WO2005012339A2 - Immunogenic protein and uses thereof - Google Patents

Abstract

This invention relates to outer layer proteins of Bacillus anthracis for use in producing an immunogenic response, which can then be applied in the detection of B.anthracis, and in the preparation of vaccines against B.anthracis infection. Antibodies which recognise these proteins and methods for the detection of B.anthracis are also described and claimed.

Description

immunogenic Protein and Uses thereof

The present invention relates to outer layer proteins of

Bacillus anthracis for use in producing an immunogenic response, which can then be applied in the detection of B. anthracis, and in the preparation of vaccines against B. anthracis infection. In addition, the invention provides antibodies which specifically recognise these proteins, and certain novel proteins themselves, together with nucleic acids encoding them.

Bacillus anthracis, the aetiological agent of anthrax, is a Gram positive, aerobic or facultative anaerobic spore forming bacterium. The outer most integument of the spores from members of the Bacillus cereus group of micro-organisms, including B. anthracis, is the exosporium. This loose fitting, balloon like structure consists of two layers (Gerhardt and Ribi, 1964); a basal layer which has a hexagonally ordered crystal lattice structure (Gerhardt and Ribi, 1964; Beaman et al . , 1971) and a second peripheral layer consisting of a nap of fine filaments termed the hairy nap (Roth and Williams, 1963; Gerhardt and Ribi, 1964; Hachisuka et al . , 1966; Moberly et al . , 1966; Kramer and Roth, 1968) .

Much of the work published on the fine structure and composition of the exosporium to date has focused upon B. cereus (Gerhardt and Ribi, 1964; Matz et al . , 1970; Beaman et al . , 1971; Charlton et al . , 1999). This layer is chemically complex, consisting of protein, amino and neutral polysaccharides, lipids and ash (Matz et al . , 1970). B. cereus exosporium antigens first appear at the engulfment stage (stage III) of sporulation (DesRosier and Lara, 1984) . B. thuringiensis (Garcia-Patrone and Tandecarz, 1995) , B. cereus (Charlton et al . , 1999) and B. anthracis (Sylvestre et al . , 2002) spores contain glycoproteins specific in their surface layers. The B. anthracis collagen-like spore surface glycoprotein (BclA) reported by Sylvestre et al (2002) is a structural component of the filaments of the hairy nap and is highly immunogenic. However, the protein composition, structure and function of the exosporium have still yet to be elucidated fully.

The exosporium may play a role in the interaction of the spore with the infected host. For example, there may be an association between the exosporium of B. anthracis spores and the macrophage (Dixon et al . , 2000; Guidi-Rontani et al . , 2001), and exosporially- located components, like the alanine racemase and inosine hydrolase reported here, may influence spore germination within the macrophage.

The applicants have identified several proteins which are tightly associated with the exosporium of B. anthracis and which appear to represent integral exosporium proteins, as they are retained after salt and detergent washes .

According to the present invention, there is provided a protein obtainable from an outer layer of B. anthracis in a pre- vegetative state, which protein is either (a) retained in said layer after washing with a salt solution, followed by washing with detergent, and/or (b) retained in a supernatant of said layer which supernatant is obtained after having first removed the exosporium from said B. anthracis in a pre-vegetative state; wherein said protein has one or more regions specific to B. anthracis, or a variant of said protein, or a fragment of any of these, for use in producing an immunogenic response to B. anthracis .

In the context of the present invention, the expression "has one or more regions specific to B. anthracis" refers to any protein which can be used to positively differentiate the Bacillus anthracis species from another Bacillus species. Such proteins could include those proteins that are only present in B. anthracis and are not identified in other Bacillus species. These proteins are known as proteins specific to B. anthracis . Alternatively such a protein could include those proteins which have one or more amino acid sequences with an identical or close homology to a protein naturally present in B. anthracis but wherein the proteins are also sufficiently different from proteins naturally present in other Bacillus species to enable differentiation between the species. It is likely that such a protein has a marked difference in sequence identity to that obtainable in a similar manner from another Bacillus species. For example such a protein may have less than 90% sequence identity, preferably less than 80% sequence identity and more preferably less than 70% sequence identity to any protein obtainable in a similar manner from those of another Bacillus species. It is to be understood herein that such proteins also include variants of such proteins, or a fragment of any of these.

For the avoidance of doubt the term protein when used herein is considered to embrace those compounds which are also known in the art as glycoproteins .

In the context of the present invention, the expression "outer layer" refers to any layers found in the surface region of B. anthracis including the spore coat layers and in particular the exosporium.

In addition, the expression "variant" as used herein refers to sequences of amino acids which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably variants will be at least 80% identical, preferably at least 90% identical, and more preferably at least 95% identical to the base sequence.

Identity in this instance can be judged for example using the algorithm of Lipman-Pearson, with Ktuple:2, gap penalty: 4, Gap Length Penalty: 12, standard PAM scoring matrix (Lipman, D.J. and Pearson, W.R., Rapid and Sensitive Protein Similarity Searches, Science, 1985, vol. 227, 1435-1441).

The term "fragment thereof" refers to any portion of the given amino acid sequence which has the same activity as the complete amino acid sequence. Fragments will suitably comprise at least 5 and preferably at least 10 consecutive amino acids from the basic sequence, or combinations of these fragments.

Proteins of the invention are particularly useful for raising antibodies, which are useful in the detection of B. anthracis . The use of proteins which are present in the spore state is helpful in that it allows for rapid detection of B. anthracis, without the need for extensive culturing of the cells.

