KR20070085457A - Vaccines, compositions and methods based on staphylococcus aureus iron-regulated surface determinants isda, isdb, and isdc - Google Patents

Abstract

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

Description

Vaccines, compositions and methods for surface-determinants ISDD, ISDD, and ISCD-based vaccines, which are controlled by iron of Staphylococcus aureus

Background

Except for Lactobacillus (Archibald (1983) FEMS Microbiol. Lett. 19: 29-32) and Borrelia burgdorferi (Posey and Gherardini (2000) Science 288: 1651-1653) Iron is absolutely necessary for growth. Despite being the fourth most abundant element in the earth's crust, iron is often a growth limiting nutrient. At aerobic environments and physiological pH, iron is in the trivalent iron (Fe ³⁺ ) state and forms insoluble hydroxides and oxyhydroxide precipitates. Mammals overcome iron limitations by containing high-affinity iron-binding glycoproteins such as transferrin and lactoferrin, which dissolve and deliver iron to host cells (Weinberg (1999) Emerg. Infect. Dis. 5: 346-352). . This result further limits free extracellular iron, and thus the concentration of free iron in the human body is measured at ^10-18 M, several levels lower than required to support productive bacterial infections (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 sequestration is an important innate defense against bacterial infections (Skaar and Schneewind (2004) Microbes and Infect. 6: 390-397).

To overcome iron limitations, bacteria have evolved into several mechanisms for obtaining these essential nutrients. For example, Pasteurellaceae members may express receptors for the recognition of iron-loaded forms of transferrin and lactoferrin (Gray-Owen and Schryvers, (1996) Trends Microbiol. 4: 185-91 ). One of the most common iron acquisition mechanisms is through low molecular weight, high affinity iron chelating agents called iron traps, and cognate cell envelope receptors that actively refractory ferric-capture complexes. Many iron traps can successfully compete with transferrin and lactoferrin for host iron. In addition, the expression of the ferric-iron capture body uptake system was characterized by E. coli (Williams (1979) Infect. Immun. 26: 925-932), Vibrio anguillarum (Crosa et al. , (1980) Infect. Immun. 27: 897-902), Erwinia chrysanthemi (Enard et al., (1988) J. Bacteriol. 170: 2419-2426) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) (Meyer et al., (1996) Infect. Immun. 64: 518-523) is an important virulence factor.

Staphylococcus aureus is known as sstABCD (Morrissey et al., (2000) Infect. Immun. 68: 6281-6288), sirABC (Heinrichs et al., (1999) J. Bacteriol. 181: 1436 -1443; Dale et al., (2004) J. Bacteriol., In press), fhuCBG (Sebulsky et al., (2000) J. Bacteriol. 182: 4394-4400), and sbn (Dale et al., ( 2004) Infect. Immun. 72: 29-37) have ABC carriers regulated by several different irons, including those encoded by operons. Carried substrate, but not known to the sst and sir system, and fhuD1 fhuD2: fhuCBG gene related to (Sebulsky and Heinrichs (2001) J. Bacteriol 183. 4994-5000) is iron (III) - a-hydroxy roksa formate complexes Related to acquisition. Several members of Staphylococcus, including a number of strains of plasma coagulase-negative Staphylococcus (CoNS) and Staphylococcus aureus, produce iron traps. Staphyloferrin A (Konetschny-Rapp et al., (1990) Eur. J. Biochem. 191: 65-74; Meiwes et al., (1990) FEMS Microbiol. Lett, two of these iron traps 67: 201-206) and Staphyloferrin B (Dreschel et al., (1993) BioMetals. 6: 185-192; Haag et al., (1994) FEMS Microbiol. Lett. 115: 125-130) The carboxylate class, but the third aureochelin (Courcol et al., (1997) Infect. Immun. 65: 1944-1948), has not been chemically characterized. In addition, the additional iron capture encoded by the sbn operon appears to be specific for Staphylococcus aureus and may be an important determinant for the toxicity of Staphylococcus aureus compared to other CoNS strains. (Dale et al., (2004) Infect. Immun. 72: 29-37).

However, the most abundant source of iron in the human body is sequestered in heme-containing proteins (hemex proteins). Heme is a ring molecule containing a single iron atom bonded to the four ring nitrogen atoms of porphyrin. Heme proteins are responsible for a number of cellular functions, and hemoglobin and myoglobin are the heme-containing proteins most abundant in mammals. Iron is essential for the survival of bacterial pathogens, and bacteria have obtained several mechanisms for utilizing iron from free heme and heme proteins. In particular, Staphylococcus aureus can obtain iron from heme or heme protein via a surface determinant (Isd) system controlled by iron. The Isd system includes one or more cytosolic proteins that can extract iron from heme as well as cell surface proteins that can bind and transport heme across the bacterial cell wall (Mazmanian et al., (2003) Science 299: 906- 909; Clarke et al., (2004) Mol. Microbiol. 51: 1509-1519). In addition, Isd proteins, in particular IsdA, are not only fibrinogen and fibronectin (Clarke et al., (2004) Mol. Microbiol. 51: 1509-1519) but also transferrin (Taylor and Heinrichs (2002) Mol. Microbiol. 43: 1603-1614 ) And Hemin (Mazmanian et al., (2003) Science 299: 906-909), and seem to bind to a wide range of extracellular matrix proteins.

Staphylococcus aureus is a predominant human pathogen that causes widespread infections from mild skin and wound infections to more severe sequelae, such as endocarditis, osteomyelitis and sepsis (Archer (1998) Clin. Infect. Dis. 26 : 1179-1181). Staphylococcus aureus' s ability to penetrate multiple tissues to form colonies is characterized by several virulence factors, such as fibronectin binding proteins, elastin binding proteins and collagen binding proteins that aid tissue attachment, and tissue destruction and bacterial transmission It may be due to its ability to express multiple exotoxins and proteases that cause them. The ability of these bacteria to acquire iron during in vivo growth also appears to be important for their pathogenesis, and several research groups have characterized several different genes of products related to binding and / or transport of host iron compounds (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).

For the first time, penicillin was used to treat the worst Staphylococcus aureus infection. However, the emergence of penicillin resistant strains of Staphylococcus aureus reduced the efficacy of penicillin in the treatment of Staphylococcus infections, and most strains of Staphylococcus aureus encountered in hospital infection today are penicillin Does not react to Penicillin resistant strains of Staphylococcus aureus produce beta-lactamase, which converts penicillin to pencillinoic acid to destroy antibiotic activity. Moreover, beta-lactamase encoding genes are often episomally proliferated for plasmids, which is often one of several genes that together confer multidrug resistance to episomal components.

Methicillin, introduced in the 1960s, overcomes most of the problems of penicillin resistance in Staphylococcus aureus. These compounds preserve the portion of penicillin responsible for antibiotic activity and modify or convert other portions that make penicillin an excellent substrate for inactivating lactase. However, methicillin resistance has appeared in Staphylococcus aureus, along with a number of other antibiotics including aminoglycosides, tetracyclines, chloramphenicol, macrolides and lincosamide to Staphylococcus aureus. Resistance to the effects appeared. In fact, methicillin resistant strains of Staphylococcus aureus generally have increased drug resistance. Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the world's most important hospital pathogens and has become a serious infection control problem. Today, many strains are multi-resistant to virtually all antibiotics except vancomycin type glycoprotein antibiotics. Drug resistance of Staphylococcus aureus infection has a significant therapeutic difficulty that is worse than when no new therapeutic agent is developed. Thus, there is an unmet medical need for new and effective therapeutic agents for treating Staphylococcus aureus infections.

Summary of the Invention

The present invention is based, at least in part, on the identification and characterization of Isd (iron controlled surface determinants) proteins IsdA, IsdB and IsdC, which are from heme and heme proteins of Staphylococcus aureus. It is part of the Isd system related to iron internalization. IsdA, IsdB and IsdC are expressed on the cell surface of Staphylococcus aureus, which is important for limited growth and survival by iron in vivo. As a result, IsdA, IsdB and IsdC proteins are attractive vaccine targets whose inhibition can lead to impaired bacterial growth in vivo. In addition, IsdA, IsdB and IsdC proteins are attractive drug targets that can be used in screening assays to identify Staphylococcus aureus specific antibiotics.

In one embodiment, the invention features an Isd protein based vaccine. In one exemplary embodiment, the Isd based vaccine comprises an IsdA polypeptide and a pharmaceutically acceptable carrier. In one embodiment, the IsdA polypeptide comprises the full length amino acid sequence of SEQ ID NO: 3. In another embodiment, the IsdA polypeptide is a peptide 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 certain embodiments, the IsdB polypeptide comprises the full length amino acid sequence of SEQ ID NO: 6. In another embodiment, the IsdB polypeptide is a peptide of at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO: 6. In another embodiment, the Isd vaccine comprises an IsdC polypeptide and a pharmaceutically acceptable carrier. In certain 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 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 in an injectable formulation and may further comprise an adjuvant.

In another embodiment, the invention features novel antibiotics comprising antibodies, antisense nucleic acids, and siRNAs that inhibit iron uptake in Staphylococcus aureus. The present invention features antibodies against IsdA, IsdB and / or IsdC. In certain embodiments, an antibody against an IsdA polypeptide can be generated against the full length recombinant amino acid sequence of SEQ ID NO: 3, or at least 5, 10, 15, 20, 25, 30, 35, 40 of SEQ ID NO: 3. For peptides of 45, 50 or 50 amino acids. Antibodies to the IsdB polypeptide may be generated against the full length recombinant amino acid sequence of SEQ ID NO: 6, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 of SEQ ID NO: 6 Can be generated for peptides of amino acids. Antibodies to the IsdC polypeptide may be generated against the full length recombinant amino acid sequence of SEQ ID NO: 9, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 of SEQ ID NO: 9 Can be generated for peptides of amino acids. Antibodies to the Isd polypeptide may be monoclonal or polyclonal. Antibodies against the Isd polypeptide may be formulated in an injectable formulation and administered as an anti-bacterial therapeutic.

In one further aspect, the invention features a screening assay to identify agents that inhibit or otherwise interfere with the expression level and / or function of any Isd protein. In one exemplary embodiment, the invention features a screening assay for agents that inhibit the expression and / or function of IsdA. In one embodiment, the assay is a binding assay and the agent that binds to and interferes with its biochemical function is a candidate Staphylococcus aureus specific antibiotic. In another embodiment, the assay is an expression assay and the agent that reduces the expression level of an Isd polypeptide is a candidate Staphylococcus aureus specific antibiotic.

In one further embodiment, the Isd protein includes but is not limited to Staphylococcus aureus, Corynebacterium diphtheriae , Listeria monocytogenes , and Bacillus anthracis It can be expressed in gram-positive bacteria without limitation. Thus, vaccines and inhibitors targeting the Isd proteins described herein can be used to treat many toxic Gram-positive bacterial strains that cause disease in mammals.

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

Brief description of the drawings

1 shows corresponding amino acids for (A) the nucleic acid sequence encoding IsdA (SEQ ID NO: 1), (B) the complementary sequence of SEQ ID NO: 1 (SEQ ID NO: 2), and (C) IsdA The sequence (SEQ ID NO: 3) is shown.

Figure 2 shows the corresponding amino acids for (A) the nucleic acid sequence encoding IsdB (SEQ ID NO: 4), (B) the complementary sequence of SEQ ID NO: 4 (SEQ ID NO: 5), and (C) IsdB. The sequence (SEQ ID NO: 6) is shown.

Figure 3 shows the corresponding amino acids for (A) the nucleic acid sequence encoding IsdC (SEQ ID NO: 7), (B) the complementary sequence of SEQ ID NO: 7 (SEQ ID NO: 8), and (C) IsdC. The sequence (SEQ ID NO: 9) is shown.

4 is an SDS-PAGE gel showing total cell lysate from Staphylococcus culture in iron-rich medium and iron-depleted medium.

5 is a graph showing Staphylococcus number recovered from mouse kidneys 6 days after injection of 10 ⁷ bacteria into the tail veins of mice.

FIG. 6 shows increased hydrogen peroxide (H ₂ O ₂ ) in wild-type Staphylococcus aureus Isd protein bound to heme compared to Staphylococcus aureus with knocked-out isdA , isdB, and isdC It is a table indicating that it can survive under the conditions of (√: shows more than 90% bacterial survival).

7A and 7B show SDS showing proteins from wild type and isdA knockout Staphylococcus aureus (A) stained with Coomassie (for total protein) and (B) stained with TMBZ (tetramethylbenzidine) -PAGE gel.

Detailed description of the invention

1. Overview

As described herein, the internalization of iron through the uptake of heme is a toxic property that can be attenuated when the isd genes such as isdA , isdB and isdC are knocked out. In addition, as described herein, the heme bound Isd protein may play an additional role in promoting Staphylococcus survival in the host. Heme-bound Isd proteins that appear to act as oxidation buffers to protect cells form the deleterious effects of free radicals. Thus, while mutations deficient in Isd protein expression are more sensitive to hydrogen peroxide administration, wild-type Staphylococcus aureus can survive at higher concentrations of hydrogen peroxide.

The Isd protein described herein is essential for Staphylococcus aureus infection in vivo and is highly expressed during Staphylococcus aureus infection. After Staphylococcus aureus enters the host, it encounters an iron constrained environment, after which the Isd protein expression is upregulated. In iron-restricted hosts, Isd expression appears to remain upregulated as Staphylococcus aureus uses iron. In particular, IsdA described herein is immunodominant, in which a 1: 4000 dilution of serum from convalescent patients (ie, Staphylococcus aureus infected patients) is positive with 4 micrograms of purified IsdA protein in Western immunoblot. Because it reacts. Thus, Isd protein is an attractive target in vaccine development. Antigen peptides of the Isd protein can be used as vaccine targets to produce an effective immune response against Staphylococcus aureus. In addition, inhibiting Isd protein function using Isd specific antibodies, antisense RNAs, siRNAs or small molecule inhibitors would be an effective means of attenuating the toxicity of Staphylococcus aureus.

The Isd protein described herein is expressed in Staphylococcus aureus. In further embodiments, the Isd protein may be expressed in other Gram-positive bacteria. Non-limiting examples of Gram-positive pathogens expressing the Isd protein include Staphylococcus aureus, Corynebacterium diphtherinee, Listeria monocytogenes, and Bacillus atlassis. Thus, vaccines and inhibitors targeting the Isd proteins described herein can be used to treat other toxic Gram-positive bacterial strains that cause disease in mammals.

2. Definition

For convenience, the meanings of the specific terms and expressions 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 “a” and “an” are used to mean one or more (ie, at least one) grammatical objects of such articles. For example, "element" means one or more than one element.

As used herein, the term "adjuvant" refers to a substance that induces an enhanced immune response when used with a particular antigen.

The term "agent" refers to chemical compounds, mixtures of chemical compounds, biological macromolecules (eg, nucleic acids, antibodies, proteins or portions thereof, such as peptides), or bacteria, plants, mycelium or animals (especially mammals) cells or tissues. It is used to denote extracts made from biological materials such as. The screening assays described herein can identify agents. The agent may be an inhibitor or antagonist of Isd mediated iron absorption of Staphylococcus aureus. The activity of the agent may make it suitable as a "therapeutic agent" which is a biological, physiological or pharmacologically active substance (or substances) which acts locally or systemically on a subject.

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

As used herein, the term "antibody" refers to immunoglobulins and any antigen-binding portion of an immunoglobulin (e.g., IgG, IgD, IgA, IgM and IgE), i. ") Means a polypeptide containing an antigen binding site. The antibody may comprise one or more heavy chains (H) and one or more light chains (L) interconnected by one or more disulfide bonds. 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" includes especially monoclonal and polyclonal antibodies. "Polyclonal antibody" means an antibody derived from the serum of an animal immunized with an antigen or antigens. "Monoclonal antibody" means an antibody produced by a monoclonal of hybridoma cells. Techniques for generating monoclonal antibodies include hybridoma technology (see Kohler & Milstein (1975) Nature 256: 495-497); Trioma technology; Human B-cell hybridoma technology (see Kohler et al. (1983) Immunol. Today 4:72), EBV hybridoma technology (see Cole et al. , 1985 In: Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, Inc., pp. 77-96) and phage display.

Polyclonal or monoclonal antibodies can be further engineered or modified to produce chimeric or humanized antibodies. A "chimeric antibody" is encoded by an immunoglobulin gene produced by genetic engineering, and the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. For example, a substantial portion of the variable (V) segment of a gene from a mouse monoclonal antibody as obtained herein can be linked to a substantial portion of a human constant (C) segment. Such chimeric antibodies will be less antigenic for humans than mouse monoclonal antibodies.

As used herein, the term “humanized antibody” (HuAb) refers to a chimeric antibody having a framework region that is substantially the same (ie, at least 85%) as the human framework, with CDRs from non-human antibodies, Wherein any constant region has at least about 85-90%, preferably about 95% polypeptide sequence identity to a human immunoglobulin constant region. See, eg, PCT Publication WO 90/07861 and European Patent No. 0451216. Perhaps all parts of the HuAb except the CDRs are substantially identical to the corresponding parts of one or more native human immunoglobulin sequences. The term "framework region" as used herein is described in KaBAT et al. (1987) Sequences of Proteins of Immunologic Interset , 4 ^th Ed., US Dept. Heath and Human Services] means portions of immunoglobulin light and heavy chain variable regions that are relatively conserved (ie, not CDR) of a single species of different immunoglobulins. Human constant region DNA sequences can be isolated from a variety of human cells according to well known procedures, but are preferably isolated from infinitely proliferating B cells. The variable regions or CDRs for generating humanized antibodies can be derived from monoclonal antibodies capable of binding antigens and will be generated in any convenient mammalian source, including mice, rats, rabbits or other vertebrates. .

The term "antibody" also includes antibody fragments. Examples of antibody fragments include Fab, Fab ', Fab'-SH, F (ab') ₂ , and Fv fragments; Without limitation a single chain Fv (scFv) molecule, a single chain polypeptide containing only one light chain variable domain, or a fragment thereof containing three CDRs of a light chain variable domain without bound heavy chain molecules, and (3) a single heavy chain variable region And diabodies having a primary structure composed of uninterrupted sequences of one of the contiguous amino acid residues, including the containing single chain polypeptide, or fragments thereof containing the three CDRs of the heavy chain variable region without bound light chain molecules. Antibody fragments; There are multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain (s) may contain any constant domain sequence found in the non-Fc region of the complete antibody (eg, CH1 of the IgG isotype) and / or found in the complete antibody May contain any hinge region sequence and / or contain a leucine zipper sequence fused to or located in the hinge region sequence or the constant domain sequence of the heavy chain (s). Suitable leucine zipper sequences include Kostelney et al. (1992) J. Immunol. 148: 1547-1553, and the jun and fos leucine zippers and the GCN4 leucine zippers disclosed in US Pat. No. 6,468,5323. Fab and F (ab ') ₂ fragments lack the Fc fragment of an intact antibody, and typically have enzymes such as papain (to generate a Fab fragment) or pepsin (to generate a F (ab') ₂ fragment). By proteolytic cleavage.

