WO2012049662A1

WO2012049662A1 - Hyperblebbing salmonella strains

Info

Publication number: WO2012049662A1
Application number: PCT/IB2011/054569
Authority: WO
Inventors: Laura Martin; Allan Saul; Christiane Gerke; Sara Sa Silva
Original assignee: Novartis Vaccines Institute For Global Health Srl
Priority date: 2010-10-15
Filing date: 2011-10-14
Publication date: 2012-04-19
Also published as: GB201017519D0

Abstract

Hyperblebbing Salmonella strains are generated by disrupting one or more components of the Tol-Pal system, such as TolR. The blebs from these strains are useful immunogens for vaccination. The individual proteins found in these blebs can also be used as immunogens.

Description

HYPERBLEBBING SALMONELLA STRAINS

TECHNICAL FIELD

This invention is in the field of immunisation against Salmonella species.

BACKGROUND ART

Salmonella are Gram-negative facultative anaerobic bacteria. Nomenclature systems have varied over the years but there are currently two recognized species: S. enterica and S. bongori [1]. Within these species there are various subspecies, including enterica, salamae, arizonae, diarizonae, houtenae, and indica. Under the new nomenclature the bacteria previously known as S. paratyphi, S. typhi, and S. typhimitri m are instead serovars within S. enterica, for example S. enterica serovar Typhimurium, abbreviated: S. Typhimurium.

The Salmonella cause a variety of diseases in many animal hosts (although S. Typhi and S. Paratyphi A are human-specific serovars), mainly via ingestion of contaminated foods. Human diseases include gastroenteritis, bacteremia, typhoid fever and paratyphoid fever.

Various approaches are being used for immunisation against Salmonella, including the use of glycoconjugates, purified proteins {e.g. from the type 3 secretion system [2]) and live recombinant bacteria [3,4].

It is an object of the invention to provide further and improved components useful in preparing Salmonella vaccines, and in particular S. enterica vaccines, including vaccines which can protect against multiple subspecies or serovars of S. enterica.

DISCLOSURE OF THE INVENTION

In one aspect, the invention uses Salmonella blebs as the immunogenic component for vaccination. Some Salmonella are known to form blebs spontaneously (e.g. the IkyD mutants of S. Typhimurium disclosed in reference 5 and various strains disclosed in reference 6), but in some circumstances a chemical treatment may be required (e.g. treatment with polymyxin B [7]). The inventors have created mutants of Salmonella in which the Tol-Pal system has been disrupted to disturb the bacterial envelope structure. During normal growth these mutants release into their culture medium large quantities of blebs which are rich in immunogenic outer membrane proteins, and these blebs can thus be used as immunogens. They can be particularly useful for raising cross-reactive responses which can protect against multiple serovars of Salmonella.

Thus the invention provides a Salmonella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins. Thus at least one protein from the natural five-protein Tol-Pal system is absent, resulting in a bacterium which, during growth in culture medium, releases greater quantities of outer membrane blebs into the medium than the same bacterium expressing all 5 Tol-Pal proteins. Preferably TolR is not expressed, but the other four proteins may be expressed. If TolA is not expressed, it is preferred that the bacterium should express no more than 3 of TolA, TolB, TolQ, TolR and Pal i.e. there is no expression of TolA and also of at least one other Tol-Pal protein. The invention also provides a Salmonella bacterium which does not express a TolR protein. The invention also provides a AtolR strain of Salmonella (particularly of a S. enterica), such as a AtolRAmsbB strain, a AtolRAwbaP strain, or a AtolR msbBAwbaP strain.

The invention also provides a Salmonella bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium.

The invention also provides a Salmonella bacterium (a) in which one or more components of its Tol- Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and (b) which does not express a native Salmonella lipopolysaccharide.

The invention also provides a method of preparing a hyperblebbing Salmonella bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's tolA, tolB, tolQ, tolR and/or pal genes. The mutating step may delete the gene. The method may also involve modification of gene(s) encoding a protein required for synthesis of the bacterium's lipopolysaccharide. Mutation of at least tolR is preferred.

The invention also provides a bleb isolated or obtainable from a bacterium of the invention. These blebs are useful as components of Salmonella vaccines.

The invention also provides a process for preparing Salmonella blebs, comprising a step of separating the blebs from a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria. Blebs prepared by this process can be used as components of Salmonella vaccines.

The invention also provides a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria. Blebs may be purified from this culture medium. The invention also provides a composition comprising blebs that, during culture of bacteria of the invention, are released into the culture medium. This composition does not comprise any living and/or whole bacteria. This composition and/or its components can be used for Salmonella vaccine preparation.

The invention also provides a composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22μηι filter of a culture medium in which a bacterium of the invention has been grown. This composition and/or its components can be used for Salmonella vaccine preparation.

The invention also provides a Salmonella bleb which includes one or more {i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51) of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648; (b) a protein comprising an amino acid sequence having at least j% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, where j is 50 or more {e.g. 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, wherein n is 7 or more {e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids {e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a signal peptide, etc. These blebs ideally do not include a native Salmonella lipopo!ysaccharide, and they may lack a native Salmonella O antigen.

641 proteins have been confirmed as present within blebs of the invention and to be immunoreactive with sera prepared against the blebs. Thus the individual proteins may be used as immunogenic components in purified form, separate from the blebs. Thus the invention also provides a bleb-free immunogenic composition comprising a bleb protein comprising: (a) one or more {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30. 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 5 1 , or 52) of amino acid sequences SEQ ID NOs 1 to 52, 60 to 355 and 366 to 648; (b) an amino acid sequence having at least j% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, where j is 50 or more {e.g. 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, wherein n is 7 or more {e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, and more preferred fragments are immunogenic fragments. Other preferred fragments lack one or more amino acids {e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of a SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a transmembrane domain, a signal peptide, etc.

Thus, for instance, the invention provides a Salmonella bleb which includes one or more of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648; (b) a protein comprising an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, wherein the fragment comprises an epitope (and, preferably, the blebs do not include a native Salmonella lipopolysaccharide and lack a native Salmonella O antigen). Similarly, the invention provides a bleb- free immunogenic composition comprising a protein comprising: (a) amino acid sequences SEQ ID NOs 1 to 52, 60 to 355 and 366 to 648; or (b) an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, said fragment comprising an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648.

Where a bleb is defined around SEQ ID NOs: 1 -52 or SEQ ID NOs: 60-355, it is ideally a S. Typhimurium bleb; where a bleb is defined around SEQ ID NOs: 356-648 it is ideally a S. Enteritidis bleb. A preferred subset within SEQ ID NOs: 1 to 52, 60-355 and 366-648 is SEQ ID NOs: 1 to 52.

The Tol-Pal system

Like many Gram-negative bacteria, the Salmonella naturally possess a Tol-Pal system which is made up of TolA, TolB, TolQ, TolR and Pal proteins. According to the invention, the natural Tol-Pal system is disrupted, thereby causing the bacterium to release greater quantities of outer membrane blebs into its culture medium during bacterial replication. Various disruptions can be made.

In some embodiments, at least one of the five Tol-Pal proteins is removed e.g. by deletion or inactivation of the gene encoding the protein. Thus the bacterium may express 0, 1 , 2, 3 or 4 of TolA, TolB, TolQ, TolR and Pal proteins. Removal of one of the five proteins can suffice, in which case the bacterium expresses only 4 of these proteins. Preferably the TolR protein is removed e.g. by inactivation of a starting strain's tolR gene. Thus the bacterium may be tolA⁺ tolB⁺ tolQ⁺ TolR Pat.

In some embodiments, the bacterium expresses all five Tol-Pal proteins, but at least one is mutated to cause hyperblebbing. For instance, the TolA, TolQ, TolR and/or Pal protein may be mutated such that the protein retains its membrane localisation but its interactions with other members of the Tol- Pal system are disrupted. The bacterium will thus retain TolA, TolQ and TolR as transmembrane proteins in the inner membrane, and Pal protein as a periplasm-facing lipoprotein in the outer membrane, but at least one of the TolA, TolQ, TolR and/or Pal proteins is mutated.

Examples of wild-type Salmonella amino acid sequences of the TolA, TolB, TolQ, TolR and Pal proteins are given in the sequence listing as SEQ ID NOs: 53 to 57, respectively. These five example sequences are from S. enterica subsp. enterica serovar Typhimurium strain LT2.

The Salmonella bacterium

The invention can be used with any of S. enterica or S. bongori, but preferably uses S. enterica. Within the S. enterica species, the invention can be used with any subspecies, including subspp. enterica, salamae, arizonae, diarizonae, houtenae, and/or indica. The invention can be used with any serovar, including but not limited to serovars Typhimurium and Enteritidis. Thus the invention can be used with any of the bacteria commonly known as S. Paratyphi, S. Typhi, S. Enteritidis, S. Typhimurium and S. Choleraesuis. It can also be used with serovars such as S. Dublin and S. Minnesota. Preferably the invention is used with the serovars that most commonly infect humans, namely S. Typhi, S. Paratyphi , S. Typhimurium and S. Enteritidis.

In addition to having a disrupted Tol-Pal system, thereby causing the bacterium to release greater quantities of outer membrane blebs into its culture medium during bacterial replication, a Salmonella of the invention can advantageously include one or more further changes relative to a wild-type strain. These changes can be used in particular to remove components from the bacterium which would be toxic or undesirable in a human vaccine.

For example, a bacterium may not express native Salmonella lipopolysaccharide (LPS), thereby reducing endotoxic activity. Various modifications can be made to prevent synthesis of native LPS, and these may disrupt the native lipid A structure, the oligosaccharide core, or the O antigen. Various mutant forms of LPS are known in Salmonella (such as the "rough" and "deep rough" mutants) and these have various genetic causes e.g. from mutations in any of rfaB, rfaC, rfaF, rfaG, rfal, rfaJ, rfbJ, rfl)P, rfaL, pmrB, pmrF, galE, etc. Absence of hexa-acylated lipid A in the LPS is preferred e.g. with a penta-acylated or tetra-acylated lipid A.