In the context of the present invention, the proteins obtainable as set out may either be structural proteins or proteins which are associated with the outer layers of the B. anthracis by adsorption or by some other means .

Proteins which are retained in the outer layers, and in particular are integral with the outer layers, are more readily and reliably available for detection purposes. Furthermore, proteins that have one or more regions specific to B. anthracis, in particular those which have less than 90% identity to those of a further Bacillus species, for example B. cereus, will allow distinction between the species to be readily made. In particular proteins which are structural or integral with the outer layers such as the exosporium, are most likely to give rise to a strong immune response, which will be protective against infection by B. anthracis . Thus proteins as described above may be used in producing an immune response in a host animal, which response is protective against infection by B. anthracis .

In one embodiment of the invention, the protein is one that is retained in the outer layer, and in particular in the exosporium, after washing with salt solution, for example 1M NaCl, followed by washing with detergent. As discussed above, such proteins are believed to be structural proteins of the outer layer, or at least are integral with said layer. Such proteins can optionally be identified by first removing the exosporium layer and then washing as set out.

Examples of such proteins are proteins of SEQ ID NO 1, 2, 3, 4, 5 , 6 or 7 as shown in Table 2. A particular example of such proteins is a protein of SEQ ID NO 6 that has less than 80% identity with any protein found in B. cereus .

Another particular examples is the protein of SEQ ID NO 7, which has been designated ExsK. The ExsK protein has not previously been described as a coat or exosporium component, although a homologue (64% identity) is encoded in B. cereus ATCC genome 14579. Thus this is novel protein and forms a particular aspect of the invention, together with variants having greater than 64% identity thereto, or immunogenic fragments of any of these.

In another embodiment of the invention, the protein is one that is retained in a supernatant of the outer layer, which supernatant is obtained after having first removed the exosporium from said B. anthracis in a pre-vegetative state. The exosporium can be removed by any means known to one skilled in the art . For example the exosporium can be removed by sonicating the sample, using a bead beater, using a French press or other well know methods. Proteins obtained in this manner are not believed to be structural proteins of the outer layer, but rather to be proteins which are adsorbed onto the outer layers of said B. anthracis .

A particular protein which is obtainable from an outer layer of B. anthracis and has one or more regions specific to B. anthracis, in particular those which have less than 80% identity to any protein obtainable from B. cereus is the protein which is designated ExsL as defined in Table 1. Although this protein does not appear to be retained in exosporium washed as described herein, it is present in the outer layers and has only 42% identity to a protein encoded by a gene in the B. cereus genome.

Proteins of the invention may be prepared using conventional methods. Although they may be isolated from B. anthracis, it is preferable that they are expressed recombinantly. For this purpose, a nucleic acid encoding the proteins is incorporated into an expression vector or plasmid, which is then used to transform a host cell. The host cell may be a prokaryotic or eukaryotic cell, but is preferably a prokaryotic cell such as E. coli . The codons utilised in the nucleic acid may be optimised for expression in the particular host cell.

Nucleic acids encoding novel proteins of the invention such as SEQ ID NO 7, as well as vectors and cells containing these form a further aspect of the invention.

Once produced, the proteins of the invention can be used to produce binding moieties comprising an antibody or a binding fragment thereof, which specifically recognises a protein according to any one of the preceding claims . These form yet a further aspect of the invention.

Antibodies or binding fragments thereof may be polyclonal or monoclonal, which may be produced using conventional methods. For instance, polyclonal antibodies may be generated by immunisation of an animal (such as a rabbit, rat, goat, horse, sheep etc) with immunogenic proteins or immunogenic subunits or fragments thereof, to raise antisera, from which antibodies may be purified.

Monoclonal antibodies may be obtained by fusing spleen cells from an immunised animal with myeloma cells, and selecting hybridoma cells which secrete suitable antibodies.

Antibody binding fragments include F(ab'>2, F(ab)₂, Fab or Fab' fragments, as well as recombinant antibodies, such as single chain (sc) antibodies FV, VH or VK fragments, but they may also comprise deletion mutants of an antibody sequence. Acronyms used here are well known in the art. They are suitably derived from polyclonal or monoclonal antibodies using conventional methods such as enzymatic digestion with enzymes such as papain or pepsin (to produce Fab and F(ab')₂ fragments respectively). Alternatively, they may be generated using conventional recombinant DNA technology.

Once obtained, the binding moieties are useful in detecting B. anthracis. Thus the invention further provides a method for detecting the presence of B. anthracis which method comprises contacting a sample suspected of containing B. anthracis cells with a binding moiety as described above, and detecting binding therebetween.

Detection methods used include conventional immunological methods for example ELISA, surface plasmon resonance and the like.

The sample is suitably an enviromental sample, suspected of containing B. anthracis spores. These do not have to be cultured to form vegetative cells but can be used directly in the detection method. Suitably the binding moiety is immobilised on a solid support, for example on an ELISA plate, but other forms of support, for example membranes such as those utilised in conventional "dip- stick" tests may also be employed.

Detection of a complex between a protein within a spore in the sample, and a binding moiety as described above can be detected using conventional methods, in particular immunological methods such as ELISA methods. Assay formats may take various forms including "sandwich" or "competitive" types.

