WO2010043842A1

WO2010043842A1 - Staphylococcus protein essential for growth

Info

Publication number: WO2010043842A1
Application number: PCT/GB2009/002291
Authority: WO
Inventors: Simon J. Foster; Victoria Fairclough
Original assignee: University Of Sheffield
Priority date: 2008-10-13
Filing date: 2009-09-29
Publication date: 2010-04-22
Also published as: GB0818627D0

Abstract

The invention relates to antigenic polypeptides expressed by Staphylococcus spp, vaccines comprising the antigenic polypeptides and therapeutic antibodies directed to the antigenic polypeptides.

Description

STAPHYLOCOCCUS PROTEIN ESSENTIAL FOR GROWTH

Field of the Invention

The invention relates to nucleic acids and proteins from Staphylococcus spp.

Background to the Invention

Staphylococcus aureus causes several diseases by various pathogenic mechanisms. The most frequent and serious of these diseases are bacteremia and its complications in hospitalized patients. In particular, Staphylococcus aureus can cause wound infections and infections associated with catheters and prosthetic devices. Serious infections associated with Staphylococcus aureus include bacteremia, osteomyelitis, invasive endocarditis and septicemia. Staphylococci have developed very sophisticated mechanisms for inducing diseases in humans, including both intracellular and extracellular factors. For instance, Staphylococcus aureus possesses several surface antigens that facilitate its survival in the blood stream by helping the bacteria to evade phagocytic killing by the host leukocytes. These surface antigens include cell wall components such as teichoic acid, protein A, and capsular polysaccharides (CPS). Due in part to the versatility of these bacteria and their ability to produce extracellular products that enhance virulence and pathogenicity, staphylococcal bacteremia and its complications continue to be serious and frequently observed nosocomial infections.

Antibiotics such as penicillin have been used successfully against both staphylococcal and enterococcal infections in humans, but more recently the effectiveness of such antibiotics has been thwarted by the ability of bacteria to develop resistance. For example, shortly after the introduction of methicillin, the first semisynthetic penicillin, strains of methicillin-resistant Staphylococcus aureus (MRSA) were isolated. Antibiotic resistance among staphylococcal isolates from nosocomial infections continues to increase in frequency, and resistant Staphylococcus aureus strains continue to cause epidemics in hospitals in spite of developed preventive procedures and extensive research into bacterial epidemiology and antibiotic development. Enterococci resistant to vancomycin started to appear in 1988 and have now become commonplace among hospital-acquired infections. Although methicillin-resistant Staphylococcus aureus organisms with intermediate resistance to vancomycin have been identified in some centers, it was only relatively recently that three Staphylococcus aureus strains with complete resistance to vancomycin were reported. This suggests that the probable conjugal transfer of vancomycin resistance from Enterococci to Staphylococci has become a reality, and dissemination of these strains could eventually lead to the widespread development of organisms that are more difficult to eradicate. The problem is compounded by multiple antibiotic resistance in hospital strains, which severely limits the choice of therapy.

The initial efficacy of antibiotics in treating and curing staphylococcal infections drew attention away from immunological approaches for dealing with these infections. Although multiple antibiotic-resistant strains of Staphylococcus aureus have emerged, other strategies such as vaccines have not been developed. In addition, passive immunization has been tested for use in immune-compromised individuals, such as neonates, who are at increased risk for contracting these bacterial infections. The data failed to support a solid conclusion in recommending the use of passive immunization in this population. Baker et al., New Engl. J. Med. 35:213-219 (1992); Fanaroff et al., New Engl. J. Med. 330:1107-1113 (1994).

During the process of any infection the virulence determinants made by Staphylococcus aureus are produced in response to environmental and physiological stimuli. These stimuli will be dependent on the niche within the body and will change as the infection progresses. Little is known of the conditions in vivo and it is likely that some components are produced solely in this environment. These are therefore potential vaccine components, which could not be discovered by previous techniques.

Many vaccines are produced by inactivated or attenuated pathogens which are injected into an individual. The immunised individual responds by producing both a humoral (antibody) and cellular (cytolytic T cells, CTL's) response. For example, hepatitis vaccines are made by heat inactivating the virus and treating it with a cross linking agent such as formaldehyde. An example of an attenuated pathogen useful as a vaccine is represented by polio vaccines which are produced by attenuating a live pathogen.