One useful strain does not express an active MsbB enzyme (an acyltransferase involved in secondary acylation of lipid A; also known as LpxM or WaaN), as msbB mutants produce LPS with reduced toxicity. Another useful strain does not express an active HtrB enzyme (another acyltransferase involved in secondary acylation of lipid A; also known as LpxL or WaaM), as htrB mutants produce LPS with reduced toxicity. Mutation of MsbB is preferred to HtrB because the latter can result in temperature sensitivity in Salmonella [8]. Absence of O antigen in LPS is useful, thereby avoiding a serospecific response. "Rough" Salmonella mutants which lack all or part of the O antigen are known in the art. One useful strain does not express an active WbaP enzyme (a transferase involved in the initiation reaction for synthesis of O antigen; also known as RfbP) as wbaP mutants (rough) can lack O antigen [9].

Preferred strains are inactivated for both msbB and wbaP. Some useful strains have penta- or tetra-acylated LPS which includes attached O antigen. More generally, though, preferred strains have penta- or tetra-acylated LPS which lacks attached O antigen. A S. enterica strain with tolR, msbB and wbaP knockouts is useful.

A Salmonella of the invention may hyper-express a Salmonella protein. For instance, expression of an immunogenic outer membrane protein can be increased by providing a second copy (chromosomal or episomal) or by providing the endogenous gene with a stronger promoter (e.g. a constitutive or inducible promoter) or by inactivating a repressor.

A Salmonella of the invention may express one or more heterologous proteins e.g. proteins which are not naturally found in Salmonella. If the heterologous protein is an outer membrane protein then blebs from the strain can be used as a delivery system for presenting non-Salmonella antigens to the immune system.

Salmonella bacteria of the invention can be prepared conveniently from wild-type or other starting strains using conventional techniques of mutagenesis. Inactivation of a gene can be achieved in various ways e.g. by deletion or mutation in its promoter, by deletion or mutation of its start codon, by introduction of a premature stop codon, by deletion of the complete coding region, by knockout, etc. Isogenic knockout mutants are preferred. In the resulting Salmonella bacterium mRNA encoding the desired gene is absent and/or its translation is inhibited (e.g. to less than 1% of wild-type levels).

A Salmonella bacterium of the invention may contain a marker gene in place of the inactivated gene e.g. an antibiotic resistance marker. This can be achieved using homologous recombination. Preferably, though, unmarked deletions (i. e. deletion without introduction of a marker gene) are used.

Some Salmonella strains possess a virulence plasmid which mediates virulence properties [ 10]. In some embodiments a Salmonella of the invention possesses a virulence plasmid; in other embodiments it does not possess a virulence plasmid.

Culture conditions for growing Salmonella are well known in the art. For example, they may be grown using an organic nitrogen source (such as amino acid mixtures e.g. containing Ala, Arg, Asn, Asp; casamino acids may be used), glycerol as a carbon source, etc. Inclusion of L-aspartic acid in the medium is particularly useful and may function as both a nitrogen and carbon source.

Salmonella of the invention may be grown under iron- limiting conditions as this has may up-regulate iron-regulated proteins which are immunogenic and highly-conserved among Salmonella spp. For instance, the bacteria may be grown in the presence of a compound such as desferal or 2,2'-dipyridyl or 8-hydroxyquinoline.

Blebs and hyperblebbing

Salmonella bacteria of the invention are, relative to their corresponding wild-type strains, hyperblebbing i.e. they release into their culture medium larger quantities of blebs than the wild-type strain. These blebs are useful as components of Salmonella vaccines.

The blebs typically have a diameter of 35- 120 nm, and sometimes 30-120 nm e.g. 50 nm diameter. Diameters in the range of 30-50 nm are commonly seen, and these are useful blebs of the invention.

The blebs are released spontaneously during bacterial growth and can be purified from the culture medium. The purification ideally involves separating the blebs from living and/or intact Salmonella bacteria e.g. by size-based filtration using a filter, such as a 0.22μηι filter, which allows the blebs to pass through but which does not allow intact bacteria to pass through [1 1], or by using low speed centrifugation to pellet cells while leaving blebs in suspension.

Thus, unlike the culture medium, bleb-containing compositions of the invention will generally be substantially free from whole bacteria, whether living or dead. The size of the blebs means that they can readily be separated from whole bacteria by filtration e.g. as typically used for filter sterilisation. Although blebs will pass through a standard 0.22μιη filters, these can rapidly become clogged by other material, and so it may be useful to perform sequential steps of filter sterilisation through a series of filters of decreasing pore size before using a 0.22μηι filter. Examples of preceding filters would be those with pore size of 0.8μπι, 0.45μηι, etc.

Separation of spontaneously-released blebs from the culture medium is more convenient than methods which involve deliberate disruption of the outer membrane (e.g. by detergent treatment or sonication) to cause outer membrane vesicles to form. Moreover, they are substantially free from inner membrane and cytoplasmic contamination. Blebs of the invention contain lipids and proteins. The protein content of the blebs has been analysed, and they include the proteins listed in Tables 1 to 3 and discussed below.

Salmonella outer membrane proteins

Tables 1 to 3 list the GenBank name and GI number for 641 proteins which were detected in Salmonella blebs of the invention. These 641 proteins may be used as immunogenic components in purified form, separate from blebs. 52 proteins were detected in a AtolR knockout of S. Typhimurium strain SL 1344; 296 in a AtolRAwbaP knockout of S. Typhimurium strain SL 1344; and 293 in a AtolRAwbaP knockout of S. Enteritidis strain PI 25109.

Polypeptides can be prepared by various means e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression or from Salmonella culture), etc. Heterologous expression in an E.coli host is a preferred expression route.

Polypeptides of the invention may be attached or immobilised to a solid support. Polypeptides of the invention may comprise a detectable label e.g. a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques. Polypeptides can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, disulfide bridges, etc.). Polypeptides are preferably Salmonella polypeptides.

Polypeptides are preferably prepared in substantially pure or substantially isolated form (i.e. substantially free from other Salmonella or host cell polypeptides) or substantially isolated form. In general, the polypeptides are provided in a non-naturally occurring environment e.g. they are separated from their naturally-occurring environment. In certain embodiments, the subject polypeptide is present in a composition that is enriched for the polypeptide as compared to a control. As such, purified polypeptide is provided, whereby purified is meant that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, where by substantially free is meant that less than 50%, usually less than 30% and more usually less than 10% of the composition is made up of other expressed polypeptides.

The term "polypeptide" refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, 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 labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can occur as single chains or associated chains. Pharmaceutical compositions

The invention provides a pharmaceutical composition comprising (a) blebs of the invention and (b) a pharmaceutically acceptable carrier. The invention also provides a process for preparing such a composition, comprising the step of admixing blebs of the invention with a pharmaceutically acceptable carrier.

The invention also provides a pharmaceutical composition comprising (a) the bleb-free immunogenic composition defined above and (b) a pharmaceutically acceptable carrier.

The immunogenic composition may include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like (e.g. stabilisers, preservatives), can also be present in such vehicles. A thorough discussion of suitable carriers is available in ref. 12.

Salmonella infections can affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. Administration via skin patch is also possible. To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml e.g. about 10+2 mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc. The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Compositions of the invention may be isotonic with respect to humans.

Due to the particulate nature of blebs a final vaccine product may be a suspension with a cloudy appearance. This appearance means that microbial contamination is not readily visible, and so the vaccine may contain an antimicrobial agent. This is particularly important when the vaccine is packaged in multidose containers. Preferred antimicrobials for inclusion are 2-phenoxyethanol and thimerosal. It is preferred, however, not to use mercurial preservatives {e.g. thimerosal), and it is preferred that the composition contains less than about 25 ng/ml mercury. More preferably, the composition is mercury- free. To improve thermal stability, a composition may include a temperature protective agent. As described in reference 13, a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0°C. Thus the composition can be stored below 0°C, but above its freezing point, to inhibit thermal breakdown. The temperature protective agent also permits freezing of the composition while protecting any mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40°C. A starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1 -80% by volume of the final mixture. Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components {e.g. antigen and adjuvant) in the composition. Examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG). Suitable PEGs may have an average molecular weight ranging from 200-20,000 Da. In a preferred embodiment, the polyethylene glycol can have an average molecular weight of about 300 Da ('PEG-300').

Compositions of the invention can include immunogens in addition to a bleb of the invention. For example, a composition can include a combination of blebs from at least two different Salmonella species, subspecies or serovars. Another useful composition can include a combination of Salmonella blebs of the invention with blebs from another bacterial genus e.g. a combination of Salmonella blebs and Shigella blebs (such as those disclosed in reference 14 e.g. from a AtolR or AtolRAmsbB strain of Shigella). For example, the invention provides a composition comprising blebs from a S. Typhimurium and also from (i) Shigella sonnei and/or (ii) Shigella flexneri. Ideally all blebs in such a combination are prepared from AtolR strains. Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other of other specified components, as needed. By 'immunologically effective amount', it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Previous work with vesicle vaccines (e.g. for meningococcus) offers pharmaceutical, posological and formulation guidance for administering blebs. The concentration of blebs in compositions of the invention will generally be between 10 and 500 μg/ml, preferably between 25 and 200 g/ml, and more preferably about 50 μg/ml or about 100 μg/ml (expressed in terms of total protein in the blebs). As shown in the examples, however, lower doses can be effective for seroconversion. Thus the concentration of blebs in compositions of the invention can be in the range of 1 ng/ml to 10 μg/ml, or 1 ng/ml to 1 μg/ml, or 0.5 μg/ml to 50 μg/ml. A dosage volume of 0.5ml is typical for injection.

The composition may be administered in conjunction with other immunoregulatory agents.

Adjuvants which may be used in compositions of the invention (particularly in bleb-free compositions) include, but are not limited to: A. Mineral-containing compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides

(e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 18], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt.

The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)₃, by infrared (I ) spectroscopy, in particular by the presence of an adsorption band at 1070cm^"1 and a strong shoulder at 3090-3100cm^"1 [chapter 9 of ref. 18] . The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pi of aluminium hydroxide adjuvants is typically about 1 1 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a P0₄/A1 molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO₄ by the presence of hydroxyl groups. For example, an I spectrum band at 3164cm^"1 (e.g. at 200°C) indicates the presence of structural hydroxyls [ch. 9 of ref. 18].

The P(VA1³⁺ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1 .2, and more preferably 0.95+0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO₄/AI molar ratio between 0.84 and 0.92, included at 0.6mg Al³⁺/ml. The aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5- 20μιη (e.g. about 5- 10μηι) after any antigen adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary. The suspensions are preferably sterile and pyrogen-free. A suspension may include free aqueous phosphate ions e.g. present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The suspensions may also comprise sodium chloride.