In a typical sandwich assay, the binding moiety is immobilised on a support, such as an ELISA plate, where is it contacted with a sample suspected of containing anthrax spores. Where present, these spores will bind the binding moiety and so become immobilised in their turn. The support is then separated from the sample, for example by washing. The presence of spores on the support can then be detected by application of secondary antibodies or binding fragments thereof, which bind to the spore, and are detectable, for example because they are labelled for instance with a visible label such as a fluorescent label, or a radiolabel, but preferably that they can be developed to produce a visible signal. A particular example of a secondary antibody is an antibody or binding fragment, that carries an enzymatic label, such as horseradish peroxidase, which can then be utilised to produce a signal by addition of the enzyme substrate, using conventional ELISA methodology. Secondary antibodies used in this way may also comprise binding moieties in accordance with the invention.

In a particular competitive assay format, the binding moiety of the invention is immobilised on a support. In this instance, a protein which binds said binding moiety in competition to the spores is added to the sample prior to contact with the support. Any spores present within the sample will compete with this protein for binding to the immobilised binding moiety. Thus, the absence of peptide on the support is indicative of the presence of spores in the sample.

In this case, the competing protein is suitably labelled so that it may be readily detected, for instance using a visible label such as a fluorescent or radiolabel. Alternatively, it may be detected using a secondary antibody or a binding fragment thereof, such as those discussed above in relation to sandwich assays, which binds the protein.

The proteins of the invention may also have pharmaceutical application as vaccines, as they may produce a protective immune response in a host animal, such as a human to whom they are administered. Methods of treatment in this way form yet a further aspect of the invention.

For pharmaceutical application, the proteins are suitably administered in the form of a pharmaceutical composition. Thus the invention further provides a pharmaceutical composition comprising a protein as described above in combination with a pharmaceutically acceptable carrier.

Compositions of the invention may further comprise pharmaceutically acceptable carriers or excipients as are well known in the art. They may be solid or liquid carriers depending upon the intended mode of administration.

Any desired mode of administration may be used to deliver the exosporium proteins for the purposes of inducing immunological responses in human or animal recipients. Particularly, although not exclusively, compositions of the invention will be intended for parenteral, including intramuscular, subcutaneous, intradermal, intraperitoneal and intravenous administration, or non parenteral including intranasal, inhalation, oral, buccal, epidermal, transcutaneous , ocular-topical, vaginal, rectal administration. The exact means of administration can be readily determined by one skilled in the art and will depend upon factors such as the nature of the toxin being treated, and the nature of the patient. It is preferred that compositions of the invention are intended for oral, intravenous or intranasal administration, or for administration by inhalation.

Compositions for parenteral administration will suitably be in the form of sterile solutions or suspensions which may contain suitable adjuvants (for example Alhydrogel™) and / or other excipients. Immunomodulators , such as cytokines, chemokines, synthetic or natural molecules of bacterial origin or any other immunomodulatory materials may also be included. Alternatively, the outer spore surface protein (s) may be formulated into emulsions, biodegradable microspheres or liposomes or any other suitable delivery system.

It is envisaged that the outer spore surface proteins may be administered to vaccines by nonparenteral (intranasal, inhalational, oral, buccal, epidermal, transcutaneous, ocular- topical, vaginal, rectal) routes. For nonparenteral delivery, antigens may be formulated into biodegradable microspheres or liposomes or any other suitable delivery system. Adjuvants, for example those derived from enterotoxins, may be included in the composition. Cytokines, chemokines, synthetic or natural molecules of bacterial origin or any other immunomodulatory molecule (s) may also be included to amplify / modify the immunological response following nonparenteral delivery of the outer spore surface proteins. Oral compositions may be in the form of tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs.

Compositions for the delivery of the formulation into the respiratory tract may be in the form of a finely divided dry powder . Compositions of the invention may comprise other components such as preservative agents, inert diluents, granulating and disintegrating agents, binding agents, lubricating agents, anti-oxidants as well as colouring, sweetening or flavouring agents, depending upon the nature of the composition.

Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

The relative amounts of pharmaceutically acceptable carrier to the protein in a formulation will vary depending upon factors such as the particular route of administration. Generally however, compositions will comprise from about 1 to about 98 percent by weight of pharmaceutically acceptable carrier, and preferably from 5 to 90 percent by weight of pharmaceutically acceptable carrier.

The size of the dose for therapeutic purposes of a composition of the invention will naturally vary according to the nature and severity of the condition, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. Generally however, patients are given from 0.5 mg to 75 mg per kg body weight of the immunogenic protein.

Thus in a further aspect, the invention provides the use of a protein as described above in the preparation of a medicament for prophylactic or therapeutic vaccination against B. anthracis . As described hereinafter, the applicants isolated and identified loosely adsorbed and likely integral exosporium proteins from spores of B. anthracis wild type Ames and B. anthracis pX02- plasmid derivative. Proteins were identified from either their amino-terminal sequence and/or Nanospray MS/MS data which provides information on N-terminal, mid-chain and C-terminal sequence with equal probability. These techniques which generate unequivocal identification of sequence were adopted because of the limited resolution of the ID gels of exosporium material. As only selected bands were examined, and some material did not enter the gel, only a small proportion of the total proteins in the exosporium have been identified so far. All of the proteins identified are encoded chromosomally.

There are no obvious differences in gross exosporium profile between the wild type and cured strains. Therefore the cured strains represent good models for studying the exosporium from spores of B. anthracis .

Two proteins, ExsK (SEQ ID NO 7) and ExsF (BxpB) (SEQ ID NO 3), have no homologues of identified function and no homologues in B. subtilis, the paradigm sporeformer. However, ExsF, which has been described recently by others (Steichen et al . , 2003; Todd et al . , 2003) is also present in both B. cereus and B. anthracis, and is more strongly conserved (97% identity) .