If the antibody preferably binds to one antigen more than most other antigens, then the antibody specifically binds to that antigen or epitope of the antigen. For example, an antibody may have less than about 50%, 20%, 10%, 5%, 1% or 0.1% cross-reactivity to one or more other epitopes.

The term "conservative substitution" refers to a change between amino acids of largely similar molecular properties. For example, interchanges within the aliphatic groups alanine, valine, leucine and isoleucine may be considered conservative. Sometimes, substitution of glycine for one of these may also be considered conservative. Other conservative interchanges are in the aliphatic groups aspartate and glutamate; In the amide groups asparagine and glutamine; In the hydroxyl groups serine and threonine; In the aromatic groups phenylalanine, tyrosine and tryptophan; In the basic groups lysine, arginine and histidine; And effectual exchange in the sulfur containing groups methionine and cysteine. Sometimes, substitutions in methionine and leucine can also be considered conservative. Preferred conservative substituents include aspartate-glutamate; Arapargin-glutamine; Valine-leucine-isoleucine; Alanine-valine; Valine-leucine-isoleucine-methionine; Phenylalanine-tyrosine; Phenylalanine-tyrosine-tryptophan; Lysine-arginine; And histidine-lysine-arginine.

An "effective amount" is an amount sufficient to provide a clinical result that is beneficial or desired in treatment. An effective amount can be administered to a patient in one or more doses. With respect to treatment, the effective amount is an amount sufficient to reduce the infection of the patient. Several factors are usually considered when determining the appropriate dosage to achieve an effective amount. Such factors include the age, sex and weight of the patient, the disease to be treated, the severity of the disease and the form and effective concentration of the agent administered.

The term “epitope” refers to the region of an antigen to which an antibody preferentially and specifically binds. Monoclonal antibodies preferentially bind to a single specific epitope of a molecule, which can be defined as a molecule. The epitope of a particular protein may consist of a limited number of amino acid residues, such as 5-15 residues, linear or nonlinear in the protein.

As used in describing nucleic acid or nucleotide sequences, "equivalent" means a nucleotide sequence that encodes a functionally equivalent polypeptide. Equivalent nucleotide sequences will include different sequences by one or more nucleotide substitutions, additions, or deletions, such as allelic variation; It will therefore contain different sequences due to the degradation of the genetic code. For example, nucleic acid variations can include those produced by nucleotide substitutions, deletions or additions. Substitutions, deletions or additions may involve one or more nucleotides. The variation can be changed in the coding region, the non-coding region, or both. Alterations in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions.

"Homologous" or alternatively "identity" means sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing the positions of each sequence that can be aligned for comparison. If the position of the compared sequence is occupied by the same base or amino acid, the molecule is homologous at that position. The degree of homology between sequences is a function of the number of homology positions shared by the match or sequence. The term "% identity" refers to sequence identity between two amino acid sequences or two nucleotide sequences. Identity can be determined by comparing the position of each sequence that can be aligned for comparison. If the same base or amino acid occupies the equivalent position of the compared sequence, the molecules are identical at that position; When equivalent sites are occupied by identical or similar amino acid residues (such as similar steric and / or electronic properties), the molecules may be said to be homologous (similar) at that position. Indications as percentages of homology, similarity or identity are referred to as a function of the same or similar amino acid number at positions shared by the compared sequences. Various sorting algorithms and / or programs are available and include FASTA, BLAST or ENTREZ. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) And can be used, for example, with default settings. ENTREZ is available through National Center for Biotechnology Information, National Library of Medicine, and National Center of Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, and Md. In one embodiment, the percent identity of two sequences can be determined by a GCG program with a gap weight of 1, eg, each amino acid gap is weighed as if it is a single amino acid or a nucleotide mismatch between two sequences. Other alignment techniques are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequesce Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Preferably, an alignment program is used to align the sequences, allowing for gaps in the sequences. Smith-Waterman is a type of algorithm that allows spacing in sequence alignment. References Meth. Mol. Biol . 70: 173-187 (1997). GAP programs using the Needleman and Wunsch alignment method can also be used to align sequences. An alternative search method uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses the Smith-Waterman algorithm to score sequences on large parallel computers. This approach improves the ability to capture away related matches, and particularly tolerates small spacing and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases. Databases of individual sequences are described in Methods in Enzymology, ed. Doolittle, detailed above. Databases include Genbank, EMBL, and the Japanese DNA Database (DDBJ).

As used herein, the term "infection" refers to the invasion and proliferation of microorganisms, such as Staphylococcus aureus, in body tissues, which is clinically unclear or competitive metabolism, toxins, intracellular replication or antigenic antibodies. The reaction can lead to local cellular damage. Infection can be local, asymptomatic, and temporary if the body's defense mechanisms are in effect. Local infections can persist and spread by expansion, resulting in acute, subacute or chronic clinical infections or disease states. Local infection may be systemic when microorganisms access lymphatic or vascular systems.

As used herein, the term “iron-controlled surface determinant system” or “Isd system” speculatively includes a number of genes encoded by five different transcription units that together encode a heme absorption system. Aureus means Isd locus. The five transcription units are isdA , isdB , isdCDEFsrtBisdG , isdH and isdI . Transcription of the isd gene is regulated by surrounding iron upon regulation of the Fur promoter. Four of the proteins encoded by the Isd locus, IsdA, IsdB, IsdC and IsdH, are covalently attached to the cell wall by an amide bond between the polypeptide chain and the C-terminus of the peptidoglycan. IsdA, IsdB and IsdH have been characterized as having a C-terminal sorting signal referred to as the LPXTG motif (ie, the motif recognized by Sortase A). IsdC has been characterized as having a C-terminal classification signal referred to as the NPQTN motif (ie, the motif recognized by Sortase B of Staphylococcus aureus). IsdA, IsdB, and IsdH are anchored to the cell wall by sorbase A (srtA), a membrane-attached transpeptidase that degrades cell surface proteins in the LPXTG motif and catalyzes the formation of amide bonds between polypeptides and peptidoglycans. IsdC is anchored to the cell wall by sorbase B (srtB), a transpeptidase similar to sorbase A. Other proteins encoded by the Isd locus that may be involved in extracting iron from heme include putative membrane translocation factors, IsdD, IsdE and IsdF, and cytoplasmic heme-iron binding proteins IsdG and IsdI.

As used herein, “IsdA polypeptide” means surface determinant A, which is regulated by iron. The sequence of an IsdA polypeptide is described in SEQ ID NO: 3 and encoded by SEQ ID NO: 1. The term also includes 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 inducing an immune response in a subject.

As used herein, “IsdB polypeptide” refers to the surface determinant B controlled by iron. The sequence of an IsdB polypeptide is described in SEQ ID NO: 6 and encoded by SEQ ID NO: 4. The term also includes any fragment, variant, analog, agonist, chemical derivative, functional derivative or functional fragment of an IsdB polypeptide. An "IsdB immunogen" is an IsdB polypeptide capable of inducing an immune response in a subject.

As used herein, “IsdC polypeptide” refers to the surface determinant C controlled by iron. The sequence of an IsdC polypeptide is described in SEQ ID NO: 9 and encoded by SEQ ID NO: 7. The term also includes 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 inducing an immune response in a subject.

"Label" and "detectable label" include radioisotopes, fluoropores, chemiluminescent molecules, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (eg biotin or hapten), and the like. It means a detectable molecule that is not limited to one. "Fluorophore" means a substance or part thereof that can exhibit fluorescence in the detectable range. Specific examples of suitable labels include fluorescein, rhodamine, monocyst, umbeliferon, texas red, luminol, NADPH, alpha-galactosidase or beta-galactosidase and horseradish peroxidase.

As used herein, the term “mutation” refers to a gene that encodes a mutant protein. As used herein, the term “mutation” refers to a protein that does not perform its usual or normal physiological function. Staphylococcus aureus polypeptide mutations can be produced by amino acid substitutions, deletions or additions. Substitutions, deletions or additions may involve one or more residues. Particularly preferred among these are substitutions, additions and deletions which alter the properties and activities of the Staphylococcus aureus protein of the present invention.

The terms "polynucleotide" and "nucleic acid" are used interchangeably. These mean polymer forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or noncoding regions of genes or gene fragments, loci defined by binding assays, exons, introns, messenger RNA (mRNAs), transfer RNAs, ribosomal RNAs, ribozymes, cDNAs, Antisense nucleic acids, recombinant polynucleotides. Branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogues. If there is a modification to the nucleotide structure, it may be provided before or after assembly of the polymer. Polynucleotides may be further modified after polymerisation, such as by conjugation with a labeling component. The term "recombinant" polynucleotide refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin that is not naturally occurring or bound to another polynucleotide in an unnatural alignment. "Oligonucleotide" means a single standard polynucleotide having less than about 100 nucleotides, such as 75, 50, 25, or less than 10 nucleotides.

The terms "polypeptide", "peptide" and "protein" (if short chain) are used interchangeably herein to refer to polymers of amino acids. The polymer can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. The term also includes amino acid polymers modified by any other manipulation, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or conjugation with a labeling component. The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids, including both glycine and D or L optical isomers, and amino acid analogs and peptidomimetic.

The term "small molecule" means 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, peptidomimetic, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of mycelia, bacteria or algae extracts that can be screened with chemical and / or biological mixtures, often with any of the assays of the present invention. The term “small organic molecule” refers to a small molecule that is often identified as an organic or medicinal compound and does not include only molecules that are nucleic acids, peptides or polypeptides.

The term “specific hybridization” means specific nucleic acid binding that is detectable. Polynucleotides, oligonucleotides, and nucleic acids of the invention are selectively hybridized to nucleic acid strands under hybridization and wash conditions that minimize detectable amounts of detectable binding to nonspecific nucleic acids. The stringent conditions known in the art and discussed herein can be used to achieve selective hybridization conditions. In general, nucleic acid sequence homology between polynucleotides, oligonucleotides and nucleic acids of interest and at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% of the present invention , 98%, 99% or more. In certain instances, hybridization and wash conditions are performed under stringent conditions in accordance with conventional hybridization procedures and the hybridization procedures further described herein.

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

Various techniques are available for measuring Tm. Typically, G-C base pairs are evaluated to contribute about 3 ° C. to Tm in the double strand, whereas A-T base pairs are estimated to contribute about 2 ° C., and Tm is below the theoretical maximum of about 80-100 ° C. However, more sophisticated models of Tm are available that take into account G-C stacking interactions, solvent effects, desired assay temperatures, and the like. For example, a probe may have the formula Td = ((((((3x # GC) + (2x # AT)) x37) -562) / # bp) -5 (where #GC, #AT and #bp are each double The number of guanine-cytosine base pairs, the number of adenine-thymine base pairs, and the number of total base pairs associated with the formation of the strands).

Hybridization can be performed at 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 can be increased, for example, from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C. or 65 ° C. to control the stringency of the reaction. The hybridization reaction may also include another agent that affects stringency, for example hybridization carried out in the presence of 50% formamide increases the stringency of hybridization at a defined temperature.

After the hybridization reaction, a single washing step or two or more washing steps can be performed at the same or different salinities and temperatures. For example, the temperature of the wash may be increased from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C. or higher to control stringency. The washing step can be carried out in the presence of a detergent, for example 0.1 or 0.2% SDS. For example, after hybridization, two wash steps at 65 ° C. each for about 20 minutes in 2 × SSC, 0.1% SDS, and optionally two additional wash steps at 65 ° C. each for about 20 minutes in 0.2 × SSC, 0.1% SDS, respectively. Can be followed.

Exemplary stringent hybridization conditions include 50% formamide, 10 x Denhardt (0.2% picol, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin) and 200 μg / ml denatured carrier DNA, eg For example, after overnight hybridization at 65 ° C. in a solution containing or consisting of sheared salmon sperm DNA, 2 wash steps at 65 ° C. for about 20 minutes at 2 × SSC, 0.1% SDS, and 0.2 × SSC, 0.1% Two wash steps each at 65 ° C. for about 20 minutes in SDS.

Hybridization may consist of hybridizing two nucleic acids in a solution, or one nucleic acid in a solution, to a nucleic acid attached to a solid support, such as a filter. If one nucleic acid is present in the solid support, a preliminary hybridization step may be performed prior to hybridization. Preliminary hybridization can be performed (without complementary polynucleotide strands) for at least about 1 hour, 3 hours or 10 hours at the same solution and at the same temperature as the hybridization solution.

Appropriately stringent conditions are known to those skilled in the art or can be determined experimentally by one skilled in the art. See, eg, Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y; S. Agrawal (ed.) Methods in Molecular Biology, volume 20; Tijssen (1993) Laboratory Techniques in biochemistry and molecular biology-hybridization with nucleic acid probes, e.g., 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., Biochem. 31: 12083 (1992).

The term “substantially homologous” when used in reference to a nucleic acid or amino acid sequence, refers to sequences that are substantially identical or similar in sequence to each other, resulting in usefulness of retention of one or more biological (including immunological) activities by generating form identity. Means. The term does not mean general evolution of the sequence.

"Subject" means a male or female mammal, including a human.

A “variant” of an Isd polypeptide means a molecule substantially similar to IsdA, IsdB or IsdC. Variant peptides can be covalently prepared by direct chemical synthesis of variant peptides using methods well known in the art. Variants of the Isd polypeptide may further comprise, for example, deletions, insertions or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can also be made so that the final construct has the desired activity. These variants generate DNA encoding the variants and then express the DNA in recombinant cell culture, thereby making site specific mutagenesis of the nucleotides in the DNA encoding the peptide molecule (Adelman et al., DNA 2: 183 (1983). ) Is illustrated. Typically, the variants exhibit the same qualitative biological activity as the wild type Isd polypeptide. It is known in the art that all possible single amino acid substitutions of known polypeptides can also be synthesized (Geysen et al., Proc. Nat. Acad. Sci. (USA) 18: 3998-4002 (1984)). Although the effects of various substitutions are not always added, two advantageous substitutions at a single residue position in an Isd polypeptide or a single neutral substitution can be safely combined without losing any Isd protein activity. Methods of making degraded polypeptides are described in Rutter, U.S. Pat. No. 5,010,175; 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 WO86 / 00991. In the design of substitution methods, those skilled in the art will determine which residues are to be changed and which amino acids or amino acid classes are appropriate substitutions. Those skilled in the art will also consult the study of sequence variation in family or naturally occurring homologous proteins. Certain amino acid substitutions are more frequently tolerated than other amino acid substitutions, and they are often associated with similarities in size and charge between the original amino acid and its replacement. Insertion or deletion of amino acids can also be made as described above. Substitutions are preferably conservative, which is described in Schulz et al., Principle of Protein Structure (Springer- Verlag, New York (1978)), which is incorporated herein by reference in its entirety; and Creighton, Proteins: Structure and Molecular Properties (W. H. Freeman & Co., San Francisco (1983)).

A “chemical derivative” of an Isd polypeptide may contain additional chemical moieties that are not usually part of the IsdA, IsdB or IsdC amino acid sequence. Such chemical modifications can be introduced into the Isd polypeptide by reacting the target amino acid residue of the polypeptide with an organic inducer capable of reacting with the selected side chain or terminal residue. The amino terminal residue can be reacted with succinic anhydride or other carboxylic anhydride. Other suitable reagents for deriving alpha-amino containing moieties include amido-esters such as methyl picolinimide; Pyridoxal phosphate; Pyridoxal; Chloroborohydride; Trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; And transaminase-catalase reacted with glyoxylate. In particular, specific modifications of the tyrosyl residues themselves regarding the introduction of spectroscopic labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane have been extensively studied. Most often, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Carboxyl side groups, such as aspartyl or glutamyl, have 1-cyclohexyl-3- [2-morpholinyl- (4-ethyl)] carbodiimide or 1-ethyl-3- (4-azonia-4, And may optionally be modified by reaction with a carbodiimide (R'NCN-R ') such as 4-dimethylpentyl) carbodiimide. Moreover, arpartyl and glutamyl residues can be converted to arparaginyl and glutamiminyl residues by reaction with ammonium ions.

A "vector" is a self-replicating nucleic acid molecule that delivers an inserted nucleic acid molecule between host cells and / or between host cells. The term includes vectors that primarily function to insert nucleic acid molecules into cells, replication vectors that function primarily for replication of nucleic acids, and expression vectors that function for transcription and / or translation of DNA or RNA. Also included are vectors that provide one or more of the above functions. As used herein, an “expression vector” is defined as a polynucleotide that can be transcribed and translated into polypeptide (s) when introduced into a suitable host cell. An "expression system" generally includes a suitable host cell composed of an expression vector that can function to provide the desired expression product.

3. Isd  gene

The three genes isdA , isdB and isdC of the Isd locus encode cell surface proteins that are covalently attached to the Staphylococcus cell wall. 1-3 provide nucleic acid sequences of isdA (SEQ ID NO: 1), isdB (SEQ ID NO: 4) and isdC (SEQ ID NO: 7).

The nucleic acid of the invention may consist of or consist essentially of any of the isd nucleotide sequences described herein. The other nucleic acid also comprises, consists of, or consists essentially of a nucleotide sequence that is at least about 70%, 80%, 90%, 95%, 98% or 99% identical or homologous to the isd gene. Substantially homologous sequences can be identified using stringent hybridization conditions.

Isolated nucleic acids that differ from the nucleic acids of the invention due to degradation in the genetic code are also within the scope of the invention. For example, many nucleic acids are designed by one or more triplets. Codons specifying the same amino acid, or synonyms (eg, CAU and CAC are synonyms for histidine) can 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 result in changes in the amino acid sequence of the polypeptides of the invention. Those skilled in the art will appreciate that such alterations in one or more nucleotides (less than 1% to about 3 or 5% or possibly more nucleotides) of a nucleic acid encoding a particular protein of the invention may be present in a given species due to natural allelic variation. I will understand. Any and all such nucleotide variations and the resulting amino acid polymorphisms are within the scope of this invention.

Nucleic acids encoding proteins having an amino acid sequence evolutionarily related to a polypeptide disclosed herein are provided wherein “evolutionally related” refers to different amino acids naturally occurring (eg, by allelic variation or differential splicing). As well as proteins with sequences, it refers to mutant variants of the proteins of the invention derived, for example, by combinatorial mutagenesis.

Also provided are fragments of a polynucleotide of the invention that encode a biologically active portion of a subject polypeptide. As used herein, a fragment of a nucleic acid encoding an active fragment of a polypeptide disclosed herein is a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of the polypeptide of the invention, wherein the fragment of the full length Isd protein disclosed herein By a nucleotide sequence that encodes a given polypeptide that retains at least a portion of its biological activity, or alternatively functions as a modulator of the biological activity of the full-length protein. For example, the fragment includes a polypeptide containing the domain of the full-length protein from which the polypeptide mediates the interaction of the protein with another molecule (eg, polypeptide, DNA, RNA, etc.).