In one embodiment, an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2 : 1 e.g. >5 : 1 , >6 : 1 , >7 : 1 , >8 : 1 , >9 : 1 , etc.

The concentration of Al⁺⁺⁺ in a composition for administration to a patient is preferably less than lOmg/ml e.g. <5 mg/ml, <4 mg/ml, <3 mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and lmg/ml. A maximum of <0.85mg/dose is preferred. B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 18; see also ref. 15] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A) may also be used.

Various suitable oil-in-water emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion are generally less than 5 μηι in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization.

The invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6, 10, 15, 19,23-hexamethyl-2,6,10, 14, 18,22-tetracosahexaene. Other preferred oils are the tocopherols (see below). Oil in water emulsions comprising sqlauene are particularly preferred. Mixtures of oils can be used. Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxy ethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l ,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. As mentioned above, detergents such as Tween 80 may contribute to the thermal stability seen in the examples below.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1 %, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%. High levels of surfactants should be avoided, however, as these may affect the stability and/or integrity of bacterial blebs.

Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:

• A submicron emulsion of squalene, Tween 80, and Span 85. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as 'MF59' [15- 17], as described in more detail in Chapter 10 of ref. 18 and chapter 12 of ref. 19. The MF59 emulsion advantageously includes citrate ions e.g. lOmM sodium citrate buffer.

• An emulsion comprising squalene, an a-tocopherol, and polysorbate 80. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably <1 (e.g. 0.90) as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2, or at a weight ratio of about 1 1 :5. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL-a-tocopherol and 5ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.

• An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer. An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X- 100) and a tocopherol (e.g. an a-tocopherol succinate). The emulsion may include these three components at a mass ratio of about 75 : 1 1 : 10 (e.g. 750μg/ml polysorbate 80, 1 10μ§/ι^η1 Triton X-100 and 100μg/ml a-tocopherol succinate), and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic™ L 121 "). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-1 " adjuvant [20] (0.05- 1% Thr-MDP, 5% squalane, 2.5% Pluronic L 121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [21] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.

An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene ( 12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or ' Span 80'). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [22]. The emulsion may also include one or more of: alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized.

An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non- ionic surfactant. As described in reference 23, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl inositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.

A submicron oil-in-water emulsion of a non-metabolisable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 24, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.

An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25].

An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25]. • An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [26].

Antigens and adjuvants in a composition will typically be in admixture at the time of delivery to a patient. The emulsions may be mixed with antigen during manufacture, or extemporaneously, at the time of delivery. Thus the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. The antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (e.g. between 5: l and 1 :5, or 10: 1 and 1 : 10) but is generally about 1 : 1.

C. Saponin formulations [chapter 22 of ref. 181

Saponin formulations may also be used as adjuvants in the invention. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria offwianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21 , as well as lipid formulations, such as ISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21 , QH-A, QH-B and QH-C. Preferably, the saponin is QS21 . A method of production of QS21 is disclosed in ref. 27. Saponin formulations may also comprise a sterol, such as cholesterol [28].

Combinations of saponins and cholesterols can be used to form un ique particles called immunostimulating complexs (ISCOMs; see chapter 23 of ref. 18; also refs 29 & 30). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid of additional detergent [31].

A review of the development of saponin based adjuvants can be found in refs. 32 & 33.

D. Bacterial or microbial derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 34. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22μιη membrane [34]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [35,36].

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM- 174 is described for example in refs. 37 & 38. Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. References 39, 40 and 41 disclose possible analog substitutions e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in refs. 42-47.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [48]. The CpG sequence may be specific for inducing a Thl immune response, such as a CpG- A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs. 49-51. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, refs. 52-54. A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-3 1™ [55-57]. Thus an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g. between 15-40 nucleotides) including at least one (and preferably multiple) Cpl motifs (i.e. a cytosine linked to an inosine to form a dinucleotide), and (ii) a polycationic polymer, such as an oligopeptide (e.g. between 5-20 amino acids) including at least one (and preferably multiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC)i₃-3' (SEQ ID NO : 58). The polycationic polymer may be a peptide comprising 1 1-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 59). This combination of SEQ ID NOs: 58 and 59 provides the IC-31™ adjuvant.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 58 and as parenteral adjuvants in ref. 59. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G 192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 60-67. A useful CT mutant is or CT-E29H [68]. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 69, specifically incorporated herein by reference in its entirety. E. Human immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-1 2 [70], etc.) [71], interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor. A preferred immunomodulator is IL-12.

F. Bioadhesives and Mucoadhesives

B ioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [72] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [73].

G. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ~100nm to ~150μιη in diameter, more preferably ~200nm to ~30μιη in diameter, and most preferably ~500nm to -Ι Ομηι in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid) , a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes (Chapters 13 & 14 of ref. 18)

Examples of liposome formulations suitable for use as adjuvants are described in refs. 74-76.

I. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues (e.g. "Resiquimod 3M"), described further in refs. 77 and 78.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [79]; (2) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g.

3dMPL) [80]; (3) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) + a cholesterol;

(4) a saponin (e.g. QS21) + 3dMPL + IL- 12 (optionally + a sterol) [81 ]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [82]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L 121 , and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL). Other substances that act as immuno stimulating agents are disclosed in chapter 7 of ref. 18.

An aluminium hydroxide adjuvant is useful, and antigens are generally adsorbed to this salt. Oil-in- water emulsions comprising squalene, with submicron oil droplets, are also preferred, particularly in the elderly. Useful adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & an aluminium salt, or resiquimod & an aluminium salt. A combination of an aluminium salt and 3dMPL may be used.

Immunisation

In addition to providing immunogenic compositions as described above, the invention also provides a method for raising an immune response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The immune response will typically include an antibody response. The immune response is preferably a protective immune response. The invention also provides compositions of the invention for use in such methods. Protection can be measured in vivo by various techniques e.g. by immunising test animals, administering a lethal dose of Salmonella, and assessing survival. Suitable models are available in the art e.g. the chicken challenge model of reference 83, the murine models of references 84 & 85, or the murine model used herein. In addition to providing immunogenic compositions as described above, the invention also provides a method for raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The antibody response is preferably a protective antibody response. The invention also provides compositions of the invention for use in such methods. The invention also provides a method for protecting a mammal against a Salmonella infection and/or disease {e.g. against gastroenteritis, typhoid fever and/or paratyphoid fever; or against bacteremia), comprising administering to the mammal an immunogenic composition of the invention.

The invention provides compositions of the invention for use as medicaments {e.g. as immunogenic compositions or as vaccines). It also provides the use of vesicles of the invention in the manufacture of a medicament for preventing a Salmonella infection in a mammal e.g. for preventing gastroenteritis, typhoid fever and/or paratyphoid fever (or bacteremia). It also provides the use of a bleb protein (as defined above) in the manufacture of a bleb-free medicament for preventing a Salmonella infection in a mammal e.g. for preventing gastroenteritis, typhoid fever and/or paratyphoid fever (or bacteremia). The mammal is preferably a human. The human may be an adult or, preferably, a child. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant); where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. The uses and methods are particularly useful for preventing/treating diseases including, but not limited to, gastroenteritis, typhoid fever, bacteremia and/or paratyphoid fever.

Efficacy of therapeutic treatment can be tested by monitoring Salmonella infection after administration of the composition of the invention. Efficacy of prophylactic treatment can be tested by monitoring immune responses against immunogenic proteins in the blebs or other antigens after administration of the composition. Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age) and then determining standard serological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made.

Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. Sublingual or buccal administration is also possible. Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is about 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4- 16 weeks), and between priming and boosting, can be routinely determined.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The term "about" in relation to a numerical value x is optional and means, for example, +10%.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of reference 86. A preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is well known and is disclosed in reference 87.

"GI" numbering is used above. A GI number, or "Genlnfo Identifier", is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases. The GI number bears no resemblance to the accession number of the sequence record. When a sequence is updated (e.g. for correction, or to add more annotation or information) then it receives a new GI number. Thus the sequence associated with a given GI number is never changed.

Where the invention concerns an "epitope", this epitope may be a B-cell epitope and/or a T-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [88,89] or similar methods), or they can be predicted (e.g. using the Jameson- Wolf antigenic index [90], matrix-based approaches [91], MAPITOPE [92], TEPITOPE [93,94], neural networks [95], OptiMer & EpiMer [96, 97] , ADEPT [98], Tsites [99], hydrophilicity [100], antigenic index [ 101 ] or the methods disclosed in references 102-103, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as "antigenic determinants".

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows SDS-PAGE results. The four lanes are MW marker, medium alone, wild-type LT2 and AtolR LT2.

Figure 2 shows mean absorbance in ELISA assays of Salmonella bleb-specific IgG, in all cases using 1 : 10000 dilutions of sera. In Figure 2A the 3 groups of bars show results after different doses of LT2 AtolR blebs (0.1 , 1 , 10μg from left to right). Each group has a bar for 3 different time points (days 0, 14, 32 from left to right). In Figures 2B and 2C ELISA analyses were performed using as coating blebs from SL 1344 AtolRAwbaP without O antigen (2B) or blebs from SL1344 AtolR with O antigen (2C); the scatter plots show results using sera from mice immunised subcutaneously with blebs from five different SL 1344 mutants (from left to right: AtolR; AtolRAwbaP; AtolRAwbaPAmsbB O. ^g; AtolRAwbaPAmsbB AtolRAwbaPAmsbB 10 μg), and each group includes results for three different time points: day 0 (circles; pre-immune sera), day 20 (triangles; post first immunization), day 45 (squares; post second immunization). Horizontal bars show geometric means for the three time points.

Figure 3 shows a competitive ELISA in the presence of exogenous LPS, where sera raised against blebs from LT2 AtolR were mixed with different amounts of LPS ^g/mL on x-axis) from S. Typhimurium and the remaining IgG response to blebs from S. Typhimurium LT2 AtolR was analyzed by ELISA. Plates were coated with LT2 AtolR blebs.

Figure 4 shows (A) 2D SDS-PAGE of SL1344 AtolR blebs, with pH from 3-11 on x-axis and MW from 10-250 kDa on y-axis; and (B) a 2D immunoblot of SL 1344 AtolR blebs probed with serum raised against LT2 AtolR blebs.