Thus, although it may be particularly useful as a vaccine, its use in detecting specifically B. anthracis is less pronounced.

Three of the exosporium proteins which the applicants isolated were related to outer spore coat proteins of B. subtilis spores, CotB (Donovan et al . , 1997), CotY (Zhang et al . , 1993) and a CotY homologue designated ExsY. CotB is exposed on the spore surface of B. subtilis (Donovan et al . , 1987; Driks, 1999), and has even been used as an anchor for non-native antigens (Isticato et al . , 2001). Its location in B. anthracis will be determined using immunological techniques . ExsY is required for exosporium assembly (M. Johnson, S.J. Todd and A. Moir, unpublished) . The cotY gene forms part of a complex operon in B. subtilis which consists of five genes, cotVWXYZ. it is hypothesised that the complex transcriptional pattern of this cluster is necessary to maintain an optimal amount of each protein for correct assembly of the spore coat in B. subtilis (Zhang et al . , 1994) There are no ORFs homologous to Cot V, W or X proteins in the B. anthracis genome.

The two enzymes, alanine racemase and the inosine preferring nucleoside hydrolase were present on the washed exosporium and as such represent proteins which are tightly associated to this layer. These two enzymes have also been shown to be present in washed B. cereus UM20.1 exosporium (Todd et al , 2003). Alanine racemase has been detected in exosporium of B. anthracis (Steichen et al . , 2003) and inosine hydrolase in alkali- extracted material from B. anthracis spores (Lai et al . , 2003). Both proteins represent enzymes of potential importance to the spore. Both alanine and inosine are potential germinants for B. anthracis, although additional components are required for the inosine response (Hills, 1949; Ireland and Hanna, 2002; Titball and Manchee, 1987). These enzymes may be involved in metabolism of germinants and presumably moderate the rate of spore germination.

B. anthracis vegetative cells produce a proteinaceous paracrystalline sheath termed the S-layer which completely covers the cell surface. The S-layer protein EAl is expressed during stationary phase of growth (Mignot et al . , 2002) and its presence in the exosporium preparations is likely to result from co-purification of the two lattice like structures, exosporium and S-layer, during the exosporium removal and purification procedure. The extent of cross-contamination with EAl varied in different preparations. The presence of BclA in unwashed and washed exosporium of B. anthracis and B. cereus exosporium was suggested by immunoblotting with anti- spore antibodies, but this protein was not amenable to MS/MS sequencing. Difficulties in dissociating proteins e.g. ExsY (SEQ ID NO 5), CotY (SEQ ID NO 4) and ExsF (SEQ ID NO 3) suggest that they are involved in relatively stable multimeric complexes along with the glycoprotein(s) in the exosporium.

Unwashed exosporium contains, in addition to the demonstrated salt and detergent-resistant components, other components, some described here that may contribute to the in vivo activities of the exosporium. Some of these proteins may not be exosporium- bound, but may be located between the exosporium and the rest of the spore, and associate during isolation.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings, a brief description of which follows.

Figure 1 Transmission electron micrographs of B. anthracis spores and exosporium

Negatively stained samples of (A) B. anthracis UM23C12 spores sonicated for 6 minutes at 12 μm and (B) Filtered, salt -washed exosporium from sonicated B. anthracis wild type Ames spores.

Figure 2 Comparison of the protein profiles of unwashed and salt/detergent washed B. anthracis exosporium

A. Proteins in exosporium extracts of B. anthracis wild type Ames were separated by SDS-PAGE on a 10% NuPAGE Bis-Tris gel, and stained with Colloidal blue. Lane 2, unwashed exosporium,- Lane 3, proteins in the supernatant of the centrifuged, unwashed, exosporium extract; Lane 4, fully washed exosporium extract. B. Proteins in an unwashed B. anthracis Ames exosporium preparation (Lane 1) were separated by SDS-PAGE on a 10% Tris- glycine gel and stained with Colloidal blue stain. Selected bands, indicated by arrows, were subjected to DE-MALDI-TOF-MS, peptide sequences obtained by Nanospray-MS/MS, and unequivocal protein assignments made.

Figure 3 Identification of selected proteins from washed B. anthracis exosporium Proteins from exosporium extracts were separated on a 16% Tricine gel, and stained with Colloidal blue. Lane 1, SigmaMarker low range molecular weight marker; Lanes 2 and 3; B. anthracis UM23C12, unwashed and washed respectively; Lanes 4 and 5, B. anthracis pX01⁺/pX02^" Ames, unwashed and washed respectively; Lanes 6 and 7, B. anthracis wild type Ames, unwashed and washed respectively. Selected bands were subjected to DE-MALDI-TOF-MS and a number of peptides identified by Nanospray-MS/MS. The number in the brackets following the proteins identified relates to the number assigned in Table 2.

Figure 4 Alkali-extracted proteins from B. anthracis UM23C12 spores

Spores of B. anthracis UM23C12 were incubated in the alkaline extraction buffer of Sylvestre et al . , (2002) and the extracted proteins were separated on a 12% Tris-glycine gel. Extraction for 15 min (Lane 2) and 1 hour (Lane 3) . SigmaMarker broad range molecular weight marker is in Lane 1.