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

Nucleic acid sequences encoding Isd polypeptides provided herein can be obtained from mRNA or genomic DNA from any organism according to the protocols disclosed herein and protocols known to those skilled in the art. CDNA encoding the polypeptides of the invention can be obtained, for example, by isolating whole mRNA from organisms such as bacteria, viruses, mammals and the like. Double stranded cDNA can then be prepared from whole mRNA and subsequently inserted into a suitable plasmid or bacteriophage vector using any of a number of known techniques. Genes encoding polypeptides of the invention can also be cloned using polymerase chain reaction techniques established in accordance with the nucleotide sequence information provided herein. In one aspect, a method of amplifying a nucleic acid or fragment thereof of the present invention comprises the steps of (a) providing a pair of single standard oligonucleotides each of at least 8 nucleotides in length and complementary to the nucleic acid sequence of the present invention, wherein A sequence complementary to an oligonucleotide is separated by at least 10 nucleotides; (b) contacting the oligonucleotide with a sample comprising the nucleic acid of the invention under conditions that allow for amplification of the nucleic acid by allowing amplification of the region located between the pair of oligonucleotides.

Isd proteins can be expressed from methods of producing recombinant vectors, host cells containing the recombinant vectors, and encoded Staphylococcus aureus polypeptides. Suitable vectors can be introduced into host cells using well known techniques such as infection, transduction, transfection, transfection, electroporation and transformation. The vector can be, for example, a phage, plasmid, virus or retroviral vector. Retroviral vectors may be capable of performing replication or may be replication defective. In the latter case, virus propagation will generally occur only by complementing the host cell.

The vector may contain markers selective for propagation in the host. Generally, plasmid vectors are introduced into precipitates, such as calcium phosphate precipitates, or complexes with charged lipids. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into a host cell.

Preferred vectors include cis-functional regulatory regions for the polynucleotides of interest. Suitable trans-acting factors can be supplied by the host, by the complementing vector, or by the vector itself upon introduction into the host.

In certain embodiments, the vector provides specific expression that may be inducible and / or cell-type specific. Particularly preferred among these vectors are those that can be induced by easy environmental factors such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, baculoviruses, papovaviruses, vaccinia viruses, adenoviruses. , Vectors derived from viruses such as foul fox virus, false madman viruses, and retroviruses, and vectors derived from combinations thereof such as cosmids and phagemids.

DNA insertion should be operably linked to suitable promoters, to mention a few, phage lambda PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters and promoters of retroviral LTR. Other suitable promoters will be known to those skilled in the art. The expression construct will further contain a site for transcription initiation, termination and a ribosomal binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably comprise a translation initiation site in the initiation and termination codons (UAA, UGA or UAG) suitably positioned at the ends of the polypeptide to be translated.

As described, the expression vector will preferably comprise one or more optional markers. Such markers include neomycin resistance for dihydrofolate reductase or eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of appropriate hosts include Escherichia coli (E.coli), Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) bacteria cells such as cell; Mycelium 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, but is not limited thereto. Suitable culture media and conditions for the host cells described above are known in the art.

Preferred vectors for use in bacteria include pQE70, pQE60 and pQE9, pQE10 available from Qiagen; PBS vectors, phagescript vectors, bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; Vectors of the pET series available from Novagen; And ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred eukaryotic vectors include 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 promoters, lambda PR and PL promoters, trp promoters and xyI / tet chimeric promoters. Suitable eukaryotic promoters include promoters of retroviral LTRs such as CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoter of Loose Sarcoma Virus (RSV), and mouse metallothionein-I promoter. There is a metallothionein promoter.

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

Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, typically from 10 to 300 nucleotides, that act to increase the transcriptional activity of the promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer located on the late side of the origin of replication in 100 to 270 nucleotides, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers.

Secreted signals suitable for expressed polypeptides, eg, amino acid sequence KDEL (SEQ ID NO: 33), can be included to secrete the translated polypeptide into the lumen, periplasmic space or extracellular environment of the endoplasmic reticulum. The signal may be endogenous to the polypeptide or may be a heterologous signal.

The coding sequence for the polypeptide of interest can be incorporated as part of a fusion gene comprising a nucleotide sequence encoding a different polypeptide. The invention encodes a fusion protein comprising a heterologous polypeptide by considering an isolated nucleic acid comprising the nucleic acid of the invention and one or more heterologous sequences encoding a heterologous peptide bound in the frame to the nucleotide sequence of the nucleic acid of the invention. Heterologous polypeptides may be fused to (a) the C-terminus of the polypeptide encoded by the nucleic acid of the invention, (b) the N-terminus of the polypeptide, or (c) the C-terminus and N-terminus of the polypeptide. In certain instances, the heterologous sequence encodes a polypeptide that allows for detection, isolation, solubilization and / or stabilization of the polypeptide fused thereto. In still other embodiments, the heterologous sequence is a poly-His tag, myc, HA, GST, Protein A, Protein G, chamodulin-binding peptide, thioredoxin, maltose-binding protein, poly arginine, poly His-Asp And a polypeptide selected from the group consisting of FLAG, a portion of an immunoglobulin protein, and a transcytotic peptide.

Fusion expression systems may be useful where it is desirable to produce immunogenic fragments of the polypeptides of the invention. For example, the VP6 capsid protein of rotavirus may be used as an immunological carrier protein for a portion of a polypeptide in the form of a monomer or virus particle. The nucleic acid sequence corresponding to the portion of the polypeptide of the invention from which the antibody is produced is a coding sequence for a later vaccinia virus structural protein to produce a series of recombinant viruses that express a fusion protein comprising a portion of the protein as part of the virion. It may be incorporated into a fusion gene construct comprising. Hepatitis B surface antigen can also be used for this role. Similarly, immunogenicity can be improved by generating fusion proteins containing portions of the polypeptides of the invention and chimeric constructs encoding the poliovirus capsid proteins (see, 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).

Fusion proteins can facilitate the expression and / or purification of the protein. For example, a polypeptide of the invention can be produced as a glutathione-S-transferase (GST) fusion protein. Such GST fusion proteins can be used to simplify the purification of polypeptides of the invention, such as by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology , eds. Ausubel et al. (NY: John) Wiley & Sons, 1991). In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly- (His) / entokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, comprises an affinity chromatography with a Ni ²⁺ metal resin. Purification of the fusion protein expressed by chromatography can be allowed. Purification leader sequences may then be subsequently removed with enterokinase to provide purified proteins (see, eg, Hochuli et al. (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: 8972).

Techniques for making fusion genes are well known. Essentially applying blunt-ended or staggered-ended ends for ligation, restriction enzyme digestion to provide suitable ends, filling of suitable adherent ends, alkaline phosphatase treatment to avoid undesired binding and enzymatic ligation Binding of various DNA fragments encoding different polypeptide sequences is performed according to conventional techniques. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that cause complementary overhangs between two consecutive gene fragments that can subsequently be annealed to generate chimeric gene sequences (see, eg, Current Protocols in Molecular Biology , eds.Ausubel et al., John Wiley & Sons: 1992).

In another embodiment, the nucleic acids of the invention are placed on a solid surface, including plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubing, containers, capillaries, pads, slices, and the like. Can be fixed. Nucleic acids of the invention can be immobilized on a chip as part of an array. The array may comprise one or more polynucleotides of the invention described herein. In one embodiment, the chip comprises one or more polynucleotides of the invention as part of an array of nucleotide sequences.

Another aspect relates to the use of a nucleic acid of the invention in an "antisense therapy". As used herein, antisense therapies refer to administration or in situ administration of an oligonucleotide probe or derivative thereof that specifically hybridizes to cellular mRNA and / or genomic DNA encoding one of the polypeptides of the invention or otherwise binds under cellular conditions. By inhibiting the expression of the polypeptide, for example by inhibiting transcription and / or translation. Binding may be by conventional base pair complementarity, or through specific interactions in the major groove of the double helix, for example when binding to a DNA double strand. In general, antisense therapies refer to the range of techniques generally applicable in the art and include any therapy that relies on specific binding to oligonucleotide sequences.

Oligonucleotides may be conjugated to another molecule such as a peptide, hybridization triggered crosslinker vehicle, hybridization triggered cleavage agent, and the like. The antisense molecule may be a "peptide nucleic acid" (PNA). PNA means an antisense molecule or anti-genetic agent comprising oligonucleotides of at least about 5 nucleotides in length bound to the peptide backbone of an amino acid residue ending in lysine. Terminal lysine imparts solubility to the composition. PNAs preferentially bind to complementary single standard DNA or RNA and stop transcript stretches and can be PEGylated to extend their lifespan in cells.

Antisense constructs of the invention may be delivered as expression plasmids that, when transcribed into cells, produce RNA that is complementary to at least a unique portion of the mRNA encoding a polypeptide of the invention. Alternatively, the antisense construct may be an oligonucleotide probe that is generated ex vivo and, when introduced into a cell, hybridizes with the mRNA and / or genomic sequence encoding the polypeptide of the invention to cause inhibition of expression. The oligonucleotide probe may be a modified oligonucleotide that is resistant to endogenous nucleases such as exonucleases and / or endonucleases and therefore stable in vivo. Exemplary nucleic acid molecules used as antisense oligonucleotides include phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see US Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). In addition, general approaches to construct oligomers useful in antisense therapies are described, for example, in van der Krol et al. (1998) Biotechniques 6: 958-976; And Stein et al. (1998) Cancer Res 48: 2659-2668.

In a further aspect, a double stranded small interfering RNA (siRNA), and a method of administering the same are provided. siRNA reduces or blocks gene expression. Without wishing to be bound by theory, it is generally contemplated that siRNAs inhibit gene expression by mediating sequence specific mRNA degradation. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing process initiated by double stranded RNA (dsRNA) that is homologous to sequences for silent genes, particularly in animals and plants (Elbashir et al. Nature 2001; 411 (6836). ): 494-8). Thus, it is understood that siRNAs and long dsRNAs having substantial sequence identity with all or a portion of the polynucleotides of the present invention can be used to inhibit expression of the nucleic acids of the present invention.

Alternatively, siRNAs that reduce or block the expression of Isd polypeptides disclosed herein can be determined by testing multiple siRNA constructs for a target gene. Such siRNAs for target genes can be chemically synthesized. The nucleotide sequence of the individual RNA strand is selected such that the strand has a region of complementarity for the target gene to be inhibited (ie, the nucleotide sequence complementary to the region of the mRNA transcript formed during expression of the target gene, or processing thereof). Product, or regions of (+) strand virus). Synthesizing the RNA strand can include solid-phase synthesis, where the ends and ends of the individual nucleotides are linked through the formation of internucleotide nucleotide 3'-5 'phosphodiester bonds in successive synthetic cycles.

Provided herein is an siRNA molecule comprising a nucleotide sequence consisting essentially of the isd nucleic acid sequences disclosed herein. The siRNA molecule may comprise two strands, each strand comprising at least essentially a nucleotide sequence that is complementary to each other, one of which is essentially identical to the sequence of the target gene. Sequences that essentially match the sequence of the target gene are referred to as "sense target sequences" and here the sequences that are essentially complementary are referred to as "antisense target sequences" of siRNA. The sense and antisense target sequences may comprise about 15 to about 30 consecutive nucleotides; About 19 to about 25 consecutive nucleotides; About 19 to about 23 consecutive nucleotides or about 19, 20, 21, 22, or 23 nucleotides in length. The length of the sense and antisense sequences is determined such that siRNAs with sense and antisense target sequences of this length preferably do not significantly induce host interferon responses and can inhibit expression of the target gene.

siRNA target sequences can be predicted using any algorithm provided on mmcmanus' world wide web with the extension web.mit.edu/mmcmanus/www/home1.2files/siRNAS.

The sense target sequence may be essentially 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 complement thereof. It may also be essentially complementary to the region comprising the boundary between the two genetic 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 can be based on the nucleotide base composition. With regard to accessibility of target nucleic acids by siRNAs, this is described, for example, in Lee et al. (2002) Nature Biotech. 19: 500. The approach involves the use of oligonucleotides complementary to the target nucleic acid as probes, for example, to determine substrate accessibility in cell extracts. After forming the double strand with the oligonucleotide probe, the substrate is sensitive to RNase H. Thus, for example, the degree of sensitivity of RNase H to a given probe, as determined by PCR, reflects the accessibility of the selected site and is an estimate of how well the corresponding siRNA will perform in inhibiting transcription from the target gene. Can be Algorithms for identifying antisense oligonucleotides may be used to identify primers for polymerase chain reaction (PCR) assays or to identify first target sequences.

The sense and antisense target sequences are preferably sufficiently complementary so that siRNAs comprising both sequences can inhibit expression of the target gene, ie mediate RNA interference. For example, the sequences can be sufficiently complementary, such as to hybridize under desired conditions in a cell. Thus, the sense and antisense target sequences are at least about 95%, 97%, 98%, 99% or 100% identical, and may differ by at least 5, 4, 3, 2, 1 or 0 nucleotides, for example.

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

siRNA may also include sequences in addition to sense and antisense sequences. For example, an siRNA can be an RNA double strand consisting of two strands of RNA, where one or more strands have a 3 'overhang. The other strand may be blunt-ended or have an overhang. In embodiments in which the RNA molecule is double stranded and both strands comprise an overhang, the length of the overhang may be the same or different for each strand. In certain embodiments, siRNAs comprise sense and antisense sequences, each of which is on one RNA strand and is paired with from about 1 to about 3, in particular about 2 nucleotides, at both 3 'ends of the RNA It consists of about 19-25 nucleotides with overhangs. To further enhance the stability of the RNAs of the invention, 3 'overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs, such as uridine 2 nucleotide 3 'overhangs by 2'-deoxythymidine, is allowed and this does not affect the efficacy of RNAi. The absence of 2 ′ hydroxyl can significantly improve nuclease resistance of overhangs, at least in tissue culture medium. The RNA strand of the siRNA may have 5 'phosphate and 3' hydroxyl groups.

In one embodiment, siRNA molecules comprise two strands of RNA forming a double strand. In another embodiment, the siRNA molecule consists of one RNA strand forming a hairpin loop, wherein the sense and antisense target sequences are hybridized and the sequence between the two target sequences essentially forms a loop of hairpin structure. to be. The spacer sequence may be any combination of nucleotides and may form a hairpin structure of any length when two complementary oligonucleotides are joined by a spacer having such sequence, wherein at least a portion of the spacer is a closed end of the hairpin To form a loop. For example, the spacer sequence may comprise 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 can be any sequence as long as it does not interfere with the formation of the hairpin structure. In particular, it is preferred that the spacer sequence is not a sequence having any significant homology to the first or second target sequence, as this may interfere with the formation of the hairpin structure. The spacer sequence is also preferably not similar to other sequences, such as the genomic sequence of the cell into which the nucleic acid sequence is to be introduced, since this may lead to undesirable consequences in the cell.

Those skilled in the art will understand that when referring to nucleic acids such as RNA, the RNA may comprise or consist of naturally occurring nucleotides or nucleotide derivatives that provide greater stability to such nucleic acids, for example. Any derivative is acceptable if the nucleic acid can act in the desired format. For example, siRNA may include nucleotide derivatives if the siRNA can still inhibit expression of the target gene.

For example, siRNAs may include one or more modified bases and / or backbones that have been modified for stability or for other reasons. For example, the phosphodiester bonds of native RNA can be modified to include one or more of nitrogen or sulfur heteroatoms. Moreover, for example, siRNAs comprising a flaky base such as inosine or a modified base such as tritylated base may be used in the present invention. It will be appreciated that a wide variety of modifications can be made to RNA to accomplish many useful purposes known to those skilled in the art. As used herein, the term siRNA includes chemically, enzymatically or metabolically modified forms of siRNA when it is derived from an endogenous template.

There is no limit to the way in which siRNA can be synthesized. Thus, it can be synthesized in vitro or in vivo using manual and / or automated processes. In vitro synthesis can be chemical or enzymatic, for example, using cloned RNA polymerase (eg, T3, T7, SP6) for transcription of DNA (or cDNA) templates, or a mixture of both. siRNA can also be prepared by chemically synthesizing each of the two strands and hybridizing the two strands to form a double strand. In vivo, siRNAs can be synthesized using recombinant techniques well known in the art (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning; Volumes I and II ( DN Clover ed. 1985); Oligonucelotide 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); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (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 (Wu and Grossman, and Wu, eds., Respectively), Mayer and Walker, eds. (1987) , Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), Scopes (1987), Protein Purification: P rinciples and Practice, Second Edition (Springer-Verlag, N.Y.) and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell eds 1986). For example, bacterial cells can be transformed with an expression vector comprising a DNA template from which siRNA is derived.

If synthesized outside the cell, the siRNA can be purified prior to introduction into the cell. Purification may be by extraction with a solvent (such as resin / chloroform) or resin, precipitation (eg in ethanol), electrophoresis, chromatography or a combination thereof. However, purification may result in loss of siRNA and may be minimized or not performed at all. The siRNA may be dried for storage or dissolved in an aqueous solution which may include salts that promote annealing and / or stabilization of buffers or RNA strands.

The double stranded structure can be formed by a single self-complementary RNA strand or two separate complementary RNA strands.

It is known that mammalian cells can respond to extracellular siRNA and have a transport mechanism for dsRNA (Asher et al. (1969) Nature 223 715-717). Thus, siRNA can be administered extracellularly or orally to the mammalian circulation into the cosmic, interstitial space. Oral introduction methods include an engineered approach in which RNA is directly mixed with mammalian nutrients, as well as engineered to express species RNA used as nutrients, and then fed to diseased mammals. For example, food bacteria such as Lactococcus lactis can be transformed to generate dsRNA (see WO93 / 17117, WO97 / 14806). Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are sites where RNA can be injected.

RNA can be introduced intracellularly into cells. Physical methods of introducing nucleic acids may also be used in this regard. siRNA can be administered using the microinjection technique disclosed in Zernicka-Goetz et al. (1997) Development 124, 1133-1137 and Wianny et al. (1998) Chromosoma 107, 430-439.

Another physical method of introducing nucleic acids intracellularly is impact by particles covered by siRNA, such as gene gun techniques that immobilize siRNA on gold particles and fire directly at the winding site. Thus, the present invention provides the use of siRNA in a gene gun to inhibit expression of a target gene. In addition, compositions suitable for gene total therapy comprising siRNA and gold particles are provided. An alternative physical method is the electroporation of the cell membrane in the presence of siRNA. This method allows large scale RNAi. Other methods known in the art of introducing nucleic acids into cells can be used, such as, for example, lipid-mediated carrier delivery, chemical-mediated delivery such as calcium phosphate, and the like. siRNAs may be introduced with components that perform one or more of the following activities: improving RNA uptake by cells, promoting double-annealing, stabilizing annealed strands or increasing inhibition of target genes.