Figure 5 shows SDS-PAGE analysis of samples taken (i) before the first TFF, (ii) after the first TFF, and (iii) after the second TFF. Each panel has three lanes showing, from left to right, total protein, bleb protein and soluble protein.

Figure 6 shows a 12% Bis-Tris SDS-PAGE of 10 μg of Salmonella blebs (a: SL1344Afo/i?, b: SL \ 344AtolRAwbaP, c: SL UUAtolRAwbaPAmsbB) grown in LB under iron-rich (left, a-c) or iron-limiting (right, a-c) conditions. Some iron regulated proteins are highlighted with an arrow.

Figure 7 shows SL 1344Ato/i? blebs visualized by transmission electron microscopy. The bar at the bottom-right is 1 OOnm long.

Figure 8 shows the analysis by flow cytometry of reactivity of sera raised against blebs from S. Typhimurium SL 1344 AtolR (panels A, C, E) or from SL 1344 AtolRAwbaP (panels B, D, F) with live bacteria of S. Typhimurium (wild-type in panels A & B; AwbaP mutants in panels C & D) and S. Enteritidis AwbaP mutants in panels E & F).

Figure 9 shows I D western blots with 2.5, 5 or 10 μg of blebs generated from S. Typhimurium SL1344 AtolRAwbaP (left in each blot) and S. Enteritidis P 125 109 AtolRAwbaP (right in each blot) and probed with sera raised against S. Typhimurium SL 1344 AtolRAwbaP (left blot) and SL1344 tolRAwbaPAmsbB (right blot).

Figure 10 shows bacterial counts in (A) spleen (B) liver (C) blood after challenge of mice with S. Typhimurium M525, following immunisation as follows (left to right): negative control; positive control; blebs from S. Typhimurium SL1344Afo/i?; blebs from SL 1344Afo/RA wbaP; and blebs from S. Enteritidis P 125109 AtolRAwbaP. Results are counts per gram of organ or per mL of blood. Results of each vaccinated group were compared by Mann- Whitney test to results of mice immunized with PBS as negative control: *** = p<0.0005, ** = p<0.005, * = p<0.05.

Figure 1 1 shows IgG titers to blebs from (A) SL1344 AtolR and (B) SL 1344 AtolRAwbaP in sera of mice immunized intranasally with blebs from SL 1344 AtolR ( ^g; left); SL 1344 AtolR (5\ig; middle), or SL 1344 AtolRAwbaP (1 μg; right).

MODES FOR CARRYING OUT THE INVENTION

Salmonella gene inactivation

Three main methods were used for gene inactivation: (i) restriction enzyme cloning; (ii) PCR; and (iii) P22 transduction. For restriction enzyme (RE) cloning, regions 500 bp upstream and downstream of the target gene (e.g. the tolR gene) were amplified by PCR with primers containing RE sites at the 5' and 3' end. These fragments were then digested with the respective RE and ligated to a previously RE digested pBlueScript II S (+) (pBS) to generate a plasmid containing the 500 bp upstream and downstream regions of tolR with a restriction site in the middle (e.g. pBS-tolR5'3'). This plasmid was subsequently digested with an RE and ligated to a kanamycin resistance cassette that had been amplified from pUC4K with primers corresponding to the same RE site at both the 5' and 3 ' end, to generate a plasmid containing the 500 bp upstream region, the kmR cassette, and the 500 bp downstream region (e.g. pBS-tolR5'-kmR-tolR3'). This plasmid was linearised with a RE and a linear DNA fragment was amplified by PCR to yield e.g. tolR5'kmRtolR3'. This fragment was electroporated into the LT2 strain expressing the phage λ red homologous recombination system encoded by plasmid pAJD434, and LT2 mutants where the target gene had been replaced with the kmR cassette (e.g. LT2AtolR::kari) were selected on LB plates containing kanamycin. Gene replacement was confirmed by PCR amplification of the genomic DNA of the mutant colonies using primers annealing upstream and downstream of the regions used for homologous recombination.

Inactivation of chromosomal genes by PCR was performed as in reference 104. For replacing wbaP a chloramphenicol-resistance gene was amplified by PCR from pKD3 using primers with 45 bp extensions that were homologous to the regions adjacent to the target gene. For replacing msbB a tetracycline-resistance gene was amplified by PCR from E.coli DH5 genomic DNA containing a Tnl 0 using primers with 45bp extensions that were homologous to the regions adjacent to the target gene. The PCR products were then transformed into electrocompetent strains expressing the phage λ red recombinase encoded on pSIM18 and successful recombination was checked by PCR on the transformant colonies grown on selective medium (chloramphenicol or hygromycin). Gene deletion was confirmed by PCR screening. Rough mutants (AwbaP) were further confirmed by silver stained LPS SDS-PAGE and resistance to P22 bacteriophage.

For inactivation of chromosomal genes by P22 transduction, ΙΟΟμΙ of an overnight culture of the donor strain with Ι ΟμΙ of P22 lysate. The mixture was incubated at 37°C for 20 mins, 3mL of liquid top LB agar (0.75% agar) were added to the mix, and poured over a pre-warmed LB plate for incubation for 4 hours at 37°C. Once the confluence peak was reached, top of the agar was scrapped, mixed with 3 mL of LB broth, mixed thoroughly in the presence of lOOul of chloroform, incubated for 20 min at room temperature, and the lysate containing the phage was stored at 4°C until further use. For transduction of the recipient strain, 1 OOul of an overnight culture was mixed with 1 Oul of P22 lysate, and incubated at 37°C for 15 min. 1 mL of LB broth with l OmM EGTA was added to the mix, incubated at 37°C for 1 hour, and cells were spun down at 14,000 rpm. 100 μΐ of the supernatant were plated on selective media containing l OmM EGTA. lOOul of P22 lysate were also plated as negative control. These methods provided: (i) AtolR mutants of S. Typhimurium strains LT2, SL1344, D23580 and of S. Enteritidis strain P I 25109; (ii) SL1344 strains with knockouts of wbaP, msbB, rfaG, rfaF or rfaC; (iii) a double deletion mutant AtolRAwbaP of strain SL1344; and (iv) a AtolRAwbaPAmsbB triple deletion mutant of strain SL 1344. Figure 1 shows a ID gel of TCA-precipitated culture supernatant, after 0.22μηι filtration, after growing wild-type and AtolR LT2 bacteria. A large number of protein bands were visible in the supernatant of AtolR mutant, but most of these bands were not visible in the wild type strain. Thus the mutant strain secretes many more proteins into the culture medium during growth.

Purification of blebs

Mutant strains were grown in LB or HTMC to OD 0.8 or 6.0, respectively. Bacteria were pelleted by centrifugation at 4000 x g and culture media was filtered through a 0.22μηι filter. Filtrates containing blebs were concentrated using a stirred ultrafiltration cell and ultracentrifuged (186,000 x g, 3 hours, 4°C). The bleb-containing pellet was resuspended in PBS . Total protein concentration was determined by Bradford assay. Transmission electron microscopy demonstrated that this purification procedure results in a homogeneous population of blebs -30-50 nm diameter (Figure 7).

In other work the knockout strains were grown under the following conditions: pH 7.1 , 37°C, dissolved oxygen maintained at 30% saturation by controlling agitation and setting maximum aeration. The pH was controlled by addition of 30% ammonium hydroxide. Foam was controlled by addition of 0.25g/L of PPG in the fermentation medium. The culture inoculum was 1 % of the fermenter volume. The fermentation process was stopped after 14 hours, when the culture achieved a cell concentration of 29 OD₆oonm- Culture supernatant containing vesicles was separated from the Salmonella biomass by tangential flow filtration (TFF) through a 0.22μηι pore size filter cassette with a 0.1m² filtration area. The biomass was retained on the cassette and the permeate containing the vesicles was collected. Soluble proteins in the permeate were removed from the blebs by a second microfiltration trough a 0.1 μηι pore size filter cassette (200cm² filtration area). Following a 10-fold concentration the retentate was subjected to 10 diafiltration steps against PB S and subsequently collected. To analyze protein content samples from each step of the process were ultracentrifuged (2 hours, 200,000g,) and the vesicle-containing pellet was resuspended in PBS. The protein contents of the vesicles (the pellet) and the soluble fraction (the supernatant) were analyzed by SDS-PAGE (Figure 5). All the samples were normalized to volume.

Blebs were also obtained from bacteria grown in the presence of 200μΜ of iron chelator 2,2'- dipyridyl to simulate in vivo iron-limiting conditions. Under these conditions Salmonella can upregulate the expression of certain outer membrane proteins which are highly conserved among serovars. Analysis of the resulting blebs did not show any major changes in protein composition. As shown in Figure 6 several proteins of predominantly 70-80 kDa size were induced under iron- limiting conditions, which is in accordance with the known major iron-regulated proteins, including cirA (74kDa), fepA (83 kDa), fhuA (81 kDa), fhuE (81 kDa), and tonB (79 kDa). Bleb immunogenicity

Three groups of mice were subcutaneous ly immunized twice with a two-week interval (days 0 & 14) using different amounts of llAtolR blebs (0.1 μg, 1.0 μg, 10.0 μg). To assess the immunogenicity by ELISA sera were collected at three time points (day 0, 14, 32). Flat bottom Maxisorp plates were coated with blebs overnight, washed with PBST, and incubated with sera (1 : 1 ,000 in PBS) for 2 hours. Plates were further washed with PBST and incubated with alkaline phosphatase anti-mouse IgG (1 : 10,000) for 1 hour. 4-nitrophenyl phosphate disodium salt hexahydrate substrate was added for 1 hour, and absorbance was measured (490-405nm).

As shown in Figure 2A mice displayed a dose-dependent antibody response to the llAtolR blebs. The second immunization was important for a suitable antibody response, as seen by the large increase in IgG response between days 14 and 32 at all three doses. The strong ELISA signal for 1 : 1000 dilutions of the sera indicates a strong immunogenicity of the blebs. Competitive ELISA assays in the presence of exogenous LPS from S. Typhimurium (Figure 3) were performed to assess the contribution of IgG directed to the LPS to the overall IgG response to blebs from LT2AtolR. More than 10 μg/mL of LPS in the assay were needed to reduce the signal in the ELISA, indicating that a significant portion of the IgG is directed to the LPS.

