. cerevisiae. It is an SDS-PAGE gel showing protein derived from Aureus.

Claims (24)

  A vaccine comprising a IsdA (SEQ ID NO: 3) polypeptide and a pharmaceutically acceptable carrier.   2. A vaccine according to claim 1 wherein the vaccine is in an injectable formulation.   The vaccine of claim 1 further comprising an adjuvant.   A vaccine comprising IsdB (SEQ ID NO: 6) polypeptide and a pharmaceutically acceptable carrier.   5. A vaccine according to claim 4 wherein the vaccine is in an injectable formulation.   The vaccine of claim 4 further comprising an adjuvant.   A vaccine comprising IsdC (SEQ ID NO: 9) polypeptide and a pharmaceutically acceptable carrier.   8. A vaccine according to claim 7 wherein the vaccine is in an injectable formulation.   8. The vaccine of claim 7, further comprising an adjuvant.   A pharmaceutical composition comprising an effective antibacterial amount of an antibody that binds to IsdA (SEQ ID NO: 3) and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an effective antibacterial amount of an antibody that binds to IsdB (SEQ ID NO: 6) and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an effective antibacterial amount of an antibody that binds to IsdC (SEQ ID NO: 9) and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising a nucleic acid antisense to SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an siRNA molecule comprising a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 and a pharmaceutically acceptable carrier.   Treating or preventing a disease or condition caused by or contributing to an infection of Staphylococcus aureus in said subject comprising administering to said subject an effective amount of the vaccine of any of claims 1, 4, or 7 Method.   A method of treating or preventing a disease or condition caused by or contributing to an infection of Staphylococcus aureus in a subject comprising administering an effective amount of the pharmaceutical composition of claim 10 to the subject.   A method of treating or preventing a disease or condition caused by or contributing to Staphylococcus aureus infection in a subject comprising administering an effective amount of the pharmaceutical composition of claim 11 to the subject.   A method of treating or preventing a disease or condition caused by or contributing to an infection of Staphylococcus aureus in a subject comprising administering an effective amount of the pharmaceutical composition of claim 12 to the subject.   A method of treating or preventing a disease or condition caused by or contributing to Staphylococcus aureus infection in a subject comprising administering an effective amount of the pharmaceutical composition of claim 13 to the subject.   15. A method of treating or preventing a disease or condition caused by or contributing to Staphylococcus aureus infection in a subject comprising administering an effective amount of the pharmaceutical composition of claim 14 to the subject. (I) an interaction between an Isd polypeptide and a suitable interacting molecule in the presence of an agent, and between the Isd polypeptide and the interacting molecule is possible in the absence of the agent. Contacting under conditions;
(Ii) determining the level of interaction between the Isd polypeptide and the interacting molecule, the interaction between the Isd polypeptide and the interacting molecule in the presence of the agent That the level of is different compared to the absence of the agent is an indication that the agent inhibits the interaction between the Isd polypeptide and the interacting molecule. A method of identifying an agent that binds to an Isd polypeptide and inhibits iron uptake.   24. The method of claim 21, wherein the Isd polypeptide is selected from the group consisting of Staphylococcus aureus IsdA, IsdB, and IsdC. (I) culturing a wild-type Staphylococcus aureus strain in the presence or absence of an agent;
(Ii) a step of comparing the expression of the Isd polypeptide, wherein the decrease in the expression of the Isd polypeptide in cells treated with the agent is greater than the fact that the agent is an Isd polypeptide in Staphylococcus aureus. In Staphylococcus aureus, comprising a step that is an indicator of inhibiting the expression of the peptide
A method for determining IsdA, IsdB, and IsdC polypeptides. (I) culturing a wild-type Staphylococcus aureus strain in the presence or absence of an agent;
(Ii) a step of comparing the expression of the isd nucleic acid, wherein a greater decrease in the expression of the isd nucleic acid in the cell treated with the agent is the expression of the isd nucleic acid in Staphylococcus aureus A method of identifying an agent that inhibits expression of a nucleic acid selected from the group consisting of isdA, isdB, and isdC nucleic acids in Staphylococcus aureus, comprising the step of:

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