Any known gene therapy can be used to administer RNA. Viral constructs packaged in viral particles will achieve both efficient introduction of expression constructs into cells and transcription of siRNA encoded by the expression constructs. Thus, siRNA may be provided inside the cell. Vectors comprising nucleic acids encoding one or two siRNA molecules, such as expression vectors, can be used for this purpose. The nucleic acid may further comprise an antisense sequence that is essentially complementary to the sense target sequence. The nucleic acid may further comprise a spacer sequence between the sense and antisense target sequences. The nucleic acid may further include promoters that direct expression of sense and antisense sequences in the cell, such as RNA polymerase II or III promoters and transcription termination signals. The sequences can be operably linked.

In one embodiment, the nucleic acid comprises an RNA coding region (eg, sense or antisense target sequence) operably linked to an RNA polymerase III promoter. Immediately following the RNA coding region may be a pol III terminator sequence that directs termination of RNA synthesis by pol III. The pol III terminator sequence generally has four or more contiguous thymidine ("T") residues. In a preferred embodiment, only two to three uridine ("U") residues are encoded by using a cluster of five consecutive T residues as terminators to interrupt pol III transcription at the second or third T of the DNA template. Is added to the 3 'end of the sequence. Various pol III promoters can be used in the present invention and include, for example, promoter fragments derived from H1 RNA genes or U6 snRNA genes of human or mouse origin or any other species. The pol III promoter can also be modified / engineered to incorporate other desirable properties such as the ability to be introduced by small chemical molecules or to be incorporated in a tissue-specific manner. For example, in one embodiment, the promoter can be activated by tetracycline. In another embodiment, the promoter may be activated by IPTG (lacI system).

siRNA can be provided to a cell by transforming the cell with two nucleic acids, such as a vector, each nucleic acid comprising an expression cassette, each expression cassette being a promoter, an RNA coding sequence (one sense target sequence and the other Antisense target sequence) and termination signal. Alternatively, a single nucleic acid may comprise two such expression cassettes. In another embodiment, the nucleic acid encodes a single stranded RNA comprising a sense target sequence bound to a spacer bound to an antisense target sequence. The nucleic acid may be present in a vector, such as an expression vector, such as a eukaryotic expression vector, which allows expression of intracellular sense and antisense target sequences introduced into the cell.

Vectors for generating siRNAs are described in 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. Vectors are also commercially available. For example, pSilencer is commercially available from Gene Therapy Systems, Inc. and pSUPER RNAi system is commercially available from Oligoengine.

Also provided herein are compositions comprising nucleic acids encoding one or more siRNAs or RNA coding regions of siRNAs. The composition is a pharmaceutical composition and includes a pharmaceutically acceptable carrier. The composition may also be provided as a device for administering the composition in a cell or subject. For example, the composition may be present as a syringe or stent. The composition may also include an agent that facilitates entry of the siRNA or nucleic acid into the cell.

In general, oligonucleotides can be synthesized using protocols known in the art, see, eg, Carurthers et al., Methods in Enzymology (1992) 211: 3-19; 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 emd, Biotechnol. Bioeng . (1998) 61: 33-45; And Brennan, US Pat. No. 6,001,311; Each of which is incorporated herein by reference in its entirety. In general, the synthesis of oligonucleotides includes conventional nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5 'end and phosphoramidite at the 3' end. In a non-limiting example, small scale synthesis is performed by Applied Biosystems, Inc. Ribonucleoside phosphoramidite sold by ChemGenes Corporation (Ashland Technology Center, 200 Homer Avenue, Ashland, MA 01721, USA) on an Expedite 8909 RNA synthesizer sold in Weiterstadt, Germany. Alternatively, the synthesis can be performed on a 96-well plate synthesizer, such as a device produced by Protogene (Palo Alto, Calif., USA), or by Usman et al . , 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, each of which is incorporated herein by reference in its entirety.

The nucleic acid molecules of the present invention can be synthesized separately and after synthesis, dsRNAs can be synthesized, for example, by 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 hybridization following synthesis and / or deprotection. It can synthesize | combine by fire. Nucleic acid molecules can be purified by gel electrophoresis using conventional methods, or purified by high pressure liquid chromatography (HPLC; see Wincott et al., Detailed above, the entire contents of which are incorporated herein by reference) and reproduced in water. Can be turbid.

In another embodiment, the level of a particular mRNA or polypeptide of the cell is reduced by introducing into the cell or nucleic acid encoding it that it is a ribozyme. In addition, ribozyme molecules designed to catalytically cleave mRNA transcripts can be introduced into or expressed in cells to inhibit expression of gene Y (see, eg, Sarver et al., 1990, Science 247: 1222-1225 and US). Patent No. 5,093,246). One commonly used ribozyme motif is the hammerhead, with the substrate sequence requirements for this being minimal. The design of the hammerhead ribozyme is described in Usman et al . , Current Opin. Struct. Biol . (1996) 6: 527-533. Usman also discussed the therapeutic uses of ribozymes. Ribozymes are 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 Patent No. 5,254,678 may be prepared or used as disclosed. Ribozyme cleavage of HIV-1 RNA is disclosed in US Pat. No. 5,144,019; Methods for cleaving RNA using ribozymes are disclosed in US Pat. No. 5,116,742; Methods for increasing the specificity of ribozymes are described in US Pat. No. 5,225,337 and Koizumi et al., Nucleic Acid Res . (1989) 17 : 7059-7071. The preparation and use of ribozyme fragments in hammerhead structures is also described in Koizumi et al., Nucleic Acids Res . (1989) 17 : 7059-7071. Preparation and use of ribozyme fragments in hairpin structures are described in Howrira and Burke, Nucleic Acids Res . (1992) 20: 2835. Ribozymes are also described in Daubendiek and Kool, Nat. Biotechnol . (1997) 15 (3) : 273-277 ) .

Gene expression can be reduced by targeting deoxyribonucleotide sequences that are complementary to the regulatory regions of the target gene (ie, gene promoters and / or enhancers) to form triple helix structures that inhibit transcription of the gene in target cells of the body. (See, generally, 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, an RNA aptamer may be introduced into or expressed in a cell. RNA aptamers are RNA ligands specific for proteins such as Tat and Rev RNA (Good et al. (1997) Gene Therapy 4: 45-54) and can specifically inhibit their translation.

4. Isd  Polypeptide

Staphylococcus aureus polypeptides comprising IsdA (SEQ ID NO: 3), IsdB (SEQ ID NO: 6), and IsdC (SEQ ID NO: 9) (FIG. 1-3) described herein are naturally purified. Products, products of chemical synthesis processes, and products produced by recombinant techniques from prokaryotic or eukaryotic host cells, including, for example, bacterial, enzyme, higher plant, insect, and mammalian cells. In certain embodiments, the polypeptides described herein inhibit the function of an Isd polypeptide.

The polypeptide may consist of or consist essentially of any of the amino acid sequences described herein. The other polypeptide may also comprise, consist of, or consist essentially of an amino acid sequence that is at least about 70%, 80%, 90%, 95%, 98% or 99% identical or homologous to the Isd polypeptide. For example, polypeptides that differ in sequence from the naturally occurring Isd protein and from about 1, 2, 3, 4, 5 or more amino acids may be contemplated. The difference may be a substitution such as a conservative substitution, deletion or addition. Such differences are preferably in areas that are not significantly conserved between different species. Such regions can be identified by aligning amino acid sequences from various species. Such amino acids may be substituted, for example, with those found in another species. Other amino acids that may be substituted, inserted or deleted at these or other positions can be identified by mutagenesis studies coupled with biological assays.

Proteins may also include one or more non-natural amino acids. For example, atypical amino acids or chemical amino acid analogs can be incorporated into the protein as substitutions or additions. Atypical amino acids include D-isomers of conventional amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexane Acids, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, citric acid, t-butylglycine, t-butylalanine, phenyl Designer amino acids such as glycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, beta-methyl amino acids, carpa-methyl amino acids, nalpa-methyl amino acids, and amino acid analogs generally Including but not limited to. Furthermore, the amino acid may be D (priority) or L (lefter).

Other proteins encompassed herein include proteins comprising modified amino acids. Representative proteins are derivative proteins that can be modified by any similar process having one or more biological functions of the protein from glycosylation, PEGylation, phosphorylation or derivatization thereof.

Proteins can be used as substantially pure preparations, for example where at least about 90% of the protein in the preparation is the desired protein.

Staphylococcus aureus polypeptides can be recovered and purified from recombinant cell culture by known methods such as ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography Hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography and high performance liquid chromatography (“HPLC”) can be used for purification. Proteins can be used as substantially pure preparations, for example at least about 90% or more of the proteins are the desired proteins. Compositions comprising at least about 50%, 60%, 70%, or 80% or more of the desired protein may also be used.

Proteins may or may not be denatured and, as a result, may or may not aggregate. Proteins can be denatured according to methods known in the art.

In certain embodiments, Isd polypeptides described herein can be fusion proteins containing domains that increase their solubility and / or promote their purification, identification, detection, and / or structural characterization. Representative domains include, for example, glutathione S-transferase (GST), protein A, protein G, chamodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His -Asp or FLAG fusion proteins and tags. Additionally representative domains include domains that alter protein position in vivo, such as signal peptides, type III secretion system-target peptides, transcytotic domains, nuclear position signals, and the like. In various embodiments, polypeptides of the invention may comprise one or more heterologous fusions. The polypeptide may contain multiple copies of the same fusion domain or may contain fusions for two or more different domains. Fusions can be generated at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or both at the N- and C-terminus of the polypeptide. It also includes a linker sequence between the polypeptide of the invention and the fusion domain to facilitate structuring of the fusion protein or to optimize protein expression or structural restriction of the fusion protein within the scope of the invention. In other embodiments, the polypeptide may be structured to contain a protease cleavage site between the fusion polypeptide and the polypeptide of the invention to remove the tag after protein expression or accordingly. Examples of suitable endoproteases include, for example, Factor Xa and TEV proteases. Proteins can also be fused to signal sequences. For example, when produced recombinantly, the nucleic acid encoding the peptide is linked at its 5 'end to the signal sequence so that the protein is secreted from the cell.

In certain embodiments, polypeptides of the invention can be synthesized chemically, by ribosomes in cell-free systems, or by ribosomes intracellularly. Chemical synthesis of polypeptides of the invention can be accomplished in several ways known in the art, for example, stepwise solid phase synthesis, semisynthetic through morphologically assisted re- ligation of peptide fragments, enzymatic enzymatic synthesis of cloned or synthetic peptide segments. Ligation, and chemical ligation can be performed. Natural chemical ligation utilizes chemoselective reactions of two unprotected peptide segments to produce transient thioester-linked intermediates. The transient thioester-linked intermediates then spontaneously rearrange to provide full length ligation products with natural peptide bonds at the ligation sites. Full length ligation products are chemically identical to proteins produced by cell-free synthesis. Full-length ligation products can be refolded and / or oxidized as allowed to form natural disulfide-containing protein molecules (see US Pat. Nos. 6,184,344 and 6,174,530; And Muir et al., Curl-. 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., Hit. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; Rajarathnam et al., Science (1994): vol. 264, p 90; R. E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and the Development of New therapeutics and Vaccines, J. B. 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., Chen. Phalan. Bull. (1985) 33: 184.

In certain embodiments, it may be advantageous to provide homologues of naturally occurring or experimentally derived polypeptides of the invention. Such homologues can act in limited doses as modulators to enhance or inhibit a subset of biological activity in the form of naturally occurring polypeptides. Thus, specific biological effects can be induced by treatment with limited function homologues and have fewer side effects compared to treatment with agonists or antagonists associated with all biological activities of the polypeptides of the invention. For example, antagonist homologues can be generated that interfere with the ability of the wild-type polypeptides of the invention to bind specific proteins but substantially do not interfere with the formation of complexes between the natural polypeptide and other cellular proteins.

The polypeptide may be derived from the full length polypeptide of the present invention. Isolated peptidyl moieties of such polypeptides can be obtained by screening polypeptides produced recombinantly from corresponding fragments of nucleic acids encoding such polypeptides. The fragments can also be chemically synthesized using techniques known in the art, such as conventional Merfield solid phase f-Moc or t-Boc chemistry. For example, a protein may be optionally split into fragments of the desired length without overlapping the fragments, or may be split to overlap fragments of the desired length. Such fragments may be produced (by recombination or chemical synthesis) and may be tested to identify peptidyl fragments thereof having the desired properties, eg, a dose that acts as a modulator of the polypeptides of the invention. In the described embodiments, the peptidyl moiety in the protein of the invention can be tested by expression as a thioredoxin fusion protein, for example for binding activity and inhibitory activity, each of which is an isolated of the protein of the invention Fragments [see, eg, US Pat. Nos. 5,270,181 and 5,292,646; And PCT Publication WO94 / 02502.

In other embodiments, truncated polypeptides can be prepared. Truncated polypeptides have 1 to 20 or more amino acid residues removed from each or both of the N- and C-terminus. Such truncated polypeptides can demonstrate expression, purification or characterization more modifiably than full length polypeptides. For example, a truncated polypeptide can be demonstrated to be more modifiable to crystallization than full length polypeptide to obtain high performance diffraction crystals or to obtain HSQC spectra with high sensitivity peaks and minimum overlap peaks. In addition, the use of truncated polypeptides can identify stable and active domains of full-length polypeptides that may be more modifiable in characterization.

In addition, the present invention is intended to modify the structure of the polypeptide of the present invention for the purpose of improving the therapeutic or prophylactic efficacy, or stability (eg, in vitro storage, resistance to proteolytic degradation in vivo, etc.). Can be. Such modified polypeptides, when designed to have one or more activities of naturally occurring forms of the protein, are considered as "functional equivalents" of the polypeptides described in more detail herein. Such modified polypeptides may be produced, for example, by amino acid substitutions, deletions, or additions, which substitutions may be made, in whole or in part, by conservative amino acid substitutions.

For example, isolated conservative amino acid substitutions such as replacement of leucine with isoleucine or valine, replacement of aspartate with glutamate, replacement of threonine with serine will not have a major effect on the biological activity of the resulting molecule. It is reasonable to expect. Whether a change in the amino acid sequence of a polypeptide results in a functional homologue can be readily determined by assessing the ability of the variant polypeptide to produce a response similar to a wild type protein. Polypeptides in which one or more replacements are performed can be readily tested in the same manner.

Methods of generating a set of combinatorial and truncation mutations of the polypeptides of the invention are provided, which are particularly useful for identifying potential variant sequences (eg, homologues). The purpose of screening such combinatorial libraries is, for example, to produce homologues that can modulate the activity of the polypeptides of the invention or, alternatively, have novel activities as a whole. Combinationally derived homologues can be produced with selective efficacy compared to naturally occurring proteins. Such homologues can be used in the development of therapeutic agents.

Likewise, mutagenesis can result in homologues with very different intracellular half-lives compared to the corresponding wild type protein. For example, the modified protein may be more or less stable against proteolytic degradation or other cellular processes result in the destruction of the protein or other inactivation. Such homologues, and the genes encoding them, can be used to alter protein expression by controlling the half-life of the protein. As above, such proteins can be used for the development of therapeutic agents or therapies.

In a similar manner, protein homologs can be generated by this combinatorial method and act as antagonists, which can interfere with the activity of the corresponding wild type protein.

In an exemplary embodiment of the method, the amino acid sequence for the population of protein homologues is preferably arranged to promote the highest homology. Populations of such a variation may include, for example, homologs from one or more species, or homologs from species that are the same species but differ from the mutation. The amino acids appearing at each position of the aligned sequence are selected to produce a degenerate set of combinatorial sequences. In certain embodiments, the combinatorial library is prepared in the manner of a degenerate library of genes encoding a library of polypeptides, each comprising at least a portion of a potential protein sequence. For example, a mixture of synthetic oligonucleotides is enzymatically ligated to a gene sequence such that the degenerate set of potential nucleotide sequences is expressed as an individual polypeptide or alternatively as a larger set of fusion proteins (eg phage display). To be possible.

There are many ways in which libraries of potential homologues can be generated from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, and the synthetic genes can then be ligated to the appropriate vector for expression. One purpose of the degenerate set of genes is to provide all sequences encoding the desired set of potential protein sequences in one mixture. Synthesis of degenerated oligonucleotides is well 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) Annie Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477]. Such techniques can be used in the evolution of other proteins as indicated (see, 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; US Patent Nos. 5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, protein homologs (both agonist and antagonist forms) are generated and for example alanine screening mutagenesis (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: 10832-10833; and Cunningham et al. (1989) Science 244: 1081-1085), linker screening 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 Mot Biol 7: 32-34), etc.) Screening using It can be isolated from the library. Specifically, linker scanning mutagenesis in combinatorial settings is the most preferred method for identifying truncated forms of bioactive proteins.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and cleavage and for screening cDNA libraries of gene products with specific properties. Such techniques will generally be able to be modified for rapid screening of gene libraries generated by combinatorial mutagenesis of protein homologues. The most widely used techniques for screening large gene libraries typically include cloning the gene library into a replaceable expression vector, transforming the appropriate cells into a library of obtained vectors, and detecting the desired activity in which the product is detected. Techniques for expressing combinatorial genes, under conditions that facilitate relatively easy separation of the vector encoding the gene of interest.

In the described embodiments of the screening assay, candidate combinatorial gene products appear on the surface of the cells, and the ability of a particular cell or virus particle to bind combinatorial gene products is detected by a "panning assay". For example, gene libraries can be cloned into genes for the surface membrane proteins of bacterial cells [Ladner et al., WO 88106630; Fuchs et al., (1991) Bio / Technology 9: 1370-1371; And Goward et al., (1992) TIBS 18: 136-140], wherein the resulting fusion proteins are detected by panning, for example using fluorescently labeled molecules that bind to cell surface proteins such as FITC-substrate. Calculate potential functional homologues. Cells can be visually examined and separated under fluorescence microscopy, or by fluorescence-activated cell sorter, if the morphology of the cell permits. Such methods can be used to identify substrates or other polypeptides that can interact with the polypeptides of the invention.

In a similar manner, the gene library can be expressed as a fusion protein on the surface of viral particles. For example, in filamentous phage systems, foreign peptide sequences can be expressed on the surface of an infectious phage to provide two advantages. First, because such phage can be applied to the affinity matrix at very high concentrations, many phages can be screened at once. Secondly, since each infective phage exhibits a combinatorial gene product on its surface, phage can be amplified by another round of infection when a particular phage is recovered from the affinity matrix in low yield. Almost the same group of E. coli filamentous phages M13, fd, and f1 are used almost entirely in phage display libraries, and either the phage gIII or gVIII coat proteins generate fusion proteins without disrupting the maximum packaging of viral particles. To Ladner et al., PCT Patent Publication No. WO 90/02909; Garrard et al., PCT Patent Publication No. 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 coat proteins may be used where appropriate.