However the use of attenuated organisms in vaccines for certain diseases is problematic due to the lack of knowledge regarding the pathology of the condition and the nature of the attenuation. For certain viral agents this is a particular problem since viruses, in particular retroviruses, have an error prone replication cycle which results in viable mutations in the genes which comprise the virus. This can result in alterations to antigenic determinants which have previously been used as vaccines. The development of so-called subunit vaccines (vaccines in which the immunogen is a fragment or subunit of a protein or complex expressed by a particular pathogenic organism) has been the focus of considerable medical research. The need to identify candidate molecules useful in the development of subunit vaccines is apparent not least because conventional chemotherapeutic approaches to the control of pathogenic organisms has more recently been stymied by the development of antibiotic resistance.

The present inventors have identified a protein that has been shown to be essential for growth in Staphylococcus aureus. This was unexpected as it was previously shown that the same protein is non-essential in the model organism Bacillus subtilis (Levin et al., 1999). Without wishing to be bound by theory, it is believed that the protein is required for cell division in Staphylococcus aureus.

Statements of the Invention

According to a first aspect of the invention there is provided an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1 ; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii) for use as a medicament.

In an embodiment of the invention the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence as shown in Figure 1.

In a preferred aspect of the invention the medicament is a vaccine.

The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may anneal under stringent hybridisation conditions to the nucleic acid sequence shown in Figure 1 or to its complementary strand. Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65⁰C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows seguences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may comprise the sequence set out in Figure 1 or a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, for example 98%, or 99%, identical to the nucleic acid sequence set out in Figure 1 at the nucleic acid residue level.

"Identity", as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity can be readily calculated (Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., AND Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, the term is well-known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleid Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403 (1990)).

The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may comprise of a fragment of a sequence according to the first aspect which is at least 30 bases long, for example, 40, 50, 60, 70, 80 or 90 bases in length.

The nucleic acid sequence encoding the antigenic polypeptide of the first aspect of the invention may be genomic DNA, complementary DNA (cDNA) or RNA, for example messenger RNA (mRNA).

Preferably, the antigenic polypeptide of the first aspect of the invention is expressed by a bacterium of the genus Staphylococcus, for example Staphylococcus aureus.

In a preferred embodiment of the invention, the antigenic polypeptide of the first aspect of the invention is associated with infective pathogenicity of an organism as defined herein. In a further preferred aspect of the invention the antigenic polypeptide comprises the amino acid sequence shown in Figure 2 or a variant sequence thereof.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by non- amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component.

The term "variant" as used herein includes polypeptides that may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative (similar) replacements: a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and T) phenylalanine, tyrosine and tryptophan.

Amino acid substitutions can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Amino acid substitutions are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide. Groups of functionally related amino acids within which conservative substitutions may be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; asvariantic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan.

Polypeptides of this invention may be in glycosylated or unglycosylated form, may be modified post-translationally (e.g., acetylation, and phosphorylation) or may be modified synthetically (e.g., the attachment of a labeling group).

As used herein, the term "polypeptide" means, in general terms, a plurality of amino acid residues joined together by peptide bonds. It is used interchangeably and means the same as peptide, protein, oligopeptide, or oligomer. The term "polypeptide" is also intended to include fragments, analogues and derivatives of a polypeptide wherein the fragment, analogue or derivative retains essentially the same biological activity or function as a reference protein. The term "polypeptide" also includes peptidomimetics and structural analogues of the described sequences, and those modified either naturally (e.g. post-translational modification) or chemically, including, but not exclusively, phosphorylation, glycosylation, sulfonylation and/or hydroxylation.

A "variant thereof of an antigenic polypeptide according to the invention may thus include a fragment or subunit of the antigenic polypeptide wherein the fragment or subunit is sufficient to induce an antigenic response in a recipient. Thus the present invention encompasses an antigenic polypeptide comprising an amino acid sequence as represented in Figure 2 or a fragment thereof or a variant polypeptide wherein said variant is modified by addition, deletion or substitution of at least one amino acid residue of the amino acid sequence presented in Figure 2 and wherein said variant polypeptide is sufficient to induce an antigenic response in a recipient. As used herein "a fragment of a polypeptide comprising the amino acid sequence as shown in Figure 2" includes fragments that contain between 1 and 50 amino acids, for example between 1 and 30 amino acids such as between 10 and 30 amino acids. In one embodiment the variant is an antigenic polypeptide comprising the amino acid sequence as represented in Figure 2.