Figure 5 Glycoprotein detection of proteins from unwashed and fully washed exosporium

Proteins separated by SDS PAGE were visualised using the ECL glycoprotein detection kit (Amersham) . Invitrogen Western blotting standard (Lane 1) ,- B. cereus ATCC 10876 exosporium, unwashed (Lane 2), and washed (Lane 3); B. anthracis UM23C12 exosporium unwashed (Lane 4), and washed (Lane 5 and 6); B. anthracis wild type Ames exosporium unwashed (Lane 7), and washed (Lane 8 and 9) ; and transferrrin (Lane 10) .

Figure 6 Reactivity of antispore monoclonal antibody, F3/1 Bll to the dominant exosporium antigen

Western blot of glycosylated and deglycosylated exosporium from B. cereus ATCC 10876 exosporium (Lanes 2 to 3 ) , B. anthracis UM23C12 (Lanes 4 and 5) and B. anthracis wild type Ames exosporium (Lanes 6 and 7) , probed with anti-spore monoclonal antibody F3/1 Bll. Lane 1 shows molecular weight marker and lane 8 contains ovalbumin, which was not detected by the MAb.

Bacterial strains, growth conditions and media. The Bacillus anthracis strains used were wild type Ames, a pX02 cured Ames derivative (Ames +/- 1) and UM23C12, a double cured derivative. The wild type Ames and pX02 cured Ames derivative were handled in Advisory Committee on Dangerous Pathogens (ACDP) 3 containment, both as vegetative cells and spores, whereas UM23C12 was grown at ACDP 2. The strains were grown routinely on nutrient agar, blood agar or L-agar or in nutrient broth or L-broth at 37°C.

Spore preparation. Spores of B. anthracis were prepared on New Sporulation medium (3.0 gl^_1Difco Tryptone Peptone; 6.0 gl^"1 Oxoid bacteriological peptone; 3.0 gl^"1 Oxoid yeast extract; 1.5 gl^{" x}Oxoid Lab Lemco; 1 ml 0.1% MnCl₂.4H₂0; 25 gl^"1 Difco Bacto agar) at 37°C, until the cultures contained in excess of 95% free spores. Spores were harvested by centrifugation at 10,000 x g for 10 min. and washed approximately 10 times in ice-cold sterile distilled water to remove contaminating vegetative cells and debris. Microscopic examination established that spore preparations contained less than 5% vegetative cells and debris. The washed spore preparations were stored at -20°C. Exosporium removal and purification. Spores were defrosted and centrifuged at 10,000 x g for 10 min at 4°C. Prior to sonication, pellets were resuspended to between approximately 5 xlO⁹ and 2.5 x 10¹⁰ spores per ml in 50mM Tris-HCl, 0.5mM EDTA buffer (pH7.5). All subsequent manipulations were at 4°C. To fragment and release exosporium material, spores were sonicated (amplitude 12 μm) using a Sanyo Soniprep, for seven 1 min bursts, each separated by 2 min cooling on ice. Exosporium fragments were separated from spores by pelleting the latter at 9,000 x g for 5 min. The spore pellet (s) were washed once in 50mM Tris-HCl,

0.5mM EDTA (pH 7.5) and the exosporium-containing supernatants were pooled, then centrifuged again (10,000 x g, 15 min) to pellet the remaining spores .

For all isolates of B. anthracis, any residual endospores in the exosporium-containing supernatant were removed by filtration through 0.45 and/or 0.2um low-protein binding filters (Acrodisc, Gelman Sciences) . The filtrate, containing exosporium, was subjected to a sterility check by inoculating 10 volumes of nutrient or L-broth with 10% of the exosporium preparation and incubating at 37°C with shaking (200 rpm) for 7 days. After centrifugation to concentrate any cells, the incubated material was plated on L-agar, in parallel with the appropriate controls. The plates were incubated at 37°C for 7 days and absence of growth of any colonies permitted clearance for use at a lower level of containment. Once cleared as sterile, the exosporium- containing filtrate was concentrated using an Amicon ultra- filtration cell fitted with a polyethersulfone membrane (Millipore, NWWL 10,000) or by pelleting in an ultracentrifuge at 184,000 x g for 1 hour at 4°C.

Salt and detergent washing of exosporium. The series of washes used on the exosporium was modified and simplified from a method for purifying B. subtilis spore coat fractions described by Nicholson and Setlow (1990) . The technique was optimised using B. anthracis UM23C12 exosporium and subsequently applied to exosporium from the ACDP 3 strains of B. anthracis . The exosporium was washed with IM NaCl, followed by 1 or 2 washes in TEP buffer (50mM Tris-HCl pH7.2 , lOmM EDTA, 2mM PMSF) containing 0.1% wv^"1 SDS. To remove the SDS, a final wash was in TEP buffer alone. Each wash involved resuspension of the purified exosporium followed by centrifugation in a Hettich micro centrifuge at 31,870 x g for 40 min.

Protein determination. Protein concentrations were determined using the BCA protein assay kit (Pierce) according to the Manufacturers instructions .

Alkali extraction of spore proteins. Alkali extraction was carried out on washed B. anthracis UM23C12 spores, as detailed by Sylvestre et al (2002). Spores were incubated in alkaline reducing buffer without SDS (50mM Tris-HCl, pHIO, 8M urea and 2% w^"1 β-mercaptoethanol) for 15 minutes or 1 hour, and the spores removed by centrifugation.