As the O antigen of Salmonella LPS is highly immunogenic, further immunization studies were performed to compare antibody responses to blebs derived from S. Typhimurium SL 1344 AtolR, AtolRAwbaP, and AtolRAwbaPAmsbB. A dosage of 1 μg was chosen for blebs from SL 1344 AtolR and SL 1344 AtolRAwbaP. A d o s e-ranging study was performed with blebs from SL1344 AtolRAwbaPAmsbB. Two subcutaneous immunizations on days 0 and 21 were performed using (a) O. ^g (first immunisation) + 0.1 μg (second immunisation), (b) 1 + 1 g, and (c) 10+ 1 μg. All blebs were generated under iron-limiting conditions (200μΜ dipyridyl). As shown in Figure 2B, sera raised against 1 μg or 10 μg (first immunisation) of blebs from SL1344 AtolRAwbaP and AtolRAwbaPAmsbB elicited higher IgG responses to blebs from SL 1344 AtolRAwbaP than 1 μg of blebs from SL 1344 AtolR, indicating that the removal of the O antigen from blebs enhances the IgG response to bleb proteins. ELISA assays with blebs from SL 1344 AtolR (Figure 2C) were performed as control and revealed that in vitro the O antigen shields the surface proteins from antibodies as most sera raised against blebs from SL 1344 AtolRAwbaP and AtolRAwbaPAmsbB did not show strong reactivity with blebs from SL1344 AtolR.

Blebs from S. Typhimurium SL1344 AtolR and SL 1344 AtolRAwbaP were also tested for their ability to elicit a strong antibody responses by intranasal immunization. Mice were immunized twice with 1 μg or 5μg of SL1344 AtolR blebs, or 1 μg or 5μg of AtolRAwbaP blebs (although the 5μg dose with AtolRAwbaP blebs was not well tolerated and so this experiment was stopped). IgG titers were determined by ELISA with serial dilutions of sera. As shown in Figure 1 1, both types of blebs elicited very strong antibody responses. Especially, blebs from SL 1344 AtolRAwbaP elicited very high titers against blebs without O antigen suggesting a strong response to the proteins or the LPS core. As 1 μg elicited titers above 1 :100,000 a much lower dosage is expected to results in seroconversion (4-fold increase of antibody titers) which is the criterion for a positive antibody response in humans.

Therefore the bleb approach has a strong potential to produce effective and low-cost vaccines and can be extended to different Salmonella strains towards a broad spectrum vaccine.

Bleb characterisation

Blebs were characterised by I D or 2D gel electrophoresis. For I D characterisation, 20 μg of blebs were precipitated for 1 hour in TCA 0.4% DOC, washed three times in 100% ethanol, and resuspended in reswelling buffer (7M urea, 2M thiourea, 2% CHAPS, 2% ASB- 14, 0. 1 % DTT, 20mM Tris base, and bromophenol blue) before loading onto a 10% Bis-Tris gel. The gel was run at 120 V in MOPS buffer and stained with Coomassie overnight. For 2D characterisation, 200 g of blebs were precipitated in the same way as ID for separation in the first dimension on non-linear pH 3-1 1 gradient IPG strip. Proteins were then separated in the second dimension on a 4-12% Bis-Tris gel in MOPS buffer, and stained with Coomassie Blue. A 2D SDS-PAGE of blebs derived from SL 1344 AtolR is shown in Figure 4A.

Protein spots were excised from the gel and proteins and identified using mass finger printing. 52 proteins identified in the AtolR knockout of strain SL1344 are listed in Table 1. Analysis of blebs from the S. Typhimurium SL 1344 AtolRAwbaP strain revealed the 296 proteins listed in Table 2. Similarly, Table 3 lists 293 proteins which were identified in blebs from the AtolRAwbaP mutant of S. Enteritidis.

A 2D separation of the SL1344 to/i? blebs was also analysed by immunoblot using sera obtained from mice immunised with LT2AtolR blebs, thereby identifying immunogenic proteins in the blebs. Gels were transferred to a nitrocellulose membrane using the iBlot dry transfer system, incubated with mouse anti-bleb sera (1 : 1000 in 3% milk in PBS), washed three times for 10 min in 3% milk, 0.1% Tween, PBS, incubated with secondary antibody anti-mouse HRP (1 :5000), washed three times for 10 min in 3% milk, 0.1% Tween, PBS, and developed by chemiluminescence using ImageQuant.

The 2D blot in Figure 4B shows three major groups of bands in the middle of the blot, confirming cross-reactivity of antibody between the LT2 and SL 1344 blebs. By comparison with earlier 2D-PAGE results the bands appear to correspond to outer membrane protein A. Cross-reactivity with different Salmonella serovars of sera raised against blebs

In order to assess the ability of anti-bleb antibodies to bind to live Salmonella, S. Typhimurium and S. Enteritidis wild type and AwbaP mutants were stained with sera raised against blebs from S. Typhimurium AtolR or AtolRAwbaP. Antibody binding was assessed by flow cytometry (Figure 8). As seen by ELISA (Figure 2B), the OAg of the LPS can interfere in vitro with the binding of antibodies to surface proteins and sera raised against SL1344 AtolRAwbaP did not react with WT SL 1344. In contrast, a strong signal was obtained using the wbaP mutants of S. Typhimurium. Strong binding was also observed with the heterologous S. Enteritidis P 125109 AwbaP strain indicating extensive cross reactivity of protein sera. This is in agreement with results of proteomic analysis, which shows significant overlap in protein identities between blebs from these two Salmonella strains, and the western blot analysis (Figure 9 and see below).

Cross-reactivity of sera generated against blebs from SL 1344 AtolRAwbaP was also assessed by immunoblotting. Proteins in blebs from S. Typhimurium SL1344 AtolRAwbaP and S. Enteritidis P 125109 AtolRAwbaP were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Pooled sera from mice immunised subcutaneous ly with S. Typhimurium SL1344 AtolRAwbaP blebs or SL1344 AtolRAwbaP AmsbB blebs were used to probe the membrane (Figure 9). The blots show that the strong antibody reactivity observed previously by ELISA and flow cytometry is elicited by a wide number of proteins in blebs. Moreover, the blots showed extensive cross reactivity with S. Enteritidis proteins, as seen by the similarities in the protein pattern between the two types of blebs. Slight differences can be observed in the 50-75 kDa region. Protection against S. Typhimurium by blebs in a murine challenge model

In order to assess the efficacy of blebs as a vaccine, ten C57/BL mice were immunized subcutaneously with 1 μg of blebs generated under iron-limiting conditions from S. Typhimurium SL 1344 AtolR, SL 1344 AtolRAwbaP, and S. Enteritidis PI 25109 AtolRAwbaP on days 0, 7, and 21. A negative control group was not immunised. As positive control, mice were immunized orally with 10⁹ bacteria of the live attenuated vaccine strain S. Typhimurium SL3261 (S. Typhimurium SL 1344 AaroA) on day 0. The mice were challenged on day 63 with 1.1 x l O⁶ bacteria of the wild-type strain S. Typhimurium M525 (with intermediate virulence). The mice were sacrificed 3 days after challenge and colonization of spleen, liver, and blood by M525 was determined. As shown in Figure 10A, immunization with all types of bleb significantly reduced colonization of the spleens. Importantly, immunization with blebs from heterologous S. Enteritidis also resulted in a reduction of colonization in the challenged mice, indicating that blebs without O antigen could potentially protect against multiple serovars of Salmonella.

In liver and blood (Figures 10B, I OC) no significant reduction of colonization was achieved using blebs without O antigen from either S. Typhimurium SL1344 AtolRAwbaP or S. Enteritidis P I 25109 AtolRAwbaP. However, since the challenge model has not been optimized and colony counts in liver and blood in non-immunized animals were much lower than expected from other experiments, the spleen results are more relevant.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. TABLE 1

NB: SEQ ID NO: 21 is the same as SEQ ID NO: 54; SEQ ID NO: 35 is the same as SEQ ID NO: 57.

TABLE 2

SEQ ID Gl Definition

NO:

60 301157743 conserved hypothetical protein

61 301157229 pagP antimicrobial peptide resistance and lipid A acylation protein

62 301160091 ggt gamma-glutamyltranspeptidase precursor

63 301159972 rplX 50S ribosomal subunit protein L24

64 301158148 putative L-lactate oxidase

65 301158229 rnb Exoribonuclease II

66 301158095 adhP alcohol dehydrogenase

67 301160028 damX DamX protein

68 301159549 N-acetylmuramoyl-L-alanine amidase

69 301160079 glgB 1 ,4-alpha-glucan branching enzyme

70 301157614 ompA outer membrane protein A

71 301158760 cirA colicin I receptor precursor

72 301159343 iroN TonB-dependent outer membrane siderophore receptor protein

73 301157186 fepA ferrienterobactin receptor precursor

74 301158100 ompD outer membrane protein

75 301156818 fhuA ferrichrome-iron receptor

76 301157745 fhuE outer-membrane receptor for Fe(iii)-coprogen, fe(iii)-ferrioxamine b and fe(iii)- rhodotrulic acid

77 301157024 tsx nucleoside-specific channel-forming protein tsx precursor

78 301157339 tolB tolB protein precursor

79 301159814 possible exported protein

80 301156721 surA survival protein SurA precursor

81 301159740 tolC outer membrane protein TolC precursor

82 301158008 putative exported protein

83 301158840 glpQ glycerophosphoryl diester phosphodiesterase periplasmic precursor

84 301159069 putative lipoprotein

85 301158826 ompC outer membrane protein C

86 301158114 possible ATP-binding protein

87 301157372 ybhC possible pectinesterase precursor

88 301156851 yaeT outer membrane protein precursor

89 301157848 osmE osmotically inducible lipoprotein E precursor

90 301159817 possible lipoprotein;

91 301159548 mltA membrane-bound lytic murein transglycosylase A precursor

92 301157421 ybiS putative exported protein

93 301156836 htrA protease DO precursor; heat shock protein HtrA

94 301157239 rlpA rare lipoprotein A precursor

95 301157417 ompX outer membrane protein x precursor

96 301160724 lamB maltoporin precursor

97 301159661 ansB L-asparaginase

98 301157978 slyB outer membrane lipoprotein SlyB precursor

99 301157340 pal peptidoglycan-associated lipoprotein precursor; 100 301161061 sit lytic murein transglycosylase;