According to a second aspect of the invention there is provided a vector comprising a nucleic acid sequence encoding a polypeptide according to the first aspect of the invention.

The vector of the second aspect of the invention may be a plasmid, cosmid, phage or virus based vector. The vector may include a transcription control sequence (promoter sequence) which mediates cell specific expression, for example, a cell specific, inducible or constitutive promoter sequence. The vector may be an expression vector adapted for prokaryotic or eukaryotic gene expression, for example, the vector may include one or more selectable markers and/or autonomous replication sequences which facilitate the maintenance of the vector in either a eukaryotic cell or prokaryotic host (Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). Vectors which are maintained autonomously are referred to as episomal vectors. Promoter is an art-recognised term and may include enhancer elements which are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences and is therefore position independent). Enhancer activity is responsive to trans acting transcription factors (polypeptides e.g. phosphorylated polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues which include intermediary metabolites (eg glucose, lipids), environmental effectors (eg light, heat,).

Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

The vector of the second aspect of the invention may include a transcription termination or polyadenylation sequences. This may also include an internal ribosome entry sites (IRES). The vector may include a nucleic acid sequence that is arranged in a bicistronic or multi-cistronic expression cassette.

According to a third aspect of the invention there is provided a method for the production of a recombinant antigenic polypeptide according to any previous aspect of the invention comprising:

(i) providing a cell transformed/transfected with a vector according to the second aspect of the invention; (ii) growing said cell in conditions suitable for the production of said polypeptides; and (iii) purifying said polypeptide from said cell, or its growth environment.

In a preferred aspect of the method of the third aspect, the vector encodes, and thus said recombinant polypeptide is provided with, a secretion signal to facilitate purification of said polypeptide. According to a fourth aspect of the invention there is provided a cell or cell-line transformed or transfected with the vector according to the second aspect of the invention.

In a preferred embodiment of the invention said cell is a prokaryotic cell, for example, a bacterium such as E. coli. Alternatively said cell is a eukaryotic cell, for example a yeast or other fungal cell, insect, amphibian, or mammalian cell, for example, COS, CHO cells, Bowes Melanoma and other suitable human cells, or plant cell.

According to a fifth aspect of the invention there is provided a vaccine comprising at least one antigenic polypeptide, or variant thereof, according to the first aspect of the invention. Preferably said vaccine further comprises an adjuvant/carrier.

The vaccine may comprise an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In one embodiment of the invention the vaccine comprises an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence as shown in Figure 1.

The vaccine according to the fifth aspect may be a subunit vaccine in which the immunogenic part of the vaccine is a fragment or subunit of the antigenic polypeptide according to the first aspect of the invention.

An adjuvant is a substance or procedure that augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, Freunds adjuvant, squalene, phosphate adjuvants and aluminium salts

(e.g. aluminium hydroxide or aluminium phosphate). Others may include muramyl dipeptides, liposomes. A carrier is an immunogenic molecule which, when bound to a second molecule, augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the "carrier" protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.

In yet a further aspect of the invention there is provided a method to immunise an animal against a pathogenic microbe comprising administering to said animal at least one polypeptide, or variant thereof, according to the first aspect of the invention. Preferably, the polypeptide is in the form of a vaccine according to the fifth aspect of the invention.

In a preferred method of the invention the animal is human.

Preferably the antigenic polypeptide of the first aspect, or the vaccine of the fifth aspect, of the invention can be delivered by direct injection either intravenously, intramuscularly, subcutaneously. Further still, the vaccine or antigenic polypeptide, may be taken orally. The polypeptide or vaccine may be administered in a pharmaceutically acceptable carrier, such as the various aqueous and lipid media, such as sterile saline, utilized for preparing injectables to be administered intramuscularly and subcutaneously. Conventional suspending and dispersing agents can be employed. Other means of administration, such as implants, for example a sustained low dose releasing bio- observable pellet, will be apparent to the skilled artisan.

The vaccine may be against a bacterial species of the genus Staphylococcus for example Staphylococcus aureus.