Electron microscopy. The samples were diluted in sterile distilled water, placed on formvar coated grids and examined after negative staining in 1% wv^"1 phosphotungstic acid in phosphate buffer (pH 7.2). After 15 to 30 seconds the excess phosphotungstic acid was withdrawn using filter paper. Samples were examined immediately using a Philips CM12 transmission electron microscope at an accelerating voltage of 60KV.

Gel Electrophoresis. Samples were mixed with an equal volume of sample buffer (62.5 mM Tris-HCl, pH 6.8; 10% w^"1 glycerol, 2% wv^"1 SDS, 5% w^"1 β-mercaptoethanol; 0.1% wv^"1 bromophenol blue), and boiled for 5 min, then held on ice for 2 to 10 min and centrifuged briefly (30 sec at 13,000 x g) to remove any insoluble material. The proteins were separated by SDS-PAGE in Tris-glycine buffer (24mM Tris, 192mM glycine and 0.1% wv^"1 SDS; (Laemmli, 1970)) at 125V using pre-cast gels (Invitrogen,

Paisley) , or on NuPAGE Bis-Tris pre-cast gels in MES buffer (Invitrogen) . To separate low molecular weight proteins, samples were boiled in Tricine gel sample buffer (900mM Tris- HCl, pH 8.45; 24% w^"1 glycerol; 8% wv^"1 SDS; 0.15% wv^"1 Coomassie blue G; 0.005% wv^"1 Phenol red and 5% w^"1 β-mercaptoethanol) and separated on precast 16% Tris-Tricine gels (Invitrogen) at 125V using the Tricine running buffer (lOOmM Tris; lOOmM Tricine, 0.1% wv^"1 SDS). Gels were stained with Colloidal Blue stain (Invitrogen) according to the Manufacturers instructions. For molecular weight determination, SigmaMarker™ molecular weight standards were used on both Tris-glycine and Tricine gels.

N-terminal sequencing. Proteins separated by SDS-PAGE were electrophoretically transferred onto PVDF membranes, using lOmM CAPS transfer buffer (pH 11.0) with 10% w^"1 methanol. The membranes were stained with 0.1% wv^"1 Coomassie® 250 in 40% w^"1 methanol/1% w^"1 acetic acid, destained with 50% w^"1 methanol and air-dried. N-terminal sequences were determined by automated Edman degradation in an Applied Biosystems gas phase sequencer.

In-gel digestion and Mass spectrometric analysis of protein samples. Bands identified for analysis from the SDS-PAGE gels, were excised, reduced/carboxymethylated and subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and purified by HPLC using a C_ι8 cartridge. The peptide containing fractions were collected and analysed by Delayed

Extraction-Matrix Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (DE-MALDI-TOF-MS) . Some peptides were subjected to sequencing using Nanospray-MS/MS at M-Scan Limited.

Sequence data. Sequences from protein bands were used to search preliminary sequence data for B. anthracis obtained from the Institute for Genomic Research (TIGR) website at http://www.tigr.org, using tblastn (Altschul et al . , 1997), and to search ORFs identified for B. cereus ATCC 14579 from http://www.integratedgenomics.com. The wild type Ames strain examined during this study was the parental strain for the double cured Ames strain used for genome sequencing (Read et al . , in press) .

Glycoprotein staining. Proteins separated on Tris-glycine SDS- PAGE gels were electrophoretically transferred onto PVDF membranes, stained and visualised using a Glycoprotein ECL detection kit (Amersham Pharmacia Biotech) .

Deglycosylation of exosporium. Trifluoromethanesulfonic acid (TFMS) /anisole (9:1 w^"1; 50μl) was added to 100-200 μg of the lyophilised exosporium and vortexed. The reaction was refrigerated for 2 hours and stopped by the drop wise addition of 1ml of diethylether/pyridine (1:1) prechilled to -20°C. The sample was spun at 16,000 x g for 8 min, and the pellet resuspended in 1 ml 0.1 M NH₄HC0₃. The resuspended exosporium was dialysed against 0. IM NH₄HC0₃ in 3,500 molecular weight cut off dialysis tubing at 4°C with 3 or 4 buffer changes. The dialysed material was spun at 16, 000 x g for 10 min and the pellet resuspended in 50 to lOOμl of 50 mM Tris HCl, 0.5 mM EDTA; pH7.5.

Western blotting. Proteins from SDS-PAGE were transferred onto Hybond™-C Extra membrane (Amersham Life Science) . The resulting blots were probed with anti-mouse monoclonal antibodies, F3/1 Bll at 1 - 2 μg ml^"1 or AF10 at 2 - 10 μg ml^"1. Blots were developed using the ECL Western blotting analysis system (Amersham Pharmacia Biotech) , diluting the secondary antibody according to the manufacturer's instructions (generally 1:3000 dilution) .

RESULTS

Exosporium preparation

Exosporium was isolated from fully virulent B. anthracis Ames, from a derivative lacking pX02 and from a variant of the Sterne strain, UM23CL2 lacking both pXOl and pX02. Sonication of spores for 6 or 7 minutes using the Sanyo Soniprep 150 ultrasonic disintegrator caused partial fragmentation of the exosporium without disruption of the spore (Figure 1A) . Some exosporium material was left attached to the spores . Whole spores were removed by low speed centrifugation, the supernatant was filtered (0.45 μ or 0.2 μm) to remove all remaining live spores, and the exosporium fragments were concentrated. Small fragments with hexagonal periodicity were evident by electron microscopy (Figure IB) . Because of the restrictions of working in ACDP 3 containment, exosporium was released using sonication rather than by a French Press procedure.

