WO2015140108A1 - Immunising against staphylococcal bone and joint infections - Google Patents

Abstract

Immunisation with EsxA, EsxB, FhuD2, Sta011, and/or Hla leads to significantly lower bacterial burden in joints of animals who are exposed to S.aureus infections. Thus the invention provides an effective way of preventing and/or treating S.aureus infections of bones and joints, thereby avoiding disorders such as osteomyelitis, septic arthritis, and prosthetic joint infection.

Description

IMMUNISING AGAINST STAPHYLOCOCCAL BONE AND JOINT INFECTIONS

This application claims the benefit of European patent application 14160390.2 (filed March 17 2014), the complete contents of which is hereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention relates to immunisation against infection of bone and joint infections by S. aureus. BACKGROUND ART

Staphylococcus aureus is a Gram-positive spherical bacterium. Annual US mortality exceeds that of any other infectious disease, including HIV/AIDS, and S.aureus is the leading cause of bloodstream, lower respiratory tract, skin & soft tissue infections. It is also the predominant cause of bone infections worldwide, and these infections are painful, debilitating and difficult to treat. S.aureus infections can take hold in bone marrow and/or within joints, and can lead to osteomyelitis, septic arthritis, and also to prosthetic joint infection [1].

It is an object of the invention to provide vaccines which are useful for preventing and/or treating S.aureus infections of bones and joints. DISCLOSURE OF THE INVENTION

The invention provides the use of one or more antigens for immunising a mammal to prevent or treat S.aureus infection of its bones and joints, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl l ; and Hla. The inventors have shown that immunisation with these antigens can lead to significantly lower bacterial burden in joints of animals who are exposed to S.aureus infections. Compared to control animals, knee joint washes show dxSx-S. aureus antibodies, reduced death of immune cells, and lower inflammatory responses. Thus the invention provides an effective way of preventing and/or treating S.aureus infections of bones and joints.

S.aureus antigens

The invention uses 1 , 2, 3, 4, or all 5 of the following antigens: EsxA; EsxB; FhuD2; StaOl l ; and Hla. These five antigens are already known in the art (e.g. see references 2-8) and further details are given below. A particularly useful composition includes all five of these antigens (preferably with a non-toxic mutant form of Hla).

The 'EsxA' antigen in the NCTC 8325 strain has amino acid sequence SEQ ID NO: 1 (GI: 88194063). EsxA antigens used with the present invention can elicit an antibody (e.g. when administered to a human) that recognises SEQ ID NO: 1 and/or may comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 1 ; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 1 , wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more). These EsxA polypeptides include variants of SEQ ID NO: 1. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1. 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 SEQ ID NO: 1 while retaining at least one epitope of SEQ ID NO: 1.

The 'EsxB' antigen in the NCTC 8325 strain has amino acid sequence SEQ ID NO: 2 (GI:88194070). EsxB used with the present invention can elicit an antibody (e.g. when administered to a human) that recognises SEQ ID NO: 2 and/or may comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 2; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 2, 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 or more). These EsxB polypeptides include variants of SEQ ID NO: 2. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 2. 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 SEQ ID NO: 2 while retaining at least one epitope of SEQ ID NO: 2. A useful EsxB antigen lacks the internal cysteine residue of SEQ ID NO: 2 e.g. it comprises SEQ ID NO: 35, wherein residue X at position 30 is either absent or is an amino acid residue without a free thiol group (under reducing conditions) e.g. is any natural amino acid except cysteine.

The 'FhuD2' antigen is annotated as 'ferrichrome-binding protein', and has also been studied in the literature [9]. It has also been known as 'Sta006' (e.g. in references 2-8). In the NCTC 8325 strain FhuD2 has amino acid sequence SEQ ID NO: 3 (GI: 88196199). FhuD2 used with the present invention can elicit an antibody (e.g. when administered to a human) that recognises SEQ ID NO: 3 and/or may comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 10%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 3; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 3, 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). These FhuD2 polypeptides include variants of SEQ ID NO: 3. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 3. 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 SEQ ID NO: 3 while retaining at least one epitope of SEQ ID NO: 3. The first 17 N-terminal amino acids of SEQ ID NO: 3 can usefully be omitted (to provide SEQ ID NO: 6). Mutant forms of FhuD2 are reported in reference 10. A useful FhuD2 antigen lacks the cysteine residue of SEQ ID NO: 3 e.g. it comprises SEQ ID NO: 34 and does not include any amino acid residue with a free thiol group (under reducing conditions) e.g. it is cysteine-free. A FhuD2 antigen may be lipidated e.g. with an acylated N-terminus cysteine. One useful FhuD2 sequence is SEQ ID NO: 7, which has a Met-Ala- Ser- sequence at the N-terminus; SEQ ID NO: 37 is another such sequence, but it lacks the cysteine present in SEQ ID NO: 7. The 'StaOl l' antigen has amino acid sequence SEQ ID NO: 4 (GP.88193872) in the NCTC 8325 strain. StaOl l antigens used with the invention can elicit an antibody (e.g. when administered to a human) that recognises SEQ ID NO: 4 and/or may comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 4; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 4, 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). These StaOl l polypeptides include variants of SEQ ID NO: 4. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 4. 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 SEQ ID NO: 4 while retaining at least one epitope of SEQ ID NO: 4. The first 23 N-terminal amino acids of SEQ ID NO: 4 can usefully be omitted (to provide SEQ ID NO: 33). A useful StaOl l antigen lacks the cysteine residue of SEQ ID NO: 4 e.g. it comprises SEQ ID NO: 36 and does not include any amino acid residue with a free thiol group (under reducing conditions) e.g. it is cysteine-free. A StaOl l antigen may be lipidated e.g. with an acylated N- terminus cysteine. One useful StaOl l sequence is SEQ ID NO: 8, which has a N-terminus methionine; SEQ ID NO: 39 is another such sequence, but it lacks the cysteine present in SEQ ID NO: 8. Variant forms of SEQ ID NO: 4 which may be used as or for preparing StaOl l antigens include, but are not limited to, SEQ ID NOs: 9, 10 and 1 1 with various Ile/Val/Leu substitutions (and Cys-free variants of these sequences can also be used with the invention). StaOl l can exist as a monomer or an oligomer, with Ca⁺⁺ ions favouring oligomerisation. The invention can use monomers and/or oligomers of StaOl l .