It will also be apparent that vaccines or antigenic polypeptides are effective at preventing or alleviating conditions in animals other than humans, for example and not by way of limitation, companion animals (e.g. domestic animals such as cats and dogs), livestock (e.g. cattle, sheep, pigs) and horses.

According to a further aspect of the invention there is provided an agent that binds to at least one antigenic polypeptide, or variant thereof, according to the invention. Preferably the agent is an antagonist. Preferably the agent inhibits the activity of said antigenic polypeptide. As used herein the term "inhibits" refers to a species which retards, blocks or prevents an interaction. Typically, inhibition does not result in 100% blockage but rather reduces the amount and/or speed of interaction.

Preferably the agent is an antibody or active binding fragment thereof. The antibody, or active binding fragment, may be a polyclonal antibody or a monoclonal antibody. Preferably the antibody, or active binding fragment, is a monoclonal antibody.

Antibodies or immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant. The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region. The variable region contains complementarity determining regions or CDR's which form an antigen binding pocket. The binding pockets comprise H and L variable regions which contribute to antigen recognition. It is possible to create single variable regions, so called single chain antibody variable region fragments (scFv's). If a hybridoma exists for a specific monoclonal antibody it is well within the knowledge of the skilled person to isolate scFv's from mRNA extracted from said hybridoma via RT PCR. Alternatively, phage display screening can be undertaken to identify clones expressing scFv's. Alternatively said fragments are "domain antibody fragments". Domain antibodies are the smallest binding part of an antibody (approximately 13 kDa). Examples of this technology is disclosed in US6, 248, 516, US6, 291, 158, US6.127, 197 and EP0368684 which are all incorporated by reference in their entirety.

In a preferred embodiment of the invention said antibody fragment is a single chain antibody variable region fragment.

In a further preferred embodiment of the invention said antibody is a humanised or chimeric antibody. A chimeric antibody is produced by recombinant methods to contain the variable region of an antibody with an invariant or constant region of a human antibody. A humanised antibody is produced by recombinant methods to combine the complementarity determining regions (CDRs) of an antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody. Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

In a further preferred aspect of the invention said antibody is a chimeric antibody produced by recombinant methods to contain the variable region of said antibody with an invariant or constant region of a human antibody.

In a further preferred aspect of the invention, said antibody is humanised by recombinant methods to combine the complimentarity determining regions of said antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

Preferably said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag.

Preferably said humanised monoclonal antibody to said polypeptide is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells.

Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.

In another aspect of the invention there is provided a vector comprising a nucleic acid sequence encoding the humanised or chimeric antibodies according to the invention.

In a yet further aspect of the invention, there is provided a cell or cell line which comprises the vector encoding the humanised or chimeric antibody according to the invention. The cell or cell line may be transformed or transfected with the vector encoding the humanised or chimeric antibody according to the invention.

In a yet further aspect of the invention there is provided a hybridoma cell line which produces a monoclonal antibody as hereinbefore described.

In a further aspect of the invention there is provided a method of producing monoclonal antibodies according to the invention using hybridoma cell lines according to the invention. .

In a yet further aspect of the invention there is provided a method for the production of the humanised or chimeric antibody according to the invention comprising:

(i) providing a cell transformed or transfected with a vector which comprises a nucleic acid molecule encoding the humanised or chimeric antibody according to the invention;

(ii) growing said cell in conditions suitable for the production of said antibody; and purifying said antibody from said cell, or its growth environment.

In a further aspect of the invention there is provided a method for preparing a hybridoma cell-line according to the invention comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having an amino acid sequence as represented in Figure 2, or a fragment thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.

In one embodiment, the polypeptide in method step (i) has an amino acid sequence as represented in Figure 2, or a fragment thereof.

The immunocompetent mammal may be a mouse, rat or rabbit.

The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, "Basic Facts about Hybridomas" in Compendium of Immunology V.ll ed. by Schwartz, 1981 , which are incorporated by reference.

A further aspect of the invention provides a pharmaceutical composition comprising an effective amount of at least one antigenic polypeptide, vaccine or agent according to the invention. When administered, the pharmaceutical compositions and formulations of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents. The compositions and formulations of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When antibodies are used therapeutically, one particular route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. The compositions and formulations of the invention are typically administered in effective amounts. An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response.