101 301160182 yiaD putative outer membrane protein

102 301158949 fadL long-chain fatty acid transport protein precursor

103 301156722 imp organic solvent tolerance protein precursor

104 301160472 pldA detergent-resistant phospholipase A

105 301158473 fliC flagellin

106 301157341 ybgF putative exported protein;

107 301159391 mltB membrane-bound lytic transglycosylase B precursor

108 301158369 pre tail-specific protease precursor

109 301159988 tuf elongation factor Tu

110 301160642 tuf2 elongation factor Tu

111 301159621 conserved hypothetical protein;

112 301158950 vacJ VacJ lipoprotein precursor

113 301159484 nlpD lipoprotein NlpD precursor

114 301158728 bglX periplasmic beta-glucosidase precursor

115 301160718 putative lipoprotein

116 301158679 wza putative polysaccharide export protein

117 301160822 groEL GroEL protein

118 301157763 potD spermidine/putrescine-binding periplasmic protein precursor

119 301157474 probable lipoprotein

120 301159831 conserved hypothetical protein

121 301158751 mgIB D-galactose-binding periplasmic protein precursor

122 301156852 ompH outer membrane protein OmpH precursor

123 301160152 yhjJ putative zinc-protease precursor

124 301160734 putative exported protein

125 301157756 putative exported protein

126 301159210 putative lipoprotein

127 301157056 yajG putative lipoprotein

128 301158115 probable TonB-dependent receptor YncD precursor

129 301159633 conserved hypothetical protein;

130 301160416 rbsB D-ribose-binding periplasmic protein

131 301160810 phoN nonspecific acid phosphatase precursor;

132 301157886 pps phosphoenolpyruvate synthase

133 301156871 rcsF RcsF protein

134 301157912 IppA major outer membrane lipoprotein;

135 301158478 yedD putative lipoprotein;

136 301158089 putative exported protein

137 301159996 fkpA FKBP-type peptidyl-prolyl isomerase

138 301157265 ybeJ ABC transporter periplasmic binding protein (glutamate/aspartate?)

139 301160155 yhjL putative polysaccharide biosynthesis protein subunit C

140 301157786 pagC outer membrane invasion protein (PagC)

141 301160725 malM maltose operon periplasmic protein

142 301157910 putative exported protein

143 301158852 conserved hypothetical protein

144 301159421 sitA Iron transport protein, periplasmic-binding protein

145 301159957 rplQ 50S ribosomal subunit protein L17

146 301158344 putative lipoprotein

147 301158821 eco Ecotin precursor.

148 301158576 erfK putative exported protein

149 301157076 conserved hypothetical lipoprotein

150 301158533 capsid related protein

151 301160584 putative ABC transport protein, solute-binding component

152 301157248 rlpB rare lipoprotein B precursor

153 301158996 cysP thiosulphate-binding protein precursor 154 301157793 putative substrate-binding transport protein

155 301157827 gapA glyceraldehyde 3-phosphate dehydrogenase A

156 301157880 nlpC putative lipoprotein

157 301159632 tktA transketolase

158 301158912 hisJ histidine-binding peripiasmic protein

159 301159565 SL2985 possible lipoprotein

160 301159667 mltC membrane-bound lytic murein transglycosylase C

161 301160831 blc putative lipoprotein

162 301157103 ushA UDP-sugar hydrolase

163 301160722 malE peripiasmic maltose-binding protein

164 301159350 putative exported protein

165 301160646 rplA 50S ribosomal subunit protein L1

166 301159726 sufl Sufi protein

167 301161024 mdoB putative phosphoglycerol transferase

168 301158135 putative lipoprotein

169 301157792 putative lipoprotein

170 301159046 putative exported protein

171 301160067 glpD aerobic glycerol-3-phosphate dehydrogenase

172 301160134 putative phosphatase

173 301157865 putative outer membrane protein

174 301160741 aphA class B acid phosphatase precursor

175 301160389 pstS peripiasmic phosphate-binding protein

176 301157476 putative N-acetylmuramoyl-L-alanine amidase

177 301159553 ptr protease III precursor (pitrilysin)

178 301156872 yaeC putative lipoprotein precursor

179 301160896 ytfM putative exported protein

180 301158176 hsIJ heat shock protein (hsIJ)

181 301160097 ugpB glycerol-3-phosphate-binding peripiasmic protein

182 301156781 aceF dihydrolipoamide acetyltransferase component (E2) of pyruvate dehydrogenase

183 301159908 mdh malate dehydrogenase;

184 301160397 atpD ATP synthase beta subunit

185 301159966 rpsE 30S ribosomal subunit protein S5

186 301157218 ma ribonuclease I precursor

187 301158058 outer membrane protein

188 301159594 possible lipoprotein

189 301160015 ppiA peptidyl-prolyl cis-trans isomerase

190 301158320 treA Peripiasmic trehalase precursor

191 301157864 putative outer membrane protein

192 301156976 foxA ferrioxamine B receptor precursor

193 301158068 putative secreted hydrolase

194 301157025 yajl putative lipoprotein

195 301158127 pcgL D-alanyl-D-alanine dipeptidase

196 301161041 osmY Putative peripiasmic protein

197 301158150 mdoD Glucans biosynthesis protein D precursor.

198 301157609 putative lipoprotein

199 301157195 fepB ferrienterobactin-binding peripiasmic protein precursor

200 301157722 flgl Flagellar P-ring protein precursor

201 301158302 hemM Outer-membrane lipoprotein lolB precursor.;

202 301159028 putative exported protein

203 301160751 siiC putative type-l secretion protein

204 301157660 agp glucose-1 -phosphatase precursor (GI Pase), secreted

205 301159977 rpsC 30S ribosomal subunit protein S3

206 301159989 fusA elongation factor G

207 301157542 ompF outer membrane protein F precursor 208 301157718 flgE flagellar hook protein FlgE

209 301157358 gpmA phosphoglycerate mutase 1

210 301157822 putative outer membrane protein

211 301159543 possible lipoprotein

212 301157171 outer membrane esterase

213 301160881 rpll 50s ribosomal subunit protein L9

214 301160119 putative lipoprotein

215 301156844 tsf Elongation factor Ts

216 301159867 conserved hypothetical protein

217 301156638 talB transaldolase B

218 301158292 putative invasin

219 301157845 putative exported protein

220 301158113 putative lipoprotein

221 301159983 rpIC 50S ribosomal subunit protein L3

222 301156782 IpdA dihydrolipoamide dehydrogenase

223 301159897 degQ serine protease

224 301161066 creA expressed protein of unknown function

225 301157748 putative lipoprotein

226 301158727 yehZ putative periplasmic protein

227 301158323 mltE membrane-bound lytic murein transglycosylase E

228 301157524 rpsA 30S ribosomal protein S1

229 301160169 dppA periplasmic dipeptide transport protein precursor

230 301160647 rplJ 50S ribosomal subunit protein L10

231 301160847 amiB N-acetylmuramoyl-L-alanine amidase

232 301159926 conserved hypothetical protein

233 301157691 mdoG Glucans biosynthesis protein G precursor.

234 301160593 glpK glycerol kinase

235 301159859 possible exported protein

236 301156710 putative lipoprotein

237 301156881 mltD membrane-bound lytic murein transglycosylase d precursor

238 301159511 eno Enolase

239 301157329 sucC succinyl-CoA synthetase beta chain

240 301159625 pgk phosphoglycerate kinase

241 301160401 atpF ATP synthase subunit B

242 301157415 dps DNA protection during starvation protein

243 301159662 conserved hypothetical protein

244 301158273 oppA periplasmic oligopeptide-binding protein precursor (OppA)

245 301158278 hns DNA-binding protein (histone-like protein Hlp-ll)

246 301158468 fliY cystine-binding periplasmic protein (FliY)

247 301157867 Putative DNA/RNA non-specific endonuclease

248 301157058 tig trigger factor

249 301156643 dnaK Chaperone protein dnaK

250 301158458 putative exported protein

251 301159154 predicted bacteriophage protein

252 301160645 rplK 50S ribosomal subunit protein L11

253 301160461 putative lipoprotein

254 301159024 maeB NADP-dependent malate dehydrogenase (decarboxylating)

255 301157779 icdA isocitrate dehydrogenase

256 301158854 ais hypothetical protein (protein induced by aluminum)

257 301157414 glnH glutamine-binding periplasmic protein precursor

258 301159599 dsbC thiol:disulfide interchange protein

259 301160666 hupA histone like DNA-binding protein HU-alpha (NS2) (HU-2)

260 301158319 gdhA glutamate dehydrogenase

261 301156795 yacK possible multicopper oxidase precursor 262 301159959 rpsD 30S ribosomal subunit protein S4

263 301156659 putative exported protein

264 301159929 accC biotin carboxylase

265 301160399 atpA ATP synthase alpha subunit

266 301159961 rpsM 30S ribosomal subunit protein S13

267 301156922 pagN possible outer membrane adhesin

268 301157086 acrA acriflavin resistance protein A precursor

269 301157330 sucD succinyl-CoA synthetase alpha chain

270 301157543 asnS asparaginyl-tRNA synthetase

271 301160437 rho transcription termination factor

272 301160902 fbp fructose-1,6-bisphosphatase

273 301156736 tbpA thiamine-binding periplasmic protein precursor

274 301161048 deoB phosphopentomutase

275 301159624 fba fructose 1 ,6-bisphosphate aldolase

276 301157321 gltA citrate synthase

277 301156989 yaiW putative lipoprotein

278 301158555 predicted bacteriophage protein

279 301160717 putative exported protein

280 301160821 groES GroES protein

281 301158205 mppA periplasmic murein peptide-binding protein MppA

282 301157772 phoP transcriptional regulatory protein PhoP, regulator of virulence determinants

283 301159457 invG type III secretion system secretory apparatus

284 301158804 exported protein

285 301157747 putative lipoprotein

286 301157367 modA molybdate-binding periplasmic protein precursor

287 301159641 putative outer membrane lipoprotein

288 301157060 clpX ATP-dependent dp protease ATP-binding subunit CIpX

289 301159730 possible exported protein

290 301159893 rpsl 30S ribosomal subunit protein S9

291 301159189 rseB sigma-E factor regulatory protein RseB precursor

292 301158233 osmB osmotically inducible lipoprotein B precursor

293 301158642 gnd 6-phosphogluconate dehydrogenase, decarboxylating

294 301158023 putative ABC transporter periplasmic binding protein

295 301157721 flgH Flagellar L-ring protein precursor

296 301156860 putative secreted chitinase

297 301158709 conserved hypothetical protein

298 301158896 pta phosphate acetyltransferase

299 301160265 rpmB 50S ribosomal subunit protein L28

300 301157522 putative lipoprotein

301 301159081 putative lipoprotein

302 301157054 cyoA cytochrome o ubiquinol oxidase subunit II

303 301156843 rpsB 30S ribosomal protein S2

304 301156780 aceE pyruvate dehydrogenase E1 component

305 301157208 dsbG thiohdisulfide interchange protein DsbG precursor

306 301158296 kdsA 2-dehydro-3-deoxyphosphooctonate aldolase

307 301158702 fbaB fructose-bisphosphate aldolase class I

308 301160667 SL4110 conserved hypothetical protein

309 301161049 deoD purine nucleoside phosphorylase

310 301159968 rplF 50S ribosomal subunit protein L6

311 301157873 rpl20 50S ribosomal protein L20. Chloroplast 50S ribosomal protein L20.