The polypeptides described herein can be reduced to generate mimetic, eg, peptide or non-peptide agonists, which can similarly bind the original protein to other cell partners. Such mutagenesis techniques and thioredoxin systems as described above are also useful for mapping determinants of proteins specifically involved in protein-protein interactions with other proteins. For illustrative purposes, important residues of a protein involved in molecular recognition of the matrix protein can be determined and used to generate peptidomimetic that can bind to the matrix protein. The peptidomimetics can then be used as inhibitors of the wild type protein by including the important residues necessary to bind to and interact with the wild type protein, thus preventing the protein from interacting with the substrate. For example, scanning mutagenesis to map amino acid residues of a protein involved in binding the substrate polypeptide may be applied to produce a peptidomimetic compound that mimics such residues in binding to the substrate.

For example, derivatives of the Isd protein described herein can be chemically modified peptides and peptidomimetics. Peptidomimetics are compounds based on or derived from peptides and proteins. Peptidomimetics can be obtained by structural modification of known peptide sequences using non-natural amino acids, morphological inhibition, equivalence substitution, and the like. The peptidomimetic constitutes a continuum of structural space between the peptide and the non-peptide synthetic structure; Thus peptidomimetics may be useful for contributing to the translation of peptides into non-peptide compounds that exhibit drug action chromophores and have the activity of the parent peptide.

Moreover, mimetope of the peptide can be provided. Such peptidomimetics are not hydrolyzable (e.g. increased stability to proteases or other physiological conditions that degrade the corresponding peptide), and these features as increased specificity and / or efficacy to stimulate cell differentiation. It can have For illustrative purposes, the non-hydrolyzable peptide analogues of these residues are described by benzodiazepines in Fredinger et al., In Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988], Azepine [Huffman et al., In Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988], substituted gamma lactam rings [Garvey et al., In Peptides. Chemistry and Biology, G.R. 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 cores (Nagai et al., (1985) Tetrahedron Lett 26: 647; and Sato et al., (1986) J Chem Soc Perkin Trans 1: 1231, and β-aminoalcohols [Gordon et al., (1985) Biochem Biophys Res Common 126: 419; and Dann et al., (1986) Biochem Biophys Res Commun 134: 71.

In addition to the various side chain replacements that can be performed to generate peptidomimetic, the present technology specifically contemplates the use of morphologically inhibited mimetics of the peptide secondary structure. Many alternatives have been developed for the amide linkage of peptides. Alternatives for amino bonds that are often developed include the following (i) trans-olefins, (ii) fluoroalkenes, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.

Examples of substitutes are:

In addition, peptidomimetics based on more substantial modifications of the peptide backbone can be used. Peptidomimetics belonging to this class include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).

Examples of analogs are:

Moreover, the method of combinatorial compounding is suitable for developing novel peptidomimetics. For example, one embodiment of the so-called “peptide morphing” method focuses on the random generation of a library of peptide analogs that include a wide range of peptide bond substitutions.

In an exemplary embodiment, the peptidomimetic can be derived as a retro-inverso analog of the peptide. Such retro-inverso analogs are described in Sisto et al. As described in US Pat. No. 4,522,752, it may be prepared according to methods known in the art. Retro-inverso analogs can be generated, for example, as described in WO 00/01720. For example, it will be appreciated that mixed peptides may be generated that include some normal peptide bonds. As a general technique, the sites most susceptible to proteolysis are typically changed to less susceptible amide bonds that are selective for mimetic switching. The final product, or intermediate thereof, can be purified by HPLC.

Peptides may include one or more amino acids or all amino acids that are D stereoisomers. The other peptide may comprise one or more amino acids that are reversed. The amino acid to be reversed may be the D stereoisomer. All amino acids of the peptide may be reversed and / or all amino acids may be the D stereoisomer.

In other described embodiments, the peptidomimetic may be derived as a retro-enantio analog of the peptide. Such retro-enantio analogs can be synthesized by commercially available D-amino acids (or analogs thereof) and by standard solid- or solution-phase peptide-synthesis techniques as described, for example, in WO 00/01720. The final product can be purified by HPLC to give pure retro-enantio analogs.

In another exemplary embodiment, trans-olefin derivatives can be prepared for the desired peptide. Trans-olefin analogs are described in Y.K. Shue et al. (1987) Tetrahedron Letters 28: 3225 and methods as described in WO 00/01720. It is further possible to prepare peptide analogs having several olefinic functionalities instead of amide functionalities by binding the pseudodipeptides synthesized by the methods described above to other pseudodipeptides.

Another class of peptidomimetic derivatives includes phosphonate analogs. Synthesis of such phosphonate analogs can be derived from known synthetic schemes. See, eg, 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 Chem Co. Rockland, IL, 1985).

Many other peptidomimetic structures are known in the art and can be readily adjusted for use with the desired peptidomimetic. By way of example, peptidomimetics can be described as 1-azabicyclo [4.3.0] nonane surrogates by 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 2-substituted piperazine residues as constrained amino acid analogs (William et al. (1996) J. Med. Chem. 39: 1345-1348. In another embodiment, certain amino acid residues are aryl and bi-aryl residues, such as monocyclic or bicyclic aromatic or heteroaromatic nuclei, or bi-aromatic, aromatic-heteroaromatic, or biheteroaromatic nuclei. It may be substituted by.

The desired peptidomimetics can be optimized by combinatorial synthesis techniques combined with high power screening.

In addition, other examples of mimetopes include, but are not limited to, protein based compounds, carbohydrate based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetic organic compounds, anti-idiotype antibodies, and / Or catalytic antibodies, or fragments thereof. Mimetopes can be obtained, for example, by screening libraries of natural and synthetic compounds for compounds that can inhibit cell survival and / or tumor growth. Mimetopes can also be obtained, for example, from libraries of natural and synthetic compounds, in particular chemical or combinatorial libraries (ie, libraries of compounds having the same building blocks but different in sequence or size). Mimetopes can also be obtained, for example, by rational drug design. In a reasonable drug design process, the three-dimensional structure of the compounds of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography. Three-dimensional structures can be used to predict the structure of an effective mimetope, for example by computer modeling. Predicted mimetope structures can be produced, for example, by chemical synthesis, recombinant DNA techniques, or obtained by separating mimetopes from natural sources (eg, plants, animals, and mycelium).

"Peptide, variant and analogue thereof" or "peptide and analogue thereof" are included in "peptide therapeutic agents" and are intended to include any peptide or modified form thereof described herein, such as peptidomimetic. Preferred peptide therapeutics, for example, reduce cell viability or reduce apoptosis by at least about 2 times, at least 5 times, at least 10 times, at least 30 times, or at least 100 times as measured in the assays described herein. Increase.

The activity of an Isd protein, fragment or variant thereof can be assayed by using an appropriate substrate or binding partner, or other reagent suitable for testing the suspected activity, as described below.

In another embodiment, the activity of a polypeptide can be measured by assaying the expression level of RNA and / or protein molecules. Transcription levels can be measured, for example, using northern blots, hybridization to oligonucleotide arrays, or by assaying the level of protein product produced. Translation levels can be measured, for example, by using Western blotting or by identifying detectable signals (eg, fluorescence, luminescence, enzyme activity, etc.) generated by the protein product. Depending on the particular situation, it may be desirable to examine the level of transcription and / or translation of a single gene or multiple genes.

Alternatively, it may be desirable to measure the overall DNA replication rate, transcription rate and / or translation rate in the cell. In general, such measurements can be performed by growing cells in the presence of detectable metabolites that are incorporated into the resulting DNA, RNA, or protein product. For example, DNA synthesis rate can be measured by growing cells in the presence of BrdU incorporated into newly synthesized DNA. The amount of BrdU can be determined histologically by using anti-BrdU antibodies.

In another embodiment, polypeptides of the invention can be immobilized on a solid surface including microtiter plates, slides, beads, films, and the like. Polypeptides of the invention may be immobilized on a "chip" as part of an array. Arrays with multiple addresses may include one or more polypeptides of the invention in one or more addresses. In one embodiment, the chip comprises one or more polypeptides of the invention as part of an array of polypeptide sequences.

In another embodiment, the polypeptides of the invention are plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubing, containers, capillaries, pads, slices And may be immobilized on a solid surface, including the like. Polypeptides of the invention may be immobilized on a "chip" as part of an array. Multiple addressed arrays comprise one or more polypeptides of the invention in one or more addresses. In one embodiment, the chip comprises one or more polypeptides of the invention as part of an array.

5. Isd vaccine

IsdA, IsdB and IsdC polypeptides are cell surface proteins expressed by Staphylococcus aureus and are essential for complete toxicity in vivo (shown using a mouse model of kidney hepatitis). In addition, IsdA is immunodominant like anti-IsdA antibodies detected in convalescent human serum. Thus, IsdA, IsdB and / or IsdC polypeptides can be used as vaccine therapy to treat Staphylococcus aureus infection.

IsdA, IsdB and / or IsdC polypeptides or polynucleotides can be formulated into a vaccine and administered to a subject to give an immune response to IsdA, IsdB and / or IsdC in the subject (eg, cellular or humoral). Can be derived.

Exemplary IsdA proteins for inclusion in vaccines are full length IsdA polypeptides or IsdA peptides. In certain embodiments, recombinant IsdA protein will be used in a vaccine. In alternative embodiments, the IsdB or IsdC protein used as a vaccine can be a full length IsdB or peptide fragment of IsdC, IsdB or IsdC, or a recombinant IsdB or IsdC protein.

Isd proteins that are antigenic and used as vaccines can be identified using a variety of methods. In one method, peptides containing antigenic sequences may be selected based on potential antigenicity and / or exposure of generally accepted criteria. Such criteria include hydrophilicity and relative antigenicity indices determined by surface exposure analysis of proteins. Determination of appropriate criteria is well known to those skilled in the art and described, for example, in Hop 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. The amino acid domain predicted by the above criteria to be surface exposed may preferentially be selected over the domain predicted to be more hydrophobic.

Portions of IsdA, IsdB, and / or IsdC determined to be antigenic can be chemically synthesized from individual amino acids by methods known in the art. Suitable methods for synthesizing protein fragments are described in Stuart and Young in "Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984).

Alternatively, the antigenic linear epitope (s) of IsdA, IsdB or IsdC can be identified by the mimetope assay using the corresponding Isd antibody. Briefly, in the mimetope assay, the polypeptide will be subdivided into overlapping fragments. For example, overlapping 15 amino acid peptides will be synthesized to include the full length of the full length polypeptide. Each 15 amino acid peptide will be overlapped by three amino acids. Alternatively, the 15 amino acid peptide fragments may be designed as tandems to cover the entire amino acid sequence. Each peptide is then biotinylated and can bind to streptavidin coated wells in 96-well plates. Reactivity of various sera can be detected by enzyme immunoassay (ELISA). After blocking the nonspecific binding, anti-Isd antibodies can be added to each well, followed by a second addition of a peroxidase conjugated antibody and a peroxidase substrate. The anti-Isd antibody may be an affinity purified anti-full length-recombinant IsdA or an affinity purified anti-IsdA peptide. Alternatively, the anti-Isd antibody may be against IsdB or IsdC. The optical density of each well can be read at 450 nm and averaged with double or triple wells. Average values obtained from similar ELISAs with control serum (ie, pre-immune antibodies) can be subtracted from test immunoglobulin values, and the resulting values can be plotted to determine which linear epitopes are recognized by the immunoglobulin (s). Can be.

In addition, competitive binding assays using synthetic peptides exhibiting linear epitopes can be used to determine antigen fragments. In certain embodiments, the antigenic fragments can inhibit uptake of the labeled iron.

Also provided herein is a DNA vaccine comprising a nucleotide sequence encoding an IsdA, IsdB and / or IsdC peptide. Exemplary DNA vaccines encode two or more IsdA peptides. Alternative DNA vaccines may encode two or more IsdB or IsdC peptides or any combination of two or more IsdA, IsdB or IsdC peptides. The efficacy of candidate vaccines (peptides or DNA) can be tested in appropriate animal models, such as rats, mice, guinea pigs, monkeys and baboons. The protective or positive effect of the vaccine is reflected in reduced fertility in test animals.

Nucleic acids encoding IsdA, IsdB or IsdC immunogens are obtained by polymerase chain reaction (PCR), which is amplification of genomic DNA, cDNA, RNA (eg, by RT-PCR), or gene fragments from cloned sequences Can be. PCR primers are selected based on known sequences of genes or cDNAs, which result in amplification of relatively unique fragments. Computer programs can be used to design primers for the desired specificity and optimal amplification purposes. See Oligo version 5.0 (National Biosciences). Factors applied in designing and selecting primers for amplification are described, 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 can be obtained from GenBank or other public sources. Alternatively, the nucleic acids of the present invention may also be used in the art, for example, using automated DNA synthesizers (e.g., synthesizers are commercially available from Biosearch, Applied Biosystems). It can be synthesized by known standard methods. Suitable cloning vectors for expressing Isd polypeptides in a host or cell can be constructed according to the standard techniques described above.

Alternatively, Isd immunogens can 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 may be incubated simultaneously or sequentially with one or more proteases. Alternatively or in addition, the intact Isd polypeptide may be treated with a disulfide reducing agent. The peptides can then be separated from each other by techniques known in the art, including but not limited to gel filtration chromatography, gel electrophoresis, and reversed phase HPLC.

6. Isd Antibodies and Uses thereof

To generate antibodies against IsdA, IsdB and / or IsdC, the host animal can be injected with a full length Isd polypeptide or Isd peptide. The host can be injected with peptides of different lengths containing the desired target 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 Peptide antigens of 120, 125, 130, 135, 140, 145 or 150 amino acids can be used. Alternatively, if some of the proteins define epitopes but are too short to be antigenic, some of these proteins may be conjugated to the carrier molecule to produce an antibody. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequence, TrpE, and human or bovine serum albumin. Conjugation can be performed by methods known in the art. Such a method must combine the cysteine residue of the fragment with the cysteine residue on the carrier molecule.

In addition, antibodies to three-dimensional epitopes, ie nonlinear epitopes, can also be prepared based on the crystallographic data of the protein. Antibodies obtained by injection of such epitopes can be selected for single chain antigens of IsdA, IsdB and IsdC. Antibodies prepared against Isd peptides can be tested for activity against the full length Isd protein as well as the peptide. The antibody has 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 relative to the Isd peptide and / or the full length Isd protein. May have Mars.

Suitable cells for DNA sequences and host cells for antibody expression and secretion include various types, including American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas" 5 ^th edition (1985) Rockville, Md., USA). From a source.

Polyclonal and monoclonal antibodies can be produced by methods known in the art. Monoclonal antibodies are described in Kohler and Milstein, Nature 1975; 256: 495-7; and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds. Not only can be produced by hybridomas prepared by using known procedures, including immunological methods described in Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); It can be produced by the recombinant DNA method described in Huse et al., Science (1989) 246: 1275-81.

Antibody purification methods are known in the art. See, eg, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. Purification methods include salt precipitation (eg with ammonium sulfate), ion exchange chromatography (eg, on cation or anion exchange columns operated at neutral pH and eluted with a step gradient to increase ionic strength), gels 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 functionally equivalent antibodies, including, for example, chimeric, humanized, and single chain antibodies, and fragments thereof. Methods for producing functional equivalents are described in PCT Application WO 93/21319; European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application No. 388,745; And European Patent Application EP 332,424.

Functional equivalents include polypeptides having an amino acid sequence substantially identical to the amino acid sequence of the variable or hypervariable region of the antibody of the invention. Amino acids that are "substantially identical" are at least 70% preferred for other amino acid sequences, preferably as determined by the FASTA irradiation method according to Pearson and Lipman, Proc Natl Acd Sci USA (1988) 85: 2444-8 It is defined as a sequence having at least about 80%, more preferably at least 90% homology.

Chimericized antibodies can have constant regions derived substantially or entirely from human antibody constant regions and variable regions derived substantially or entirely from sequences of variable regions from non-human mammals. Humanized antibodies may have complement determining regions (CDRs) derived substantially or entirely from corresponding human antibody regions and constant and variable regions other than CDRs derived substantially or entirely from non-human mammals.

Suitable mammals that are not humans can include any mammal from which a monoclonal antibody can be produced. Suitable examples of non-human mammals may include, for example, rabbits, rats, mice, horses, goats, or primates.

Antibodies against IsdA, IsdB or IsdC used as anti-infective agents can be prepared as described above. In other embodiments, antibodies that recognize functional Isd fragments can also be used in random peptide phage display techniques (Eidne et al., Biol Reprod. 63 (5): 1396-402 (2000)). Briefly, random peptide phage display libraries of 15 or 20 conjugates can be used to determine peptides that can interact with functional Isd peptides by competitive replacement of Fab fragments of Isd antibodies. To this end, immobilized Staphylococcus aureus cells are attached to wells in multiwell plates, and immunostaining for IsdA, IsdB or IsdC is followed by the presence and absence of unique and random peptides expressed by phage display. Can be evaluated. Once competitive peptides have been identified by amino acid sequencing, increased amounts of peptides can be synthesized and used as alternative molecular antagonists for antibodies specific for functional fragments. Another alternative is to screen small molecule libraries for the ability to competitively replace Fab fragments with functional IsdA, IsdB or IsdC fragments. Molecular antagonists identified in this manner can be used to neutralize the effects of antibodies produced by an immune response to an Isd polypeptide or polynucleotide vaccine.

In a further embodiment, an antibody against IsdA, IsdB or IsdC (whole antibody or antibody fragment) is a biocompatible material, such as polyethylene glycol molecule (PEG), according to methods well known to those skilled in the art to increase the half-life of the antibody. Can be conjugated to. See, for example, US Pat. No. 6,468,532. For example, functional PEG polymers from Nektar Therapeutics are available. Commercially available PEG derivatives include amino-PEG, PEG amino acid esters, PEG-hydrazides, PEG-thiols, PEG-succinates, carboxymethylated PEGs, PEG-propionic acids, PEG amino acids, PEG succinimidyl succinates, PEG succinimidyls Propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl ester of amino acid PEG, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tre Silates, PEG-glycidyl ethers, PEG-aldehydes, PEG vinylsulfones, PEG-maleimides, PEG-orthopyridyl-disulfides, heterofunctional PEGs, PEG vinyl derivatives, PEG silanes and PEG phosphorides, This is not restrictive. Reaction conditions for coupling such PEG derivatives will vary depending on the polypeptide, the degree of PEGylation desired, and the PEG derivative used. Some factors related to the choice of PEG derivatization include the desired point of attachment (eg lysine or cysteine R-group), the hydrolytic stability and reactivity of the derivative, the stability, the toxicity and antigenicity of the binding, the analytical suitability, etc. .

7. Pharmaceutical Composition

Purified IsdA, IsdB or IsdC polypeptides and nucleic acids are formulated to provide oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intravaginal, and aphrodisiac methods (ie, upper layers of skin using, for example, a bifurcated needle) Scratching through) or any other immunization standard pathway. Isd polypeptides may further be delivered orally as vaccines by enteric coated capsules that are soluble in the intestine and taken up by antigen presenting cells in Peyer's patches. Oral delivery of an Isd polypeptide can be supplemented by injection of an Isd polypeptide.