Effects of salt-washing the exosporium

A large number of protein bands from concentrated wild type Ames exosporium were separated by SDS-PAGE (Figure 2A, lane 2 and Figure 2B, lane 1) . The supernatant from the first centrifugation to concentrate the material after filtration (Figure 2A, lane 3) contained many of the same bands. The exosporium preparation was washed successively with salt (IM NaCl) and detergent (0.1% wv^"1 SDS) to remove loosely adsorbed proteins. The exosporium protein profile after these washing procedures (Figure 2A, lane 4) is enriched for the proteins in the original extract (lane 2) that were less strongly represented in the supernatant fraction in lane 3.

Identification of proteins in unwashed exosporium

Several protein bands from SDS-PAGE gels of unwashed exosporium preparations were identified by N-terminal sequencing and/or Nanospray MS/MS sequencing following DE-MALDI-TOF-MS. Peptide sequences were searched against predicted open reading frames (ORFs) of B. anthracis (provided by T. Read, TIGR) . An example of a profile used for analysis is in Fig 2B, and Table 1 lists the proteins identified. The S-layer protein EAl was frequently detected in protein profiles, from both unwashed and washed B. anthracis exosporium, as multiple bands of ca. 98, 75 and 66 kDa. These exosporium preparations had not been purified on density gradients, and therefore could contain contaminating S- layer material. Three of the nine proteins identified (alanine racemase, CotY and ExsY) were also identified in washed exosporium. GroEL has already been reported as an exosporium- adsorbed protein in B. cereus, removable by salt washing (Charlton et al . , 1999; Todd et al . , 2003). The Zn-dependent metalloprotease Immune inhibitor A (InhA) , a major component of unwashed exosporium preparations of B. cereus, was not present in B. anthracis exosporium.

An exhaustive study of the many other bands in the unwashed material was not undertaken- instead; the exosporium preparations were washed more stringently, to identify tightly bound or integral proteins .

Identification of proteins in the washed exosporium

Salt and detergent washed exosporium preparations from all three strains examined show a generally reproducible pattern of proteins on SDS-PAGE on a 16% acrylamide, Tris-tricine buffered gel, except for the variable EAl S-layer proteins (Figure 3, lanes 3, 5 and 7) .

A number of bands were extracted and mass spectrometry (MS/MS sequencing of tryptic peptides) provided internal sequences for all bands tested except the largest (>200kDa; Figure 3). Several protein components were identified - their amino acid sequences, predicted from the B. anthracis genome sequence, are shown in Table 2.

The strong band with apparent molecular weight of 43 kDa present in both unwashed and washed exosporium was identified from both

MS sequencing (Table 2) and direct N-terminal sequencing (Table 1) , as an alanine racemase homologue (50% amino acid identity to the B. subtilis dal gene product) . A second alanine racemase homologue encoded in the unfinished B. anthracis genome sequence has a very different N-terminal amino acid sequence (only 10 out of 21 identities with the observed N-terminus) . The band at 33 kDa (Table 2) contains a homologue of inosine- uridine preferring nucleoside hydrolases (30% amino acid identity with the paradigm protozoal enzyme of Gopaul et al . , (1996)).

The band at 17 kDa contained sequences that correspond to a protein, identifiably encoded by B. anthracis , and very recently described and designated BxpB by Steichen et al (2003). The predicted molecular weight of 17 kDa corresponds to that observed experimentally. The equivalent protein in B. cereus exosporium has been designated ExsF (Todd et al . , 2003). A related paralogue is also encoded in the B. anthracis genome sequence, sequence PVIND is the only sequence obtained present in both genes.

The band at 14 kDa contains sequences derived from at least two separate proteins. One is a homologue of (30% identity to) B. subtilis spore coat protein CotB (Table 2), of predicted size 19.4 kDa. The sequence data do not correspond to the sequence of a second, rather shorter CotB homologue, encoded in the B. anthracis genome immediately downstream of the first.

The other sequences from the 14kDa band, FEAFAP and SANLT, are present in the sequences of both ExsY and CotY. These are paralogues (84% identity) and are more distant homologues of the

B. subtilis CotZ and CotY proteins, which are present in the insoluble outer layers of the B. subtilis spore coat (Driks,

1999) .

The band at 10 kDa contains peptides from a predicted protein, which we have named ExsK. This protein has a homologue in the B. cereus ATCC 14579 genome sequence with 64% identity. Direct comparison of ExsK with B. subtilis proteins by FASTA gives the highest, though not significant score (e = 0.12) as the B. subtilis outer coat protein Cotx. ExsK has no valid homologues in B. subtilis, but has some similarity in composition near the N-terminus (a very charged region at residues 29-37, preceding a cysteine-rich region) to the equivalent region of CotX.

Peptide FEAFAP was also present in the 10 kDa band, suggesting that either or both ExsY and CotY sequences are present. The band at ca. 6.5 kDa contained a peptide sequence (SIDDDD) specific to CotY. Presumably these proteins in the 10 and 6.5 kDa positions are partial degradation/ processing products, as the predicted size of the intact proteins would be 16 to 17 kDa.

Peptide sequences were obtained from the second largest band (MFSSD and FEAFAP) , demonstrating the presence of BxpB (ExsF) and either or both of ExsY and CotY in this band. These proteins are therefore present in the gel in high molecular weight complexes, as well as in a monomeric form (BxpB/ExsF) or in a proteolytically cleaved form (ExsY/ CotY) .