312 301159389 recA RecA protein

313 301159593 conserved hypothetical protein

314 301159590 outer membrane protein (associated with virulence)

315 301158999 putative lipoprotein 316 301157913 pykF pyruvate kinase

317 301156747 mraZ conserved hypothetical protein.

318 301158716 metG Methionyl-tRNA synthetase

319 301157977 slyA Transcriptional regulator slyA.

320 301158276 adh alcohol dehydrogenase

321 301157589 sodCI bacteriophage encoded superoxide dismutase [cu-zn] 1 precursor (ec 1.15.1.1)

(sodCI)

322 301158952 pgtE outer membrane protease E

323 301159337 flaG flagellin

324 301156761 ftsZ cell division protein FtsZ

325 301157063 ppiD peptidyl-prolyl cis-trans isomerase D

326 301159233 rpl19 50S ribosomal protein L19. Chloroplast 50S ribosomal protein L19.

327 301158508 ompS1 outer membrane protein S1

328 301157433 yliB putative ABC transporter periplasmic binding protein

329 301158355 manY phosphotransferase enzyme II, C component

330 301159342 iroE putative exported protein

331 301157734 fabH 3-oxoacyl-[acyl-carrier-protein] synthase III

332 301159894 rplM 50S ribosomal subunit protein L13

333 301157507 serS seryl-tRNA synthetase

334 301159980 rplB 50S ribosomal subunit protein L2

335 301156928 yafK putative exported protein

336 301159976 rpIP 50S ribosomal subunit protein L16

337 301159888 nanA N-acetylneuraminate lyase

338 301157724 flgK flagellar hook-associated protein 1

339 301158653 rfbF glucose-1-phosphate cytidylyltransferase

340 301158004 outer membrane protein

341 301159207 clpB ClpB protein (heat shock protein f84.1)

342 301159981 rplW 50S ribosomal subunit protein L23

343 301159832 pnp polynucleotide phosphorylase

344 301158913 argT lysine-arginine-ornithine-binding periplasmic protein precursor

345 301159634 speB Agmatinase

346 301159978 rpIV 50s ribosomal protein I22

347 301160843 putative arginine-binding periplasmic protein

348 301159596 lysS Lysyl-tRNA synthetase

349 301157601 pepN aminopeptidase N

350 301159085 pepB Peptidase B

351 301160599 hsIV heat shock protein

352 301160776 yjcO putative exported protein

353 301157871 infC translation initiation factor IF-3

354 301160271 rph RNase PH

355 - ratB putative outer membrane protein (RatB)

TABLE 3

SEQ ID Gl Definition

NO:

356 207857056 copper-zinc superoxide dismutase

357 207859616 fimbrial protein precursor

358 207857291 conserved hypothetical protein

359 207856043 outer membrane esterase

360 207856094 antimicrobial peptide resistance and lipid A acylation protein

361 207859265 putative aldolase 362 207856527 flagellar hook associated protein (FliD)

363 207858047 TonB-dependent outer membrane siderophore receptor protein

364 207858445 outer membrane protein TolC precursor

365 207858671 50S ribosomal subunit protein L24

366 207856784 Exoribonuclease II

367 207856938 alcohol dehydrogenase

368 207858725 DamX protein

369 207858254 N-acetylmuramoyl-L-alanine amidase

370 207858777 1 ,4-alpha-glucan branching enzyme

371 207857687 outer membrane protein C

372 207856415 outer membrane protein A

373 207857633 colicin I receptor precursor

374 207857289 outer-membrane receptor for Fe(iii)-coprogen, fe(iii)-ferrioxamine b and fe(iii)-rhodotrulic acid

375 207857899 putative lipoprotein

376 207855894 nucleoside-specific channel-forming protein tsx precursor

377 207856192 tolB protein precursor

378 207855605 survival protein SurA precursor

379 207855846 ferrioxamine B receptor precursor

380 207857021 putative exported protein

381 207857701 glycerophosphoryl diester phosphodiesterase periplasmic precursor

382 207857928 putative lipoprotein

383 207856919 possible ATP-binding protein

384 207856224 possible pectinesterase precursor

385 207855742 outer membrane protein precursor

386 207856390 outer membrane protein F precursor

387 207857180 osmotically inducible lipoprotein E precursor

388 207858517 possible lipoprotein

389 207858253 membrane-bound lytic murein transglycosylase A precursor

390 207856273 putative exported protein

391 207855725 protease DO precursor; heat shock protein HtrA

392 207856104 rare lipoprotein A precursor

393 207856269 outer membrane protein x precursor

394 207859385 maltoporin precursor

395 207858367 L-asparaginase

396 207857051 outer membrane lipoprotein SlyB precursor

397 207856193 peptidoglycan-associated lipoprotein precursor

398 207855707 ferrichrome-iron receptor

399 207859703 lytic murein transglycosylase

400 207858882 putative outer membrane protein

401 207858596 serine protease

402 207857809 long-chain fatty acid transport protein precursor

403 207855606 organic solvent tolerance protein precursor

404 207856528 flagellin

405 207859157 detergent-resistant phospholipase A

406 207856194 putative exported protein

407 207858095 membrane-bound lytic transglycosylase B precursor

408 207856649 tail-specific protease precursor

409 207858687 elongation factor Tu

410 207858328 conserved hypothetical protein

411 207857810 VacJ lipoprotein precursor

412 207858185 lipoprotein NlpD precursor

413 207857601 periplasmic beta-glucosidase precursor

414 207856051 ferrienterobactin receptor precursor 415 207859379 putative lipoprotein

416 207857554 putative polysaccharide export protein

417 207859479 GroEL protein

418 207857271 spermidine/putrescine-binding periplasmic protein precursor

419 207859319 vitamin B12 receptor protein

420 207856324 probable lipoprotein

421 207858531 conserved hypothetical protein

422 207857624 D-galactose-binding periplasmic protein precursor

423 207855743 outer membrane protein OmpH precursor

424 207858851 putative zinc-protease precursor

425 207859396 putative exported protein

426 207857278 putative exported protein

427 207858013 putative lipoprotein

428 207855925 putative lipoprotein

429 207856933 outer membrane porin protein (ompD)

430 207857971 putative membrane protein

431 207858340 conserved hypothetical protein

432 207859107 D-ribose-binding periplasmic protein

433 207859469 nonspecific acid phosphatase precursor

434 207857144 phosphoenolpyruvate synthase

435 207855762 RcsF protein

436 207859550 2',3'-cyclic-nucleotide 2'-phosphodiesterase

437 207857117 major outer membrane lipoprotein

438 207856523 putative lipoprotein

439 207856944 exported protein

440 207858695 FKBP-type peptidyl-prolyl isomerase

441 207856130 ABC transporter periplasmic binding protein (glutamate/aspartate?)

442 207858854 putative polysaccharide biosynthesis protein subunit C

443 207857249 outer membrane invasion protein (PagC)

444 207859386 maltose operon periplasmic protein

445 207857119 putative exported protein

446 207857712 conserved hypothetical protein

447 207858124 Iron transport protein, periplasmic-binding protein

448 207857252 putative lipoprotein

449 207858656 50S ribosomal subunit protein L17

450 207856674 putative lipoprotein

451 207857682 Ecotin precursor.

452 207857450 putative exported protein

453 207855945 conserved hypothetical lipoprotein

454 207859264 putative ABC transport protein, solute-binding component

455 207856113 rare lipoprotein B precursor

456 207857857 thiosulphate-binding protein precursor

457 207857242 putative substrate-binding transport protein

458 207857209 glyceraldehyde 3-phosphate dehydrogenase A

459 207857150 putative lipoprotein

460 207858339 transketolase

461 207857772 histidine-binding periplasmic protein

462 207858270 possible lipoprotein

463 207858373 membrane-bound lytic murein transglycosylase C

464 207859488 putative lipoprotein

465 207859383 periplasmic maltose-binding protein

466 207858054 putative exported protein

467 207859328 50S ribosomal subunit protein L1

468 207858431 Sufi protein 469 207859670 putative phosphoglycerol transferase

470 207856899 putative lipoprotein

471 207857243 putative lipoprotein

472 207857907 putative exported protein

473 207858765 aerobic glycerol-3-phosphate dehydrogenase

474 207858833 putative phosphatase

475 207857163 putative outer membrane protein

476 207855759 copper homeostasis protein CutF precursor (lipoprotein nlpE)

477 207859403 class B acid phosphatase precursor

478 207859080 periplasmic phosphate-binding protein

479 207856326 putative N-acetylmuramoyl-L-alanine amidase

480 207858258 protease III precursor (pitrilysin)

481 207855763 putative lipoprotein precursor

482 207859556 putative exported protein

483 207856855 heat shock protein (hsIJ)

484 207858795 glycerol-3-phosphate-binding periplasmic protein

485 207855668 dihydrolipoamide acetyltransferase component (E2) of pyruvate dehydrogenase

486 207858607 malate dehydrogenase

487 207859088 ATP synthase beta subunit

488 207858665 30S ribosomal subunit protein S5

489 207856970 outer membrane protein

490 207858301 possible lipoprotein

491 207858713 peptidyl-prolyl cis-trans isomerase

492 207856697 Periplasmic trehalase precursor

493 207857164 putative outer membrane protein

494 207856960 putative secreted hydrolase

495 207855895 putative lipoprotein

496 207856906 D-alanyl-D-alanine dipeptidase

497 207859687 Putative periplasmic protein

498 206708683 Glucans biosynthesis protein D precursor.