When administered, the pharmaceutical preparations and formulations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Pharmaceutical compositions and formulations may be combined if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

In a further aspect of the invention there is provided the use of an antigenic polypeptide according to the first aspect of the invention in the manufacture of a medicament for the treatment or prophylaxis of a Staphylococcus (e.g. Staphylococcus aureus) infection or a StapΛy/ococcus-associated disorder. As used herein the expression "Staphylococcus infection" refers to an infection caused, or contributed to, by a bacterial pathogen belonging to a bacterial species of the genus Staphylococcus.

As used herein the expression "StapΛy/ococcus-associated disorder" refers to a disorder caused, or contributed to, by a Staphylococcus infection.

The StapΛy/ococci/s-associated disorder may be selected from the group consisting of bacteremia, septicaemia, tuberculosis, bacteria-associated food poisoning, blood infections, peritonitis, endocarditis, osteomyelitis, septicemia, skin disorders, meningitis, pneumonia, stomach ulcers, gonorrhoea, strep throat, toxic shock, necrotizing fasciitis, impetigo, histoplasmosis, Lyme disease, gastro-enteritis, dysentery and shigellosis.

Preferably the StapΛy/ococctvs-associated disorder is bacteremia or septicaemia.

A further aspect of the invention there is provided the use of antibodies according to the invention in the manufacture of a medicament for the treatment of a Staphylococcus (e.g. Staphylococcus aureus) infection or a Støpfty/ococcus-associated disorder.

In a further aspect of the invention there is provided a method of treating a patient comprising administering to the patient an antigenic polypeptide according to the first aspect of the invention, or a vaccine according to the fifth aspect of the invention, or an antibody according to the invention. Preferably the method is for the treatment of a Staphylococcus (e.g. Staphylococcus aureus) infection or a StapΛy/ococcus-associated disorder. Preferably the patient is human.

The present invention also provides the use of an antigenic polypeptide, or variant thereof, in the identification of agents which modulate the activity of said polypeptide wherein the polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1 ; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii). In a preferred use according to the invention the polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence as shown in Figure 1.

According to a further aspect of the invention there is provided a kit comprising an agent specifically reactive with a polypeptide encoded by a nucleic acid sequence as represented in Figure 1 , or a fragment or variant thereof as defined herein, or an agent specifically reactive with a polypeptide comprising an amino acid sequence as represented in any of Figure 2, or a fragment or variant thereof as defined herein.

In a preferred embodiment of the invention said kit further comprises an oligonucleotide or antibody specifically reactive with said nucleic acid molecule or said polypeptide.

Preferably said kit comprises a thermostable DNA polymerase and components required for conducting the amplification of nucleic acid. Preferably said kit includes a set of instructions for conducting said polymerase chain reaction and control nucleic acid.

In an alternative preferred embodiment of the invention said kit comprises an antibody specifically reactive with a polypeptide comprising an amino acid sequence as represented in Figure 2, or a fragment or variant thereof as defined herein.

According to a further aspect of the invention there is provided a method to screen for an agent that modulates the activity of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii); wherein the method comprises a) forming a preparation comprising a polypeptide, or sequence variant thereof, and at least one agent to be tested; b) determining the activity of said agent with respect to the activity of said polypeptide. A difference between said activity in the presence of the agent and in the absence of the agent is indicative that the agent modulates said activity. Typically the agent inhibits the activity of the polypeptide.

The amino acid sequence represented in Figure 2 can be used for the structure-based design of molecules which modulate (e.g. inhibit) the activity of the polypeptide. Such structure based design is also known as "rational drug design". The proteins can be three-dimensionally analysed by, for example, X-ray crystallography, nuclear magnetic resonance or homology modelling, all of which are well-known methods. The use of structural information in molecular modelling software systems is also encompassed by the invention. Such computer-assisted modelling and drug design may utilise information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding etc. One particular method of the invention may comprise analysing the three-dimensional structure of the protein of Figure 2 for likely binding sites of targets, synthesising a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.

In a method of the invention said agent may be an antagonist. Agents identified by the screening method of the invention may include, antibodies, small organic molecules, (for example peptides, cyclic peptides), and dominant negative variants of the polypeptides herein disclosed.