In addition, the Staphylococcus aureus anti-Isd antibodies, isd antisense nucleic acids, and siRNAs described herein can be administered by a variety of means depending on their use, as is well known in the art. For example, when the Staphylococcus aureus antagonist composition is administered orally, they may be formulated into tablets, capsules, granules, powders or syrups. Alternatively, the formulations of the invention may be administered parenterally by injection (intravenous, intramuscular or subcutaneous), instillation preparations or suppositories. For application by the ocular mucosal route, the compositions of the present invention may be formulated as eye drops or ophthalmic ointments. Such formulations may be prepared by conventional means, and if desired, the composition may be combined with any conventional additives such as excipients, binders, disintegrants, lubricants, correctors, solubilizers, suspending aids, emulsifiers or coatings. Can be mixed together.

In the formulations of the present invention, wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavors, and fragrances, preservatives, and antioxidants may be present in the formulated formulations. have.

The composition may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations are usually in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of the composition that can be combined with the carrier material to produce a single dose may depend upon the subject treated and the particular mode of administration.

Methods of preparing these formulations include the step of bringing into association the composition of the present invention with a carrier and optionally one or more accessory ingredients. Generally, formulations are prepared by uniformly and tightly associating a formulation with a liquid carrier or finely divided solid carrier, or both, and, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using flavorings, generally sucrose and acacia or tragacanth), powders, granules, or aqueous or non-aqueous As a solution or suspension in liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastel (with inert bases such as gelatin and glycerin, or sucrose and acacia), , Each contains a predetermined amount of the desired composition as the active ingredient. The compositions of the invention can also be administered as bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders and granules, etc.), the desired composition may comprise one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / Or mixed with any of the following components: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) wetting agents, such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution inhibiting agents such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) adsorbents, kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; And (10) colorants. In the case of capsules, tablets and pills, the composition may also comprise a buffer. Solid compositions of a similar type may be used in soft and hard-filled gelatin capsules as fillers by using excipients such as lactose or lactose, high molecular weight polyethylene glycols and the like.

Tablets may be made by tableting or molding, optionally with one or more accessory ingredients. Tableted tablets may be binders (eg gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (eg sodium starch glycolate or crosslinked sodium carboxymethyl cellulose), surfactants or dispersants It can be prepared by using. Molded tablets can be made by molding a mixture of the desired composition moistened with an inert liquid diluent in a suitable machine. Tablets and other solid dosage forms such as dragees, capsules, pills and granules may optionally be scored or prepared with coatings and shells such as enteric skin and other coatings known in the pharmaceutical formulation art. have.

Liquid dosage forms suitable for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the desired compositions, liquid dosage forms include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, 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 oil, corn oil, gum, olive, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, Fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof.

Suspending agents may be added to the desired composition in addition to suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan and sorbitan esters, microcrystalline cellulose, aluminum metahydroxyde, bentonite, agar and tragacanth and their It may contain a mixture.

Formulations for rectal or vaginal administration may be prepared by mixing the desired composition with one or more suitable irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates, but are solid at room temperature, It may be present as a suppository that is liquid at body temperature and therefore melts in the body cavity to release the active agent. Formulations suitable for vaginal administration also include pessary, tampon, cream, gel, paste, foam or spray formulations containing carriers known to be suitable in the art.

Dosage forms for transdermal administration of the desired compositions 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 that may be required.

Ointments, pastes, creams and gels, in addition to the desired compositions, may include excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, talc and Zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to the desired composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. The spray may further contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Compositions of the present invention may alternatively be administered by aerosol. Such aerosols can be carried out by preparing an aqueous aerosol, liposome preparation or solid particles containing the compound. Non-aqueous (eg fluorocarbon propellant) suspensions can be used. Acoustic nebulizers can be used because these sonic nebulizers provide minimal exposure of the agent to shear forces that can degrade the compounds contained in the desired composition.

In general, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the desired composition with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers depend on the requirements of the particular desired composition but are typically nonionic surfactants (Tween, Pluronic, or polyethylene glycol), nontoxic proteins such as serum albumin, sorbitan esters, Oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

In addition, Isd based vaccines can be administered parenterally as injections (intravenous, intramuscular or subcutaneous). The vaccine composition of the present invention may optionally contain one or more adjuvants. Any suitable adjuvant such as aluminum hydroxide, aluminum phosphate, plant and animal oils, etc. may be used in the amount of adjuvant depending on the nature of the particular adjuvant used. In addition, anti-infective vaccine compositions may also contain one or more stabilizers, such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin and glucose, as well as proteins such as albumin or casein, buffers such as alkalis. Metal phosphates and the like. Preferred adjuvants include the SynerVax ™ adjuvant.

Pharmaceutical compositions of the invention suitable for parenteral administration include one or more pharmaceutically acceptable isotonic, isotonic, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or antioxidants as sterile injectable solutions or dispersions immediately prior to use, Includes the desired composition with sterile powders that can be reconstituted into solutions or dispersions that can include buffers, antibiotics, solutes, suspensions or thickeners that result in formulations that are isotonic with the blood of the intended recipient.

Examples of suitable aqueous and non-aqueous carriers that can 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, vegetable oils such as olive oil, and Injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

In addition, Isd immunogens or Isd antibodies of the invention may be encapsulated in liposomes and administered via injection. A commercially available liposome delivery system is commercially available under the name Novasomes ™ from Novavax, Inc., Rockville, Md. These liposomes are specifically formulated for immunogen or antibody delivery. In an embodiment of the present invention, Novasomes ™ containing an Isd peptide or antibody molecule bound to the surface of a liposome that is positively charged to the non-phospholipid can be used.

The pharmaceutical compositions described herein include, but are not limited to, staphylo including furunculosis, chronic boil disease, pus scab, acute osteomyelitis, pneumonia, endocarditis, burn skin syndrome, toxin shock syndrome, and food poisoning. It can be used to prevent or treat a condition or disease resulting from Cocus aureus infection.

8. Exemplary Screening Assays for Isd Inhibitors

In general, agents or compounds that can reduce pathogenic pathogenesis by interfering with iron-determined surface determinants (Isd) are identified from large libraries or chemical libraries of both natural or synthetic (or semi-synthetic) extracts. Can be. Those skilled in the art of drug discovery and development will appreciate that precise sources of agents (eg test extracts or compounds) are not critical to the screening process (es) of the present invention. Thus, virtually any number of chemical extracts or compounds can be screened by using the methods described herein. Examples of such agents, extracts or compounds include, but are not limited to, plant-, mycelium-, prokaryotic- or animal-based extracts, fermented broths, and synthetic compounds, and variants of existing compounds. Various methods also include, but are not limited to, random or direct synthesis (eg, semi-synthetic or total synthesis) of any number of chemical compounds, including saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Can be used to derive. Synthetic compound libraries can be purchased from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). In addition, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Biotics: Sussex, UK, Xenova: Slough, UK, and Harbor Branch Oceangraphics Institute: Ft. Pierce. , Fla.) And Phamnamar, USA: Cambridge, Mass.). In addition, natural and synthetic libraries, if desired, are produced according to methods known in the art, for example by standard extraction and fermentation methods. In addition, if desired, any library or compound is readily modified by using standard chemical, physical, or biochemical methods.

In addition, those skilled in the field of drug discovery and development are those skilled in the art of replicating or repeating units of anti-pathogenic activity (e.g., taxonomic, biological and chemical decoupling agents, or any combination thereof), or It will be appreciated that a method for removal should be used where possible.

If the crude extract is found to have anti-pathogenic or anti-pathogenic activity, or binding activity, fractionation of the positive lead extract is necessary to isolate the chemical composition that causes the observed effect. Thus, the purpose of the extraction, fractionation, and purification procedures is the careful characterization and identification of chemical entities in crude extracts with anti-pathogenic activity. Methods for fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds that are found to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art.

Potential inhibitors of Isd encoded polypeptides can include organic molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind to and inhibit or extinguish the activity of a nucleic acid sequence or polypeptide of the invention. Efficacy antagonists also include small molecules that bind to and bind to the binding site of the polypeptide to prevent its binding to cell binding molecules, thereby inhibiting normal biological activity. Other potential antagonists include antisense molecules.

In addition, S. aureus anti-Isd antagonists identified by the screening assays described herein can be administered in a variety of ways depending on their use as described above.

8.1 Interaction Test

Purified and recombinant IsdA, IsdB and IsdC polypeptides can be used in the development of assays for screening agents that bind to the Isd gene product and cleave protein-protein interactions. Potential inhibitors or antagonists of IsdA, IsdB, or IsdC may include organic small molecules, peptides, polypeptides, peptide mimetics, and antibodies that bind to and reduce or eliminate IsdA, IsdB or IsdC.

In certain embodiments, (i) contacting the Isd polypeptide with the appropriate interacting molecule in the presence of the agent under conditions that allow for interaction between the Isd polypeptide and the interacting molecule in the absence of the agent; And (ii) determining the level of interaction between the Isd polypeptide and the interacting molecule, wherein the agent binds to the Isd polypeptide and inhibits iron uptake, wherein the agent is present in the absence of the agent. Different levels of interaction between the Isd polypeptide and the interacting molecule indicate that the agent inhibits the interaction between the Isd polypeptide and the interacting molecule.

In another embodiment, agents that disrupt the interaction between the Isd polypeptide and the interacting molecule can be identified. In one exemplary binding assay, the reaction mixture can be generated to include one or more biologically active moieties of IsdA, IsdB or IsdC, agent (s) of interest, and appropriate interacting molecules. One exemplary interaction molecule can be heme protein, hemin, transferrin, fibrinogen or fibronectin. In one exemplary embodiment, the agent of interest is an antibody against a particular Isd polypeptide. Binding of the antibody to the Isd polypeptide may inhibit the function of the Isd polypeptide in the binding heme or heme protein. Detection and quantification of the interaction of a particular Isd polypeptide with an appropriate interacting molecule provides a method for determining the efficacy of an agent that inhibits the interaction. The efficacy of an agent can be measured by generating a dose response curve from data obtained using various concentrations of test agent. Moreover, control assays can also be performed to provide a baseline for comparison. In the control assay, the interaction of a particular Isd polypeptide with an appropriate interacting molecule can be quantified in the absence of a test agent.

Interaction between a particular Isd polypeptide and an appropriate interacting molecule can be detected by various techniques. Modulation of complex formation can be quantified by immunoassay or by chromatographic detection, for example, by using detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides. Can be.

Measurement of the interaction of a particular Isd protein with an appropriate interacting molecule can be directly observed by using surface plasmon resonance technology in an optical biosensor device. This method is particularly useful for measuring interactions with larger (> 5 kDa) polypeptides and can be adjusted to screen for inhibitors of protein-protein interactions.

Alternatively, it would be desirable to immobilize a particular Isd polypeptide or appropriate interacting molecule to facilitate separation of the complex from uncomplexed forms of one or both of the proteins, and to allow automation of the assay. For example, binding of specific Isd proteins to interacting molecules in the presence and absence of candidate agents can be performed in any vessel suitable for containing the reactants. Such examples include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided having a domain that allows the protein to bind to the matrix. For example, a glutathione-S-transferase / IsdA (GST / IsdA) fusion protein may be prepared on glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates. May be required, for example, under ³⁵ S-labeled interacting molecules, and test agents, and under conditions that induce complex formation, although slightly more stringent conditions may be required, for example, And a mixture incubated at physiological conditions with respect to pH. After incubation, the beads are washed to remove any unbound label, the matrix is fixed and radiolabeled directly (beads are placed in scintillant) or the complexes are subsequently dissociated and then measured in supernatant. Alternatively, the complex is dissociated from the matrix, separated by SDS-PAGE, and the level of interacting molecules found in the bead fraction is quantified from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins and other molecules on the matrix can also be used in this assay. For example, certain Isd proteins or one of the appropriate interacting molecules can be immobilized by utilizing the conjugation of biotin and streptavidin. For example, biotinylated IsdA, IsdB or IsdC can be prepared from biotin-NHS (N-hydroxy-succinimide) by using techniques known in the art (eg biotinylation kits, Pierce Chemicals, Rockfield, Ill., USA, can be immobilized to wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, an antibody that is reactive with IsdA, IsdB or IsdC but does not interfere with the interaction between the polypeptide and the interacting molecule can be derivatized in the well of the plate, and IsdA, IsdB or IsdC is well-received by antibody conjugation. Can be collected on. As described above, the preparation of the interaction molecule and the test compound can be incubated in the polypeptide-existing wells of the plate, and the amount of complex trapped in the wells can be quantified in the presence or absence of the test agent. In addition to the methods described above for GST-fixed complexes, exemplary methods of detecting the complexes include immunodetection of complexes using antibodies reactive with the interacting molecule or enzymes that detect enzymatic activity associated with the interacting molecule. Associated assays are included.

For example, enzymes may be chemically conjugated or provided as fusion proteins with interacting molecules. For example, the interaction molecule may be chemically crosslinked or genetically fused with horseradish peroxidase, and the amount of polypeptide captured in the complex may be an enzyme, eg, 3,3'-diamino- It can be assayed with a chromogenic substrate of benzadiine tetrahydrochloride or 4-chloro-1-naphthol. Similarly, fusion proteins comprising polypeptides and glutathione-S-transferases can be provided and complex formation can be quantified by detecting GST activity with 1-chloro-2,4-dinitrobenzene. Habig et al. (1974) J. Biol. Chem. 249: 7130.

8.2 Expression Assay

In further embodiments, the antagonist of iron uptake may affect the expression of isdA , isdB and isdC nucleic acids or proteins. In this screen, Staphylococcus aureus cells can be treated with compound (s) of interest and then assayed for the effect of compound (s) on isdA , isdB and isdC nucleic acid or protein expression.

In certain embodiments, (i) culturing the wild-type Staphylococcus aureus strain in the presence or absence of an agent; And (ii) comparing the expression of the Isd polypeptide, thereby identifying an agent that inhibits the expression of the Isd polypeptide of Staphylococcus aureus, wherein the expression of the Isd polypeptide in cells treated with the agent A greater decrease of indicates that the agent inhibits the expression of the Isd polypeptide of Staphylococcus aureus.

In one alternative embodiment, (i) culturing the wild type Staphylococcus aureus in the presence or absence of an agent; And (ii) comparing the expression of the isd nucleic acid, thereby identifying an agent that inhibits the expression of the isd nucleic acid of Staphylococcus aureus, wherein the expression of the isd nucleic acid in a cell treated with the agent. A greater reduction of indicates that the agent inhibits the expression of isd nucleic acid of Staphylococcus aureus.

For example, total RNA is described by Chomczynski et al. (1987) Anal. Biochem. 162: 156-159, isolated from Staphylococcus aureus cells cultured in the presence or absence of a test agent by using any suitable technique, such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Can be. The expression of isdA , isdB or isdC can be determined by any suitable method, such as Northern blot analysis, polymerase chain reaction (PCR), reverse transcription in combination with polymerase chain reaction (RT-PCR), and ligase chain reaction. Can be assayed by reverse transcription in combination with RT-LCR.

Northern blot analysis can be performed as described in Harada et al. (1990) Cell 63: 303-312. Briefly, total RNA is prepared from Staphylococcus aureus cells cultured in the presence of a test agent. For northern blots, RNA is denatured in appropriate buffers (eg glyoxal / dimethyl sulfoxide / sodium phosphate buffer), added to agar gel electrophoresis, and transferred onto nitrocellulose filters. After RNA is bound to the filter by a UV linker, it is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon semen, SDS, and sodium phosphate buffer. The DNA sequence of Staphylococcus aureus isdA , isdB or isdC is labeled according to any suitable method (eg 32P-multiprimed DNA labeling system (Amersham)) and used as a probe. After hybridization overnight, the filter is washed and exposed to x-ray film. In addition, control tests can be performed to provide a baseline for comparison. In the control test, the expression of isdA , isdB or isdC in Staphylococcus aureus can be quantified in the absence of test agent.

Alternatively, the level of mRNA encoding IsdA, IsdB or IsdC polypeptide can be assayed, for example, by using the RT-PCR method described in Makino et al. (1990) Technique 2: 295-301. have. In summary, this method involves adding total RNA isolated from Staphylococcus aureus cells cultured in the presence of a test agent in a reaction mixture containing RT primers and appropriate buffer. After incubation for primer annealing, the mixture can be supplemented with RT buffer, dNTP, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of RNA, RT products are added to PCR using labeled primers. Alternatively, rather than label the primers, labeled dNTPs can be included in the PCR reaction mixture. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on polyacrylamide gels. After drying the gel, the radioactive activity of the appropriate band can 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. Modifications to the RT-PCR method will be apparent to those skilled in the art. Other PCR methods capable of detecting nucleic acids of the present invention can be found in PCR Primer: A Laboratory Manual (Dieffenbach et al., Cold Spring Harbor Lab Press, 1995). Control trials can be performed to provide a baseline for comparison. In the control test, the expression of isdA , isdB or isdC in Staphylococcus aureus can be quantified in the absence of test agent.

Alternatively, expression of IsdA, IsdB and IsdC polypeptides can be quantified by using antibody based methods such as immunoassay after treating Staphylococcus aureus cells with a test agent. Competitive and non-competitive assay systems, techniques such as Western blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), “sandwich” immunoassays, immunoprecipitation assays, precipitin Any suitable immunoassay can be used including reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement binding assay, immunoradiometric assay, fluorescence immunoassay and Protein A immunoassay. have.

For example, IsdA, IsdB or IsdC polypeptides can be detected in samples obtained from Staphylococcus aureus cells treated with test agents by two-step sandwich assay means. In the first step, capture reagents (eg, one of the IsdA, IsdB or IsdC antibodies) are used to capture the specific polypeptide. Capture reagents may optionally be immobilized on a solid phase. In the second step, captured markers are detected using direct or indirectly labeled detection reagents. In one embodiment, the detection reagent is an antibody. The amount of IsdA, IsdB or IsdC polypeptide present in Staphylococcus aureus cells treated with the test reagent can be calculated by reference to the amount present in Untreated Staphylococcus aureus cells.

Suitable enzyme labels include, for example, labels from oxidase groups that catalyze the production of hydrogen peroxide by reacting with a substrate. Glucose oxidase is particularly preferred because it has good stability and its substrate is readily available. The activity of the oxidase label can be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody / substrate reaction. In addition to enzymes, other suitable labels include radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H).

Examples of suitable fluorescent labels include fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, o-ftaldehyde labels, and fluorescamine A cover is included.

Examples of suitable enzyme labels include maleate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, Trios phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, Glucoamylases, and acetylcholine esterases. 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 equarin labels ( aequorin label).

example

The invention described generally may be more readily understood by reference to the following examples, which are intended to merely illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

Example 1: Expression of IsdA, IsdB and IsdC Proteins

IsdA, IsdB and IsdC proteins or fragments thereof are expressed in an environment restricted by iron as shown in FIG. 4 (Staphylococcus aureus-Fe). The SDS-PAGE gels shown in FIG. 4 indicate that the IsdA, IsdB and IsdC proteins are three of the most dominant iron regulated proteins expressed by Staphylococcus aureus. These proteins are not expressed when Staphylococcus aureus cells are cultured in iron-rich medium (Staphylococcus aureus + Fe), and therefore presumably all highly expressed in vivo.