499 207856410 putative lipoprotein

500 207856609 putative phage encoded hydrolase

501 207857311 Flagellar P-ring protein precursor

502 207856715 Outer-membrane lipoprotein lolB precursor.

503 207857887 putative exported protein

504 207859412 putative type-l secretion protein

505 207858676 30S ribosomal subunit protein S3

506 207858688 elongation factor G

507 207857315 flagellar hook protein FlgE

508 207856210 phosphoglycerate mutase 1

509 207857213 putative outer membrane protein

510 207858248 possible lipoprotein

511 207859539 50s ribosomal subunit protein L9

512 207858818 putative lipoprotein

513 207855735 Elongation factor Ts

514 207858566 conserved hypothetical protein

515 207855522 transaldolase B

516 207856725 putative invasin

517 207857183 putative exported protein

518 207856920 putative lipoprotein

519 207858682 50S ribosomal subunit protein L3

520 207855669 dihydrolipoamide dehydrogenase

521 207857814 outer membrane protease E

522 207859708 conserved hypothetical protein 523 207857286 putative lipoprotein

524 207857600 putative periplasmic protein

525 207856694 membrane-bound lytic murein transglycosylase E

526 207856372 30S ribosomal protein S1

527 207858868 periplasmic dipeptide transport protein precursor

528 207859329 50S ribosomal subunit protein L10

529 207859504 N-acetylmuramoyl-L-alanine amidase

530 207858625 conserved hypothetical protein

531 207857342 Glucans biosynthesis protein G precursor.

532 207859273 glycerol kinase

533 207858558 possible exported protein

534 207855594 putative lipoprotein

535 207855772 membrane-bound lytic murein transglycosylase d precursor

536 207858212 Enolase

537 207856182 succinyl-CoA synthetase beta chain

538 207858332 Phosphoglycerate kinase

539 207859092 ATP synthase subunit B

540 207856267 DNA protection during starvation protein

541 207858368 conserved hypothetical protein

542 207857162 Putative DNA/RNA non-specific endonuclease

543 207856743 periplasmic oligopeptide-binding protein precursor (OppA)

544 207856739 DNA-binding protein (histone-like protein Hlp-ll)

545 207856532 cystine-binding periplasmic protein (FliY)

546 207855927 trigger factor

547 207855527 Chaperone protein dnaK

548 207856541 putative exported protein;

549 207858514 possible exported protein

550 207859327 50S ribosomal subunit protein L11

551 207856060 ferrienterobactin-binding periplasmic protein precursor

552 207859148 putative lipoprotein

553 207857883 NADP-dependent malate dehydrogenase (decarboxylating)

554 207857255 isocitrate dehydrogenase

555 207857714 Ais protein

556 207856266 glutamine-binding periplasmic protein precursor

557 207858306 thiohdisulfide interchange protein

558 207859348 histone like DNA-binding protein HU-alpha (NS2) (HU-2)

559 207856698 glutamate dehydrogenase

560 207858789 gamma-glutamyltranspeptidase precursor

561 207858658 30S ribosomal subunit protein S4

562 207855543 putative exported protein

563 207858628 biotin carboxylase

564 207859090 ATP synthase alpha subunit

565 207858660 30S ribosomal subunit protein S13

566 207855791 possible outer membrane adhesin

567 207855955 acriflavin resistance protein A precursor

568 207856183 succinyl-CoA synthetase alpha chain

569 207856391 asparaginyl-tRNA synthetase

570 207859128 transcription termination factor

571 207859562 fructose-1 ,6-bisphosphatase

572 207855620 thiamine-binding periplasmic protein precursor

573 207859694 phosphopentomutase

574 207858331 fructose 1 ,6-bisphosphate aldolase

575 207856174 citrate synthase

576 207855859 putative lipoprotein 577 207859478 GroES protein

578 207856806 periplasmic murein peptide-binding protein MppA

579 207857262 transcriptional regulatory protein PhoP, regulator of virulence determinants

580 207858160 secretory protein (associated with virulence)

581 207857665 exported protein

582 207857287 putative lipoprotein

583 207856219 molybdate-binding periplasmic protein precursor

584 207858347 putative outer membrane lipoprotein

585 207855929 ATP-dependent dp protease ATP-binding subunit ClpX

586 207858435 possible exported protein

587 207858592 30S ribosomal subunit protein S9

588 207857992 sigma-E factor regulatory protein RseB precursor

589 207857516 6-phosphogluconate dehydrogenase, decarboxylating

590 207857006 putative ABC transporter periplasmic binding protein

591 207857312 Flagellar L-ring protein precursor

592 207855751 putative secreted chitinase

593 207857582 conserved hypothetical protein

594 207857756 phosphate acetyltransferase

595 207858965 50S ribosomal subunit protein L28

596 207856370 putative lipoprotein

597 207857940 putative lipoprotein

598 207855923 cytochrome o ubiquinol oxidase subunit II

599 207855734 30S ribosomal protein S2

600 207855667 pyruvate dehydrogenase E1 component

601 207856073 thiohdisulfide interchange protein DsbG precursor

602 207856721 2-dehydro-3-deoxyphosphooctonate aldolase

603 207857575 fructose-bisphosphate aldolase class I

604 207859349 conserved hypothetical protein

605 207858667 50S ribosomal subunit protein L6

606 207857157 50S ribosomal protein L20. Chloroplast 50S ribosomal protein L20.

607 207858093 RecA protein

608 207858300 conserved hypothetical protein

609 207858295 outer membrane protein (associated with virulence)

610 207857860 putative lipoprotein

611 207857116 pyruvate kinase

612 207855633 Protein mraZ.

613 207857052 Transcriptional regulator slyA.

614 207856741 alcohol dehydrogenase

615 207856621 phage encoded superoxide dismutase [cu-zn] 1 precursor (ec 1.15.1.1) (sodCI)

616 207855647 cell division protein FtsZ

617 207855932 peptidyl-prolyl cis-trans isomerase D

618 207858021 50S ribosomal protein L19. Chloroplast 50S ribosomal protein L19.

619 207856493 outer membrane protein S1

620 207858990 putative autotransported protein (MisL)

621 207856284 putative ABC transporter periplasmic binding protein

622 207857300 3-oxoacyl-[acyl-carrier-protein] synthase III

623 207858593 50S ribosomal subunit protein L13

624 207856355 seryl-tRNA synthetase

625 207858679 50S ribosomal subunit protein L2

626 207855797 putative exported protein

627 207858675 50S ribosomal subunit protein L16

628 207858587 N-acetylneuraminate lyase

629 207857309 flagellar hook-associated protein 1

630 207857528 glucose-1-phosphate cytidylyltransferase 631 207857025 outer membrane protein

632 207858010 ClpB protein (heat shock protein f84.1)

633 207858680 50S ribosomal subunit protein L23

634 207859378 putative exported protein

635 207858532 polynucleotide phosphorylase

636 207857773 lysine-arginine-ornithine-binding periplasmic protein precursor

637 207858341 Agmatinase

638 207858677 50s ribosomal protein I22

639 207859500 probable arginine-binding periplasmic protein

640 207858303 Lysyl-tRNA synthetase

641 207856402 aminopeptidase N

642 207857944 Peptidase B

643 207859279 heat shock protein

644 207859438 putative exported protein

645 207857159 Translation initiation factor IF-3. Translation initiation factor IF-3, chloroplast.

646 207858971 RNase PH

647 - putative outer membrane protein (RatB) (pseudogene)

648 - glucose-1-phosphatase precursor (GI Pase), secreted

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Claims

1. A Salmonella bacterium which does not express an active TolR protein and does not express a native Salmonella lipopolysaccharide.

2. A bacterium which is a AtolR strain of Salmonella which does not express a native Salmonella lipopolysaccharide.

3. The bacterium of claim 1 or claim 2, wherein the bacterium does not express a native Salmonella O antigen.

4. The bacterium of any preceding claim, wherein the strain is: (a) a AtolRAmsbB strain; (b) a AtolRAwbaP strain; or (c) a AtolRAwbaP AmsbB strain.

5. A Salmonella bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium.

6. A Salmonella bacterium in which one or more components of its Tol-Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and which does not express a native Salmonella lipopolysaccharide.

7. The bacterium of any preceding claim, wherein the Salmonella is a Salmonella enterica.

8. The bacterium of claim 7, wherein the Salmonella enterica is a S. enterica subsp. enterica.

9. The bacterium of claim 7 or claim 8, wherein the Salmonella enterica is a serovar Typhimurium or Typhi or Enteritidis or Paratyphi of S.entirica subsp. enterica serovor

10. A process for preparing blebs, comprising a step of separating the blebs from a culture medium comprising Salmonella bacteria which express no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins (for example, Salmonella bacteria according to any of claims 1 to 10), which have been grown under conditions which permit the release of blebs into the medium by the bacteria.

1 1. The process of claim 10, wherein the bacteria have been grown under iron-limiting conditions.

12. A method of preparing a hyperblebbing Salmonella bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's tolB, tolQ, tolR and/or pal genes.

13. A bleb isolated or obtainable from a Salmonella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins (for example, a Salmonella bacterium according to any of claims 1 to 9), or from a bacterium obtainable by the method of claim 12, or by the process of claim 10 or claim 1 1.

14. A composition comprising blebs that, during culture of a Salmonella bacterium which either expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins (for example, a Salmonella bacterium according to any of claims 1 to 9) or is obtainable by the method of claim 12, are released into the culture medium.

15. The composition of claim 14, which does not comprise any living and/or whole bacteria.

16. A composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22μπι filter of a culture medium in which has been grown (i) a Salmonella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins, (ii) a bacterium of any one of claims 1 to 9 or (iii) a bacterium obtainable by the method of claim 12.

17. Culture media comprising bacteria which have been grown under conditions which permit them to release blebs into the culture medium, wherein the bacteria are (i) Salmonella bacteria which express no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins (ii) bacteria according to any of claims 1 to 9, or (iii) bacteria obtainable by the method of claim 12.