As mentioned above, the invention also provides, in certain embodiments, "dominant negative" polypeptides derived from the polypeptides herein disclosed. A dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand. Likewise, a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphoryiate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal. Similarly, a dominant negative transcription factor which binds to another transcription factor or to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription. It will be apparent to one skilled in the art that modification to the amino acid sequence of polypeptides or agents according to the present invention could enhance the binding and/or stability of the peptide with respect to its target sequence. Modifications include, by example and not by way of limitation, acetylation and amidation. Alternatively or preferably, said modification includes the use of modified amino acids in the production of recombinant or synthetic forms of peptides. It will be apparent to one skilled in the art that modified amino acids include, for example, 4-hydroxyproline, 5-hydroxylysine, N⁶- acetyllysine, N⁶-methyllysine, N⁶,N⁶-dimethyllysine, N⁶,N⁶,N⁶-trimethyllysine, cyclohexyalanine, D-amino acids, ornithine. Other modifications include amino acids with a C_2, C₃ or C₄ alkyl R group optionally substituted by 1 , 2 or 3 substituents selected from halo ( eg F, Br, I), hydroxy or C₁-C₄ alkoxy. It will also be apparent to one skilled in the art that polypeptides could be modified by cyclisation. Cyclisation is known in the art, (see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res (2001), 57: 277-291 ; Dutta et al J. Peptide Res (2000), 8: 398-412; Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following materials, methods and figures:

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the DNA sequence of EzrA from Staphylococcus aureus;

Figure 2 shows the amino acid sequence of EzrA from Staphylococcus aureus;

Figure 3 S. aureus P_spac-ezrA strain is unable to form isolated colonies on BHI agar in the absence of IPTG

Figure 4 Effect of depletion of EzrA on growth of S. aureus. SH 1000 lacl and P_Sp_ac-ezri4 strains were grown overnight and then subcultured to OD₆₀₀ 0.4 in media with 100 μM IPTG. Cells were washed 3 times in warm BHI to remove traces of the inducer, and then diluted to OD₆₀₀ 0.001 in warm media with 0 or 1000 μM IPTG. Both strains were grown at 37°C in BHI medium containing appropriate antibiotics;

EXAMPLE

EzrA is essential for growth of Staphylococcus aureus

To prove essentiality of EzrA in Staphylococcus aureus, a conditional mutant was generated. The gene was placed under the control of the IPTG-inducible Spac promoter (Ps_pacjYansura and Henner, 1984) using pAISHI (Aish, 2003).

The 5' end of EzrA and its ribosome-binding site was amplified by PCR using high fidelity Extensor PCR Reddymix (Thermo Scientific), the oligonucleotides VRF75 and VRF76 (5¹- AAAAAAGAATTCAATGATAAATTAGGAGGAGAAGCA-S' and 5'- AAAAAAGGATCCCGGCTAATTAATGGTTCAACG-S' respectively) and SH 1000 chromosomal DNA as template. The 870 bp product was digested with EcoRI and BamHI restriction enzymes and ligated into the pAISHI vector digested with the same enzymes. The resulting plasmid, pVF23, was extracted from £. coli and transformed into Staphylococcus aureus RN4220 by electroporation.

Following transformation of pVF23 into Staphylococcus aureus RN4220, and integration of the plasmid into the chromosome via single crossover recombination, a full length copy of EzrA was placed under the control of Ps_Pac_> and the 5' portion of the gene was left under the control of the native promoter and in a transcriptional fusion with lacZ. The resulting strain was checked by PCR to confirm that the plasmid had integrated correctly and was transduced into Staphylococcus aureus SH1000 wild type. In order to prevent expression from Psp_acin the absence of IPTG, the lacl gene encoding the Lacl repressor was then introduced on a multicopy plasmid, pGL485.

The resulting strain is unable to form isolated colonies on solid media in the absence of IPTG (Figure 3) and shows a severe growth defect upon removal of the inducer in liquid culture (Figure 4). These results show that EzrA is essential for growth of Staphylococcus aureus.

Claims

1. An antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1 ; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii) for use as a medicament.

2. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the polypeptide is encoded by an isolated nucleic acid sequence as shown in Figure 1.

3. An antigenic polypeptide as claimed in any one preceding claim wherein the medicament is a vaccine.

4. An antigenic polypeptide as claimed in any preceding claim wherein the nucleic acid encoding the antigenic polypeptide anneals under stringent hybridisation conditions to the nucleic acid sequence shown in Figure 1 or to its complementary strand.