Overexpression of IsdA, IsdB and IsdC as well as IsdE following fusion to E. coli results in very colored lysates. Absorption and magnetic circular dichroism spectroscopy is used to confirm that this staining is due to the ability of proteins to utilize different forms of protoporphyrin and heme from the E. coli cytoplasm, which plays a role of protoporphyrin and heme in heme binding. Confirm.

Example 2: isd  Generation of Gene Knockout Mutations

In addition, the coding regions of isdA , isdB and isdC or portions thereof may be blocked from individually producing strains containing a single mutation in each of the isd genes. The isdA coding region was blocked by inserting a cassette encoding resistance to tetracycline. The isdB coding region was blocked by inserting a cassette encoding resistance to erythromycin. The isdC coding region was blocked by inserting a cassette encoding resistance to kanamycin. Each mutation is then described in Sebulsky et al., (2001) J. Bacteriol. 183: 4994-5000, using the phage transduction process and transfer to the same genetic background selected with appropriate resistance. In addition, strains containing mutations in which two or more isd genes are knocked out (eg, mutated strains in isdA , isdB and isdC ) can also be generated.

Example 3: Survival Study of Mouse Model of Kidney Infection

25 g of female Swiss-Webster mice were purchased from Charles River Laboratories Canada, Inc. and bred in microisolation cages. The bacteria were grown overnight in Trypric Soy Broth (TSB), harvested and washed three times with sterile saline. Preliminary experiments showed that Staphylococcus aureus Newman colonized mice in this model more than RN6390 and injected into the tail vein to obtain acute but non-fatal kidney infection. The optimal amount of Uss Newman proved 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 the inoculum on TSB. Six days after injection, mice were sacrificed and kidneys were aseptically isolated. Using a PowerGen 700 homogenizer, kidneys were homogenized in sterile PBS containing 0.1% Triton X-100 for 45 seconds and the homogenized dilutions were plated on TSB-agar to count viable bacteria. Data presented are log CFU recovered per mouse.

Results using a murine renal boil model of Staphylococcus aureus infection indicate that a strain with IsdA alone or a mutation with both IsdA, IsdB and IsdC attenuates Staphylococcus aureus toxicity. Interestingly, after 6 days of injection, recovered mutant bacteria were reduced by 90% compared to the number recovered from wild type, indicating that these proteins play an essential role in bacterial suitability during infection when expressed on bacterial cell surfaces. This also indicates that inhibition of these proteins in vivo can prevent infection by Isd-expressing bacteria (ie for Isd based vaccines) or cause the removal of Isd-expressing bacteria once the infection is initiated. .

Example 4 Survival of Staphylococcus aureus at Increasing Hydrogen Peroxide Concentration

Isd protein bound to heme appears to act as an oxidation buffer that protects Staphylococcus aureus cells from the deleterious effects of free radicals. Direct comparisons of Newman strains incubated in the presence of heme to Newman strains incubated in the presence of heme to remove IsdA, IsdB and IsdC indicate that the mutant cells cannot survive in increased concentrations of hydrogen peroxide (FIG. 6). Thus, mutations lacking the expression of several Isd proteins are more sensitive to hydrogen peroxide attack.

FIG. 7 shows wild type Staphylococcus aureus and Staphylococcus aureus isdA :: km ^c both performed on SDS-PAGE gels stained with (A) Coomassie and (B) TMBZ (tetramethylbenzidine) Expression of IsdA plus and minus in both is shown. Catalase activity associated with the heme bound form of IsdA degrades the TMBZ compound to produce a colored reaction product. Thus, heme bound IsdA can aid resistance to oxidative killing by macrophages.

Example 5: Isd Vaccine

Vaccines comprising the recombinant IsdA polypeptide can establish protective immunity against systemic and local Staphylococcus aureus infection in mice. Recombinant IsdA protein can be prepared using standard techniques. Groups of 12-15 Swiss-Webster mice (25 g) can be used for all immunization experiments and can be injected intraperitoneally (IP). Mice can be boosted with subsequent injections at various different time points. Serum can be monitored over the course of the experiment on anti-IsdA antibody titers. At about 30 days, mice can be administered intravenously with 1 × 10 ⁷ Staphylococcus aureus and monitored for an additional 7 days. We have previously shown that injections with this number of live organisms cause non-fatal kidney infections. Mice can be sacrificed at various time points after infection to monitor the number of organisms that infect kidney tissue. Passive immunization experiments can also be performed using serum obtained from previously immunized mice to test the effectiveness of infection prevention in other groups of mice. Similar immunization experiments can be performed with IsdB and IsdC polypeptides.

Example 6: IsdA, IsdB and IsdC Antibodies

A. Preparation of Monoclonal Antibodies Against Full Length Isd Proteins

BALB / c mice can be similarly boosted to native IsdA, IsdB or IsdC after about 6 weeks after initial immunization via intraperitoneal injection with full length recombinant IsdA, IsdB or IsdC. Mice can be immunized with an appropriate adjuvant. Mouse serum can be obtained about 10 days after the second injection and can be tested for nti-HRP activity via ELISA. Mice with high levels of anti-HRP activity in serum may be selected for cell fusions. Spleens can be harvested from the mice and cell suspensions can be prepared by perfusion using Dulbecco's Modified Eagle Medium (DMEM).

Spleen cell suspensions containing B-lymphocytes and macrophages can be prepared by perfusion of the spleen. The cell suspension can be washed and collected by centrifugation; Myeloma cells can also be washed in this manner. Live cells can be counted and placed in a 37 ° C. water bath. 1 mL of 50% polyethylene glycol (PEG) may be added to DMEM. Balb / c spleen cells can be fused with SP 2 / 0-Ag 14 mouse myeloma cells by PEG, and the resulting hybridomas contain hypoxanthine (H), aminopterin (A ) And thymidine (T) (HAT) selection in tissue culture medium. Viable cells can be grown in confluence. Spent culture media can be identified for antibody titer, specificity and affinity. After dilution by the slow addition of DMEM medium to PEG, cells can be incubated in PEG for 1-1.5 minutes at 37 ° C. The cells can be pelleted and 35-40 mL of DMEM containing 10% fetal calf serum can be added. The cells can then be dispensed into tissue culture plates and incubated overnight in a humidified incubator at 37 ° C. in 5% CO ₂ .

The next day, DMEM-FCS containing Hypoxyxanthin (H), Aminopterin (A) and thymidine (T) medium (HAT medium) can be added to each well. The concentration of HAT in the medium added is twice the final concentration required, ie H _final = 1 times 10 ⁻⁴ M; A _final = 4 × 10 ⁻⁷ M; And T _final = 1.6 times 10 ^-5 M.

The plate can then be incubated with HAT medium every 3-4 days over 2 weeks. The fused cells can then be cultured in DMEM-FCS containing HAT medium. The fused cells are 1/2 to 3/4 confluence on the bottom of the wells, and the tissue culture solution of the upper layer can be harvested and tested for IsdA, IsdB or IsdC specific antibodies by ELISA. Positive wells can be cloned by limiting dilution on macrophages or thymic cell feeder plates and cultured in DMEM-FCS. Cloned wells can be tested and recloned three times before statistically significant monoclonal antibodies are obtained. The spent culture medium can be tested from antibody producing clones.

B. Preparation of Polyclonal Antibodies Against Full Length Isd Proteins

Unconjugated purified recombinant IsdA, IsdB and / or IsdC can be used as the antigen for immunizing two rabbits. Briefly, 1 mg of recombinant IsdA, IsdB or IsdC can be resuspended in 1 ml of phosphate buffered saline, emulsified with an equivalent volume of Complete Freund's Adjuvant, and about 1 ml (whole Half the volume) can be injected into each peritoneal cavity. Second and third immunizations can be followed using incomplete Freund's adjuvant after two and three weeks. Serum can be tested using Enzyme Immunoassay (ELISA) to determine recombinant IsdA, IsdB or IsdC specific antibody titers. Anti-recombinant IsdA, IsdB and / or IsdC containing serum showing high titers based on 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 can be tested for their ability to attenuate the toxicity of Staphylococcus aureus infection.

Example 7: Expression Assay

Screening assays for agents that inhibit the expression of IsdA in Staphylococcus aureus will be performed as follows. Wild-type Staphylococcus aureus cells will be cultured overnight in Tryptic Soy Liquid Medium (TSB) (Difco) in the presence or absence of a test agent. After 24 hours of incubation, cells were washed in 1 × PBS (phosphate buffered saline) followed by 10 μg of Lysostar in STE (0.1 M NaCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]). It will dissolve at 37 ° C. by using lysostaphin. Cell lysates will then be transferred to anti-IsdA antibody precoated plates and incubated at room temperature for 45-60 minutes. As a control, cell lysates from untreated Staphylococcus aureus cells will be used. After washing three times with water, a secondary antibody conjugated to either alkaline phosphatase (AP) or horseradish peroxidase (HRP) will be added and incubated for one hour. The plate will then be washed to separate the binding from the free antibody complex. Chemiluminescent substrates (alkali phosphatase or Super Signal luminol solution from Pierce for horseradish peroxidase) will be used to detect bound antibodies. Microplate luminometers will be used to detect chemiluminescent signals. Absence of signals in cell lysate samples obtained from cells treated with the test agent will indicate that the test agent inhibits the expression of IsdA. Similar expression assays can also be performed for IsdB and IsdC.

Unless otherwise stated, conventional cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology techniques within the skill of the art will apply to the practice of the present invention. See, eg, 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 Patent 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 , VoIs. 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 Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

Inclusion of References

All publications and patents mentioned herein are incorporated herein by reference in their entirety, as specifically and individually indicated that each individual publication or patent is incorporated by reference. In case of inconsistency, the present application, including any definitions, will control.

Equivalent

Those skilled in the art will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein using routine experiments only. Such equivalents are intended to be covered by the appended claims.