Alkali extraction of proteins from spores

Spores of B. anthracis UM23C12 were incubated with alkaline reducing buffer without SDS, as used by Sylvestre et al (2002) to extract a fraction containing BclA, a major exosporium glycoprotein. Large bands in an SDS-PAGE protein profile of the extracted proteins (Figure 4) were subjected to MS/MS and N- terminal sequencing. The diffuse multiple band (>250kDa) in the 15 min extract contained peptides from ExsY (SVDDDS) and ExsF (MFSSD) and yielded the N-terminal sequence for glycoprotein BclA (AFDPNLVGPT) . Specific staining did confirm the presence of glycoprotein in this region of the gel (Figure 5). On more extended incubation, a smaller band at 205 kDa increased in intensity; peptides PVIND and PVELI from ExsF were both identified in this band.

Glycoprotein(s) in the exosporium

SDS-PAGE separated B. anthracis exosporium proteins were tested for glycoprotein using an ECL glycoprotein detection system (Amersham Pharmacia Biotech) and compared to those of B. cereus (Figure 5) . A large diffuse band extending from the insoluble fraction at the top of the gel, to a molecular weight of approximately 140 kDa was stained in the B. anthracis exosporium preparations. B. cereus has a second diffuse band at 70 kDa.

Monoclonal antibodies F3/1 Bll, raised against bacterial spores of B. anthracis wild type Ames (C. Redmond, unpublished) or AF10, raised against irradiated B. anthracis Sterne spores by Dr J. Kearney, were used as probes in Western blots of SDS-PAGE separated exosporium extracts. Both bound to the insoluble fraction at the top of the gel and to a band at >200kDa (Figure 6) .

Exosporium preparations were chemically deglycosylated using trifluoromethanesulfonic acid (TFMS) . Following deglycosylation, antibodies AF10 and F3/1 Blldetect protein bands at apparent molecular weights of 42 kDa in B. cereus and approximately 66 kDa in both B. anthracis Ames and UM23C12 (Figure 6) . The deglycosylated exosporium samples were analysed for the presence of glycoprotein and bands with sizes corresponding to those detected by the antibodies were stained (data not shown) . Therefore complete deglycosylation of the exosporium glycoproteins was not achieved. A BclA specific antibody (described in Sylvestre et al . , 2002) also detected proteins in identical positions to those detected by AF10 and F3/1 Bll. Therefore AF10 and F3/1 Bll must also be detecting BclA. The B. anthracis bands detected are smaller than reported for the Sterne strain in Sylvestre et al (2002),- the length of BclA has been shown to vary in different B. anthracis isolates (Steichen et al . , 2003; Sylvestre et al . , 2002; 2003). The size of the protein detected in deglycosylated anthrax exosporium by the antibodies was larger than the molecular weight of BclA (35kDa) predicted from the genome sequence of the Ames strain. References

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Table 1 Proteins identified in unwashed exosporium from B. anthracis spores

Sequences identified from: ^a Ames +/- 1 exosporium Wild type Ames exosporium

e 2 Proteins identified in washed exosporium from B. anthracis spores

ORF numbers from TIGR genome sequence

Claims

Claims

1. A a protein obtainable from an outer layer of B. anthracis in a pre-vegetative state, which protein is either (a) retained in said layer after washing with a salt solution, followed by washing with detergent, and/or (b) retained in a supernatant of said layer which supernatant is obtained after having first removed the exosporium from said B. anthracis in a pre-vegetative state; wherein said protein has one or more regions specific to B. anthracis, or a variant of said protein, or a fragment of any of these, for use in producing an immunogenic response to B. anthracis .

2 . A protein according to claim 1 for use in producing an antibody, useful in the detection of B. anthracis .

3. A protein according to claim 1 for use in producing an immune response in a host animal, which response is protective against infection by B. anthracis .

4. A protein according to any one of the preceding claims which is retained in said outer layer after washing with salt solution, followed by washing with detergent.

5. A protein according to claim 4 wherein the outer layer is the exosporium.

6. A protein according to claim 5 which is a protein of SEQ ID NO 1 , 2 , 3 , 4 , 5 , 6 or 7 as shown in Table 2 , or a variant or fragment thereof .

7. A protein according to claim 6 which is a protein of SEQ ID NO 6 or 7 , or a variant or fragment thereof .

8. A protein according to claim 1 which is retained in a supernatant of said layer which supernatant is obtained after having first removed the exosporium from said B. anthracis in a pre-vegetative state.

9. A protein according to Claim 8 wherein the protein has less than 80% sequence identity to any protein obtainable in a similar manner from another Bacillus species.

10. A protein according to claim 9 which is protein ExsL as defined in Table 1.

11. A protein comprising SEQ ID NO 7 or a variant having greater than 64% identity thereto, or immunogenic fragment thereof.

12. A protein according to claim 11 which comprises SEQ ID NO 7.

13. A binding moiety comprising an antibody or a binding fragment thereof, which specifically recognises a protein according to any one of the preceding claims .

14. A method for detecting the presence of B. anthracis which method comprises contacting a sample suspected of containing B. anthracis cells with a binding moiety according to claim 13 and detecting binding therebetween.

15. A pharmaceutical composition comprising a protein according to any one of claims 1 to 13 in combination with a pharmaceutically acceptable carrier.

16. The use of a protein according to any one of claims 1 to 13 in the preparation of a medicament for prophylactic or therapeutic vaccination against B. anthracis.

17. An isolated nucleic acid which encodes a protein according to claim 11 or claim 12.