5. An antigenic polypeptide as claimed in claim 1 wherein the antigenic polypeptide comprises the amino acid sequence shown in Figure 2 or a variant sequence thereof.

6. A vector comprising a nucleic acid sequence encoding an antigenic polypeptide as claimed in any one of claims 1 to 5.

7. A method for the production of a recombinant antigenic polypeptide as claimed in any one of claims 1 to 5 comprising:

(i) providing a cell transformed/transfected with a vector according to claim 6;

(ii) growing said cell in conditions suitable for the production of said polypeptides; and (iii) purifying said polypeptide from said cell, or its growth environment.

8. A cell or cell-line transformed or transfected with a vector according to claim 6.

9. A vaccine comprising at least one antigenic polypeptide, or variant thereof, as claimed in any one of claims 1 to 5.

10. A vaccine as claimed in claim 9 wherein the vaccine further comprises a carrier and/or adjuvant.

11. A vaccine as claimed in claim 9 or 10 wherein the vaccine is a subunit vaccine in which the immunogenic part of the vaccine is a fragment or subunit of the antigenic polypeptide according to any one of claims 1 to 5.

12. A method to immunise an animal against a pathogenic microbe comprising administering to said animal at least one antigenic polypeptide, or part thereof, according to any one of claims 1 to 5.

13. A method as claimed in claim 12 wherein the polypeptide is in the form of a vaccine according to any one of claims 9 to 11.

14. A pharmaceutical composition comprising an effective amount of at least one of the antigenic polypeptides as claimed in any one of claims 1 to 5, or a vaccine as claimed in any one of claims 9 to 11 , in combination with a pharmaceutically acceptable carrier or diluent.

15. An antibody, or active binding fragment thereof, which binds at least one antigenic polypeptide, or variant thereof, according to any one of claims 1 to 5.

16. An antibody as claimed in claim 15 wherein the antibody is a monoclonal antibody.

17. A hybridoma cell line which produces a monoclonal antibody as claimed in claim 16.

18. An antibody as claimed in claim 15 or 16 wherein the antibody is a chimeric antibody.

19. An antibody as claimed in claim 15 or 16 wherein the antibody is a humanised antibody comprising the complimentarity determining regions of said antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

20. A vector comprising a nucleic acid sequence encoding a chimeric antibody according to claim 18 or a humanised antibody according to claim 19.

21. A cell or cell line transformed or transfected with the vector of claim 20.

22. A method for the production of a humanised or chimeric antibody comprising: i) providing a cell transformed or transfected with a vector according to claim 20; ii) growing said cell in conditions suitable for the production of said antibody; and purifying said antibody from said cell, or its growth environment.

23. A method for preparing a hybridoma cell-line comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having an amino acid sequence as represented in Figure 2, or a fragment thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step

(ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.

24. Use of an antigenic polypeptide as claimed in any one of claims 1 to 5 in the manufacture of a medicament for the treatment or prophylaxis of a Staphylococcus infection or a SføpΛy/ococcus-associated disorder.

25. Use as claimed in claim 24 wherein the infection is caused by Staphylococcus aureus.

26. Use as claimed in any one of claims 24 or 25 wherein the Staphylococcus- associated disorder is selected from the group consisting of bacteremia, septicaemia, tuberculosis, bacteria-associated food poisoning, blood infections, peritonitis, endocarditis, osteomyelitis, septicemia, skin disorders, meningitis, pneumonia, stomach ulcers, gonorrhoea, strep throat, toxic shock, necrotizing fasciitis, impetigo, histoplasmosis, Lyme disease, gastro-enteritis, dysentery and shigellosis.

27. Use of an antibody as claimed in claim 18 or 19 in the manufacture of a medicament for the treatment of a Staphylococcus infection or a Staphylococcus- associated disorder.

28. A method of treating a Staphylococcus infection or StøpΛy/coccϋs-associated disorder in a patient comprising administering to the patient an antigenic polypeptide as claimed in any one of claims 1 to 5, or a vaccine as claimed in any one of claims 9 to 11 , or an antibody as claimed in claim 18 or 19.

29. Use as claimed in claim 27 or method as claimed in claim 28 wherein the infection is caused by Staphylococcus aureus.