                               SEQUENCE LISTING <110> THE UNIVERSITY OF WESTERN ONTARIO <120> STAPHYLOCOCCUS AUREUS ISD PROTEIN-BASED ANTI-INFECTIVES <130> UWA-013.25 <140> PCT / IB05 / 004126 <141> 2005-10-25 <150> 60 / 621,921 <151> 2004-10-25 <160> 12 <170> Patent In Ver. 3.3 <210> 1 <211> 1053 <212> DNA <213> Staphylococcus aureus <400> 1 atgacaaaac attatttaaa cagtaagtat caatcagaac aacgttcatc agctatgaaa 60 aagattacaa tgggtacagc atctatcatt ttaggttccc ttgtatacat aggcgcagac 120 agccaacaag tcaatgcggc aacagaagct acgaacgcaa ctaataatca aagcacacaa 180 gtttctcaag caacatcaca accaattaat ttccaagtgc aaaaagatgg ctcttcagag 240 aagtcacaca tggatgacta tatgcaacac cctggtaaag taattaaaca aaataataaa 300 tattatttcc aaaccgtgtt aaacaatgca tcattctgga aagaatacaa attttacaat 360 gcaaacaatc aagaattagc aacaactgtt gttaacgata ataaaaaagc ggatactaga 420 acaatcaatg ttgcagttga acctggatat aagagcttaa ctactaaagt acatattgtc 480 gtgccacaaa ttaattacaa tcatagatat actacgcatt tggaatttga aaaagcaatt 540 cctacattag ctgacgcagc aaaaccaaac aatgttaaac cggttcaacc aaaaccagct 600 caacctaaaa cacctactga gcaaactaaa ccagttcaac ctaaagttga aaaagttaaa 660 cctactgtaa ctacaacaag caaagttgaa gacaatcact ctactaaagt tgtaagtact 720 gacacaacaa aagatcaaac taaaacacaa actgctcata cagttaaaac agcacaaact 780 gctcaagaac aaaataaagt tcaaacacct gttaaagatg ttgcaacagc gaaatctgaa 840 agcaacaatc aagctgtaag tgataataaa tcacaacaaa ctaacaaagt tacaaaacat 900 aacgaaacgc ctaaacaagc atctaaagct aaagaattac caaaaactgg tttaacttca 960 gttgataact ttattagcac agttgccttc gcaacacttg cccttttagg ttcattatct 1020 ttattacttt tcaaaagaaa agaatctaaa taa 1053 <210> 2 <211> 1053 <212> DNA <213> Staphylococcus aureus <400> 2 ttatttagat tcttttcttt tgaaaagtaa taaagataat gaacctaaaa gggcaagtgt 60 tgcgaaggca actgtgctaa taaagttatc aactgaagtt aaaccagttt ttggtaattc 120 tttagcttta gatgcttgtt taggcgtttc gttatgtttt gtaactttgt tagtttgttg 180 tgatttatta tcacttacag cttgattgtt gctttcagat ttcgctgttg caacatcttt 240 aacaggtgtt tgaactttat tttgttcttg agcagtttgt gctgttttaa ctgtatgagc 300 agtttgtgtt ttagtttgat cttttgttgt gtcagtactt acaactttag tagagtgatt 360 gtcttcaact ttgcttgttg tagttacagt aggtttaact ttttcaactt taggttgaac 420 tggtttagtt tgctcagtag gtgttttagg ttgagctggt tttggttgaa ccggtttaac 480 attgtttggt tttgctgcgt cagctaatgt aggaattgct ttttcaaatt ccaaatgcgt 540 agtatatcta tgattgtaat taatttgtgg cacgacaata tgtactttag tagttaagct 600 cttatatcca ggttcaactg caacattgat tgttctagta tccgcttttt tattatcgtt 660 aacaacagtt gttgctaatt cttgattgtt tgcattgtaa aatttgtatt ctttccagaa 720 tgatgcattg tttaacacgg tttggaaata atatttatta ttttgtttaa ttactttacc 780 agggtgttgc atatagtcat ccatgtgtga cttctctgaa gagccatctt tttgcacttg 840 gaaattaatt ggttgtgatg ttgcttgaga aacttgtgtg ctttgattat tagttgcgtt 900 cgtagcttct gttgccgcat tgacttgttg gctgtctgcg cctatgtata caagggaacc 960 taaaatgata gatgctgtac ccattgtaat ctttttcata gctgatgaac gttgttctga 1020 ttgatactta ctgtttaaat aatgttttgt cat 1053 <210> 3 <211> 350 <212> PRT <213> Staphylococcus aureus <400> 3 Met Thr Lys His Tyr Leu Asn Ser Lys Tyr Gln Ser Glu Gln Arg Ser   1 5 10 15 Ser Ala Met Lys Lys Ile Thr Met Gly Thr Ala Ser Ile Ile Leu Gly              20 25 30 Ser Leu Val Tyr Ile Gly Ala Asp Ser Gln Gln Val Asn Ala Ala Thr          35 40 45 Glu Ala Thr Asn Ala Thr Asn Asn Gln Ser Thr Gln Val Ser Gln Ala      50 55 60 Thr Ser Gln Pro Ile Asn Phe Gln Val Gln Lys Asp Gly Ser Ser Glu  65 70 75 80 Lys Ser His Met Asp Asp Tyr Met Gln His Pro Gly Lys Val Ile Lys                  85 90 95 Gln Asn Asn Lys Tyr Tyr Phe Gln Thr Val Leu Asn Asn Ala Ser Phe             100 105 110 Trp Lys Glu Tyr Lys Phe Tyr Asn Ala Asn Asn Gln Glu Leu Ala Thr         115 120 125 Thr Val Val Asn Asp Asn Lys Lys Ala Asp Thr Arg Thr Ile Asn Val     130 135 140 Ala Val Glu Pro Gly Tyr Lys Ser Leu Thr Thr Lys Val His Ile Val 145 150 155 160 Val Pro Gln Ile Asn Tyr Asn His Arg Tyr Thr Thr His Leu Glu Phe                 165 170 175 Glu Lys Ala Ile Pro Thr Leu Ala Asp Ala Ala Lys Pro Asn Asn Val             180 185 190 Lys Pro Val Gln Pro Lys Pro Ala Gln Pro Lys Thr Pro Thr Glu Gln         195 200 205 Thr Lys Pro Val Gln Pro Lys Val Glu Lys Val Lys Pro Thr Val Thr     210 215 220 Thr Thr Ser Lys Val Glu Asp Asn His Ser Thr Lys Val Val Ser Thr 225 230 235 240 Asp Thr Thr Lys Asp Gln Thr Lys Thr Gln Thr Ala His Thr Val Lys                 245 250 255 Thr Ala Gln Thr Ala Gln Glu Gln Asn Lys Val Gln Thr Pro Val Lys             260 265 270 Asp Val Ala Thr Ala Lys Ser Glu Ser Asn Asn Gln Ala Val Ser Asp         275 280 285 Asn Lys Ser Gln Gln Thr Asn Lys Val Thr Lys His Asn Glu Thr Pro     290 295 300 Lys Gln Ala Ser Lys Ala Lys Glu Leu Pro Lys Thr Gly Leu Thr Ser 305 310 315 320 Val Asp Asn Phe Ile Ser Thr Val Ala Phe Ala Thr Leu Ala Leu Leu                 325 330 335 Gly Ser Leu Ser Leu Leu Leu Phe Lys Arg Lys Glu Ser Lys             340 345 350 <210> 4 <211> 1938 <212> DNA <213> Staphylococcus aureus <400> 4 atgaacaaac agcaaaaaga atttaaatca ttttattcaa ttagaaagtc atcactaggc 60 gttgcatctg tagcgattag tacactttta ttattaatgt caaatggcga agcacaagca 120 gcagctgaag aaacaggtgg tacaaataca gaagcacaac caaaaactga agcagttgca 180 agtccaacaa caacatctga aaaagctcca gaaactaaac cagtagctaa tgctgtctca 240 gtatctaata aagaagttga ggcccctact tctgaaacaa aagaagctaa agaagttaaa 300 gaagttaaag cccctaagga aacaaaagca gttaaaccag cagcaaaagc cactaacaat 360 acatatccta ttttgaatca ggaacttaga gaagcgatta aaaaccctgc aataaaagat 420 aaagatcata gcgcaccaaa ctctcgtcca attgattttg aaatgaaaaa agaaaatggt 480 gagcaacaat tttatcatta tgccagctct gttaaacctg ctagagttat tttcactgat 540 tcaaaaccag aaattgaatt aggattacaa tcaggtcaat tttggagaaa atttgaagtt 600 tatgaaggtg acaaaaagtt gccaattaaa ttagtatcat acgatactgt taaagattac 660 gcttacattc gcttctctgt ttcaaatgga acaaaagccg ttaaaattgt aagttcaact 720 cacttcaata acaaagaaga aaaatacgat tacacattaa tggaattcgc acaaccaatt 780 tataacagtg cagataaatt caaaactgaa gaagattata aagctgaaaa attattagcg 840 ccatataaaa aagcgaaaac actagaaaga caagtttatg aattaaataa aattcaagat 900 aaacttcctg aaaaattaaa ggctgagtac aagaagaaat tagaggatac aaagaaagct 960 ttagatgagc aagtgaaatc agctattact gaattccaaa atgtacaacc aacaaatgaa 1020 aaaatgactg atttacaaga tacaaaatat gttgtttatg aaagtgttga gaataacgaa 1080 tctatgatgg atacttttgt taaacaccct attaaaacag gtatgcttaa cggcaaaaaa 1140 tatatggtca tggaaactac taatgacgat tactggaaag atttcatggt tgaaggtcaa 1200 cgtgttagaa ctataagcaa agatgctaaa aataatacta gaacaattat tttcccatat 1260 gttgaaggta aaactctata tgatgctatc gttaaagttc acgtaaaaac gattgattat 1320 gatggacaat accatgtcag aatcgttgat aaagaagcat ttacaaaagc caataccgat 1380 aaatctaaca aaaaagaaca acaagataac tcagctaaga aggaagctac tccagctacg 1440 cctagcaaac caacaccatc acctgttgaa aaagaatcac aaaaacaaga cagccaaaaa 1500 gatgacaata aacaattacc aagtgttgaa aaagaaaatg acgcatctag tgagtcaggt 1560 aaagacaaaa cgcctgctac aaaaccaact aaaggtgaag tagaatcaag tagtacaact 1620 ccaactaagg tagtatctac gactcaaaat gttgcaaaac caacaactgc ttcatcaaaa 1680 acaacaaaag atgttgttca aacttcagca ggttctagcg aagcaaaaga tagtgctcca 1740 ttacaaaaag caaacattaa aaacacaaat gatggacaca ctcaaagcca aaacaataaa 1800 aatacacaag aaaataaagc aaaatcatta ccacaaactg gtgaagaatc aaataaagat 1860 atgacattac cattaatggc attactagct ttaagtagca tcgttgcatt cgtattacct 1920 agaaaacgta aaaactaa 1938 <210> 5 <211> 1938 <212> DNA <213> Staphylococcus aureus <400> 5 ttagttttta cgttttctag gtaatacgaa tgcaacgatg ctacttaaag ctagtaatgc 60 cattaatggt aatgtcatat ctttatttga ttcttcacca gtttgtggta atgattttgc 120 tttattttct tgtgtatttt tattgttttg gctttgagtg tgtccatcat ttgtgttttt 180 aatgtttgct ttttgtaatg gagcactatc ttttgcttcg ctagaacctg ctgaagtttg 240 aacaacatct tttgttgttt ttgatgaagc agttgttggt tttgcaacat tttgagtcgt 300 agatactacc ttagttggag ttgtactact tgattctact tcacctttag ttggttttgt 360 agcaggcgtt ttgtctttac ctgactcact agatgcgtca ttttcttttt caacacttgg 420 taattgttta ttgtcatctt tttggctgtc ttgtttttgt gattcttttt caacaggtga 480 tggtgttggt ttgctaggcg tagctggagt agcttccttc ttagctgagt tatcttgttg 540 ttcttttttg ttagatttat cggtattggc ttttgtaaat gcttctttat caacgattct 600 gacatggtat tgtccatcat aatcaatcgt ttttacgtga actttaacga tagcatcata 660 tagagtttta ccttcaacat atgggaaaat aattgttcta gtattatttt tagcatcttt 720 gcttatagtt ctaacacgtt gaccttcaac catgaaatct ttccagtaat cgtcattagt 780 agtttccatg accatatatt ttttgccgtt aagcatacct gttttaatag ggtgtttaac 840 aaaagtatcc atcatagatt cgttattctc aacactttca taaacaacat attttgtatc 900 ttgtaaatca gtcatttttt catttgttgg ttgtacattt tggaattcag taatagctga 960 tttcacttgc tcatctaaag ctttctttgt atcctctaat ttcttcttgt actcagcctt 1020 taatttttca ggaagtttat cttgaatttt atttaattca taaacttgtc tttctagtgt 1080 tttcgctttt ttatatggcg ctaataattt ttcagcttta taatcttctt cagttttgaa 1140 tttatctgca ctgttataaa ttggttgtgc gaattccatt aatgtgtaat cgtatttttc 1200 ttctttgtta ttgaagtgag ttgaacttac aattttaacg gcttttgttc catttgaaac 1260 agagaagcga atgtaagcgt aatctttaac agtatcgtat gatactaatt taattggcaa 1320 ctttttgtca ccttcataaa cttcaaattt tctccaaaat tgacctgatt gtaatcctaa 1380 ttcaatttct ggttttgaat cagtgaaaat aactctagca ggtttaacag agctggcata 1440 atgataaaat tgttgctcac cattttcttt tttcatttca aaatcaattg gacgagagtt 1500 tggtgcgcta tgatctttat cttttattgc agggttttta atcgcttctc taagttcctg 1560 attcaaaata ggatatgtat tgttagtggc ttttgctgct ggtttaactg cttttgtttc 1620 cttaggggct ttaacttctt taacttcttt agcttctttt gtttcagaag taggggcctc 1680 aacttcttta ttagatactg agacagcatt agctactggt ttagtttctg gagctttttc 1740 agatgttgtt gttggacttg caactgcttc agtttttggt tgtgcttctg tatttgtacc 1800 acctgtttct tcagctgctg cttgtgcttc gccatttgac attaataata aaagtgtact 1860 aatcgctaca gatgcaacgc ctagtgatga ctttctaatt gaataaaatg atttaaattc 1920 tttttgctgt ttgttcat 1938 <210> 6 <211> 645 <212> PRT <213> Staphylococcus aureus <400> 6 Met Asn Lys Gln Gln Lys Glu Phe Lys Ser Phe Tyr Ser Ile Arg Lys   1 5 10 15 Ser Ser Leu Gly Val Ala Ser Val Ala Ile Ser Thr Leu Leu Leu Leu              20 25 30 Met Ser Asn Gly Glu Ala Gln Ala Ala Ala Glu Glu Thr Gly Gly Thr          35 40 45 Asn Thr Glu Ala Gln Pro Lys Thr Glu Ala Val Ala Ser Pro Thr Thr      50 55 60 Thr Ser Glu Lys Ala Pro Glu Thr Lys Pro Val Ala Asn Ala Val Ser  65 70 75 80 Val Ser Asn Lys Glu Val Glu Ala Pro Thr Ser Glu Thr Lys Glu Ala                  85 90 95 Lys Glu Val Lys Glu Val Lys Ala Pro Lys Glu Thr Lys Ala Val Lys             100 105 110 Pro Ala Ala Lys Ala Thr Asn Asn Thr Tyr Pro Ile Leu Asn Gln Glu         115 120 125 Leu Arg Glu Ala Ile Lys Asn Pro Ala Ile Lys Asp Lys Asp His Ser     130 135 140 Ala Pro Asn Ser Arg Pro Ile Asp Phe Glu Met Lys Lys Glu Asn Gly 145 150 155 160 Glu Gln Gln Phe Tyr His Tyr Ala Ser Ser Val Lys Pro Ala Arg Val                 165 170 175 Ile Phe Thr Asp Ser Lys Pro Glu Ile Glu Leu Gly Leu Gln Ser Gly             180 185 190 Gln Phe Trp Arg Lys Phe Glu Val Tyr Glu Gly Asp Lys Lys Leu Pro         195 200 205 Ile Lys Leu Val Ser Tyr Asp Thr Val Lys Asp Tyr Ala Tyr Ile Arg     210 215 220 Phe Ser Val Ser Asn Gly Thr Lys Ala Val Lys Ile Val Ser Ser Thr 225 230 235 240 His Phe Asn Asn Lys Glu Glu Lys Tyr Asp Tyr Thr Leu Met Glu Phe                 245 250 255 Ala Gln Pro Ile Tyr Asn Ser Ala Asp Lys Phe Lys Thr Glu Glu Asp             260 265 270 Tyr Lys Ala Glu Lys Leu Leu Ala Pro Tyr Lys Lys Ala Lys Thr Leu         275 280 285 Glu Arg Gln Val Tyr Glu Leu Asn Lys Ile Gln Asp Lys Leu Pro Glu     290 295 300 Lys Leu Lys Ala Glu Tyr Lys Lys Lys Leu Glu Asp Thr Lys Lys Ala 305 310 315 320 Leu Asp Glu Gln Val Lys Ser Ala Ile Thr Glu Phe Gln Asn Val Gln                 325 330 335 Pro Thr Asn Glu Lys Met Thr Asp Leu Gln Asp Thr Lys Tyr Val Val             340 345 350 Tyr Glu Ser Val Glu Asn Asn Glu Ser Met Met Asp Thr Phe Val Lys         355 360 365 His Pro Ile Lys Thr Gly Met Leu Asn Gly Lys Lys Tyr Met Val Met     370 375 380 Glu Thr Thr Asn Asp Asp Tyr Trp Lys Asp Phe Met Val Glu Gly Gln 385 390 395 400 Arg Val Arg Thr Ile Ser Lys Asp Ala Lys Asn Asn Thr Arg Thr Ile                 405 410 415 Ile Phe Pro Tyr Val Glu Gly Lys Thr Leu Tyr Asp Ala Ile Val Lys             420 425 430 Val His Val Lys Thr Ile Asp Tyr Asp Gly Gln Tyr His Val Arg Ile         435 440 445 Val Asp Lys Glu Ala Phe Thr Lys Ala Asn Thr Asp Lys Ser Asn Lys     450 455 460 Lys Glu Gln Gln Asp Asn Ser Ala Lys Lys Glu Ala Thr Pro Ala Thr 465 470 475 480 Pro Ser Lys Pro Thr Pro Ser Pro Val Glu Lys Glu Ser Gln Lys Gln                 485 490 495 Asp Ser Gln Lys Asp Asp Asn Lys Gln Leu Pro Ser Val Glu Lys Glu             500 505 510 Asn Asp Ala Ser Ser Glu Ser Gly Lys Asp Lys Thr Pro Ala Thr Lys         515 520 525 Pro Thr Lys Gly Glu Val Glu Ser Ser Ser Thr Thr Pro Thr Lys Val     530 535 540 Val Ser Thr Thr Gln Asn Val Ala Lys Pro Thr Thr Ala Ser Ser Lys 545 550 555 560 Thr Thr Lys Asp Val Val Gln Thr Ser Ala Gly Ser Ser Glu Ala Lys                 565 570 575 Asp Ser Ala Pro Leu Gln Lys Ala Asn Ile Lys Asn Thr Asn Asp Gly             580 585 590 His Thr Gln Ser Gln Asn Asn Lys Asn Thr Gln Glu Asn Lys Ala Lys         595 600 605 Ser Leu Pro Gln Thr Gly Glu Glu Ser Asn Lys Asp Met Thr Leu Pro     610 615 620 Leu Met Ala Leu Leu Ala Leu Ser Ser Ile Val Ala Phe Val Leu Pro 625 630 635 640 Arg Lys Arg Lys Asn                 645 <210> 7 <211> 684 <212> DNA <213> Staphylococcus aureus <400> 7 atgaaaaata ttttaaaagt ttttaataca acgattttag cgttaattat catcatcgcg 60 acattcagta attctgcaaa tgccgcagat agcggtactt tgaattatga ggtttacaaa 120 tacaatacca atgacacgtc aattgctaat gactatttta ataaaccggc aaagtacatt 180 aagaaaaatg gtaaattgta tgttcaaata actgtcaacc acagtcattg gattactgga 240 atgagtatcg aaggacataa agaaaatatt attagtaaaa acactgccaa agatgaacgc 300 acttctgaat ttgaagtaag taagttgaac ggtaaaatag atggaaaaat tgacgtttat 360 atcgatgaaa aagtaaatgg aaagccattc aaatatgacc atcattacaa cattacatat 420 aaatttaatg gaccaactga tgtagcaggt gctaatgcac caggtaaaga tgataaaaat 480 tctgcttcag gtagtgacaa aggatctgat ggaacgacta ctggtcaaag tgaatctaat 540 agttcgaata aagacaaagt agaaaatcca caaacaaatg ctggtacacc tgcatatata 600 tatgcaatac cagttgcatc cttagcatta ttaatcgcaa tcacattgtt tgttagaaaa 660 aaatctaaag gcaatgtgga ataa 684 <210> 8 <211> 684 <212> DNA <213> Staphylococcus aureus <400> 8 ttattccaca ttgcctttag atttttttct aacaaacaat gtgattgcga ttaataatgc 60 taaggatgca actggtattg catatatata tgcaggtgta ccagcatttg tttgtggatt 120 ttctactttg tctttattcg aactattaga ttcactttga ccagtagtcg ttccatcaga 180 tcctttgtca ctacctgaag cagaattttt atcatcttta cctggtgcat tagcacctgc 240 tacatcagtt ggtccattaa atttatatgt aatgttgtaa tgatggtcat atttgaatgg 300 ctttccattt actttttcat cgatataaac gtcaattttt ccatctattt taccgttcaa 360 cttacttact tcaaattcag aagtgcgttc atctttggca gtgtttttac taataatatt 420 ttctttatgt ccttcgatac tcattccagt aatccaatga ctgtggttga cagttatttg 480 aacatacaat ttaccatttt tcttaatgta ctttgccggt ttattaaaat agtcattagc 540 aattgacgtg tcattggtat tgtatttgta aacctcataa ttcaaagtac cgctatctgc 600 ggcatttgca gaattactga atgtcgcgat gatgataatt aacgctaaaa tcgttgtatt 660 aaaaactttt aaaatatttt tcat 684 <210> 9 <211> 227 <212> PRT <213> Staphylococcus aureus <400> 9 Met Lys Asn Ile Leu Lys Val Phe Asn Thr Thr Ile Leu Ala Leu Ile   1 5 10 15 Ile Ile Ile Ala Thr Phe Ser Asn Ser Ala Asn Ala Ala Asp Ser Gly              20 25 30 Thr Leu Asn Tyr Glu Val Tyr Lys Tyr Asn Thr Asn Asp Thr Ser Ile          35 40 45 Ala Asn Asp Tyr Phe Asn Lys Pro Ala Lys Tyr Ile Lys Lys Asn Gly      50 55 60 Lys Leu Tyr Val Gln Ile Thr Val Asn His Ser His Trp Ile Thr Gly  65 70 75 80 Met Ser Ile Glu Gly His Lys Glu Asn Ile Ile Ser Lys Asn Thr Ala                  85 90 95 Lys Asp Glu Arg Thr Ser Glu Phe Glu Val Ser Lys Leu Asn Gly Lys             100 105 110 Ile Asp Gly Lys Ile Asp Val Tyr Ile Asp Glu Lys Val Asn Gly Lys         115 120 125 Pro Phe Lys Tyr Asp His His Tyr Asn Ile Thr Tyr Lys Phe Asn Gly     130 135 140 Pro Thr Asp Val Ala Gly Ala Asn Ala Pro Gly Lys Asp Asp Lys Asn 145 150 155 160 Ser Ala Ser Gly Ser Asp Lys Gly Ser Asp Gly Thr Thr Thr Gly Gln                 165 170 175 Ser Glu Ser Asn Ser Ser Asn Lys Asp Lys Val Glu Asn Pro Gln Thr             180 185 190 Asn Ala Gly Thr Pro Ala Tyr Ile Tyr Ala Ile Pro Val Ala Ser Leu         195 200 205 Ala Leu Leu Ile Ala Ile Thr Leu Phe Val Arg Lys Lys Ser Lys Gly     210 215 220 Asn val glu 225 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide motif <220> <221> MOD_RES <222> (3) Variable amino acid residue <400> 10 Leu Pro Xaa Thr Gly   1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide motif <400> 11 Asn Pro Gln Thr Asn   1 5 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 12 Lys Asp Glu Leu   One  

Claims (24)

A vaccine comprising an IsdA (SEQ ID NO: 3) polypeptide and a pharmaceutically acceptable carrier. The vaccine of claim 1, wherein the vaccine is in an injectable formulation. The vaccine of claim 1, further comprising an adjuvant. IsdB (SEQ ID NO: 6) A vaccine comprising a polypeptide and a pharmaceutically acceptable carrier. The vaccine of claim 4 which is present in the injectable formulation. The vaccine of claim 4 further comprising an adjuvant. A vaccine comprising an IsdC (SEQ ID NO: 9) polypeptide and a pharmaceutically acceptable carrier. 8. The vaccine of 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 antibody that binds to an anti-bacterial effective amount of IsdA (SEQ ID NO: 3) and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody that binds to an anti-bacterial effective amount of IsdB (SEQ ID NO: 6) and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody that binds to an anti-bacterial effective amount of IsdC (SEQ ID NO: 9) and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a nucleic acid that is 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. A subject caused or caused by an infection of Staphylococcus aureus of a subject, comprising administering to the subject an effective amount of the vaccine of claim 1. How to treat or prevent a disease or condition. A method of treating or preventing a disease or condition caused by or caused by an infection of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 10. A method of treating or preventing a disease or condition caused by or caused by an infection of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 11. A method of treating or preventing a disease or condition caused by or caused by an infection of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 12. A method of treating or preventing a disease or condition caused by or caused by an infection of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 13. A method for treating or preventing a disease or condition caused by or caused by an infection of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 14. (i) contacting the Isd polypeptide with the appropriate interacting molecule in the presence of the agent under conditions that allow for interaction between the Isd polypeptide and the interacting molecule in the absence of the agent; And (ii) identifying an agent that binds to the Isd polypeptide and inhibits iron absorption, including determining a level of interaction between the Isd polypeptide and the interacting molecule, And a different level of interaction between the Isd polypeptide and the interacting molecule in the presence of the agent in the absence of the agent indicates that the agent inhibits the interaction between the Isd polypeptide and the interacting molecule. The method of claim 21, wherein the Isd polypeptide is selected from the group consisting of Staphylococcus aureus IsdA, IsdB, and IsdC. (i) culturing the wild type Staphylococcus aureus strain in the presence or absence of an agent; And (ii) comparing the expression of the Isd polypeptide, the method of identifying an agent that inhibits the expression of a polypeptide selected from the group consisting of IsdA, IsdB and IsdC polypeptides of Staphylococcus aureus, the agent A greater reduction in the expression of an Isd polypeptide in a cell treated with indicates that the agent inhibits the expression of an Isd polypeptide of Staphylococcus aureus. (i) culturing the wild type Staphylococcus aureus in the presence or absence of an agent; And (ii) comparing the expression of the isd nucleic acid, the method of identifying an agent that inhibits the expression of a nucleic acid selected from the group consisting of isdA , isdB and isdC nucleic acids of Staphylococcus aureus, the method of zero than the large reduction in the process of the isd nucleic acid in cells expressing the effect that I indicating that inhibits the expression of nucleic acid of isd Stability Philo nose kusu aureus.

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