The 'Hla' antigen is the 'alpha-hemolysin precursor' also known as 'alpha toxin' or simply 'hemolysin'. In the NCTC 8325 strain Hla has amino acid sequence SEQ ID NO: 5 (GI: 88194865). Hla is an important virulence determinant produced by most strains of S.aureus, having pore-forming and haemolytic activity. Anti-Hla antibodies can neutralise the detrimental effects of the toxin in animal models, and Hla is particularly useful for protecting against pneumonia.

Useful Hla antigens can elicit an antibody (e.g. when administered to a human) that recognises SEQ ID NO: 5 and/or may comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 5; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 5, 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). These Hla antigens include variants of SEQ ID NO: 5. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 5. 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 SEQ ID NO: 5 while retaining at least one epitope of SEQ ID NO: 5. The first 26 N-terminal amino acids of SEQ ID NO: 5 can usefully be omitted (e.g. to give SEQ ID NO: 12). Truncation at the C- terminus can also be used e.g. leaving only 50 amino acids (residues 27-76 of SEQ ID NO: 5) [1 1].

Hla's toxicity can be avoided by chemical inactivation (e.g. using formaldehyde, glutaraldehyde or other cross-linking reagents). Instead, however, it is preferred to use mutant forms of Hla which remove its toxic activity while retaining its immunogenicity. Such detoxified mutants are already known in the art. A preferred Hla antigen is a mutant S.aureus hemolysin having a mutation at residue 61 of SEQ ID NO: 5, which is residue 35 of the mature antigen (i.e. after omitting the first 26 N-terminal amino acids = residue 35 of SEQ ID NO: 12). Thus residue 61 may not be histidine, and may instead be e.g. lie, Val or preferably Leu. A His-Arg mutation at this position can also be used. For example, SEQ ID NO: 13 is the mature mutant Hla-H35L sequence (i.e. SEQ ID NO: 12 with a H35L mutation) and a useful Hla antigen comprises SEQ ID NO: 13. Another useful mutation replaces a long loop with a short sequence e.g. to replace the 39mer at residues 136-174 of SEQ ID NO: 5 with a tetramer such as PSGS (SEQ ID NO: 14), as in SEQ ID NO: 15 (which also includes the H35L mutation) and SEQ ID NO: 16 (which does not include the H35L mutation). Another useful mutation replaces residue Y101 e.g. with a leucine (SEQ ID NO: 17). Another useful mutation replaces residue D152 e.g. with a leucine (SEQ ID NO: 18). Another useful mutant replaces residues H35 and Y101 e.g. with a leucine (SEQ ID NO: 19). Another useful mutant replaces residues H35 and D152 e.g. with a leucine (SEQ ID NO: 20).

Further useful Hla antigens are disclosed in references 12 and 13. SEQ ID NOs: 21 , 22 & 23 are three useful fragments of SEQ ID NO: 5 ('Hla₂7-76 \ 'Hla₂₇-89 ' and 'Hla₂₇-₇9 ' , respectively). SEQ ID NOs: 24, 25 & 26 are the corresponding fragments from SEQ ID NO: 13.

One useful Hla sequence is SEQ ID NO: 27. It has a N-terminal Met, then an Ala-Ser dipeptide from the expression vector, then SEQ ID NO: 13 (from NCTC8325 strain). Where a composition includes both EsxA and EsxB antigens, these may be present as a single polypeptide (i.e. as a fusion polypeptide). Thus a single polypeptide can elicit antibodies (e.g. when administered to a human) that recognise both SEQ ID NO: 1 and SEQ ID NO: 2. The single polypeptide can include: (i) a first polypeptide sequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 1 and/or comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 1 , as defined above for EsxA; and (ii) a second polypeptide sequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), 99.5%) or more) to SEQ ID NO: 2 and/or comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 2, as defined above for EsxB. The first and second polypeptide sequences can be in either order, N- to C- terminus. SEQ ID NOs: 28 ('EsxAB') and 29 ('EsxBA') are examples of such polypeptides, both having hexapeptide linkers AS GGGS (SEQ ID NO: 30). Another 'EsxAB' hybrid comprises SEQ ID NO: 31 , which may be provided with a N-terminus methionine (e.g. SEQ ID NO: 32). A useful variant of EsxAB lacks the internal cysteine residue of EsxB e.g. it comprises SEQ ID NO: 40 wherein residue X at position 132 is either absent or is an amino acid residue without a free thiol group (under reducing conditions) e.g. is any natural amino acid except cysteine. Thus a preferred EsxAB antigen for use with the invention has amino acid sequence SEQ ID NO: 38.

Thus a useful polypeptide comprises an amino acid sequence (a) having 80% or more identity (e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 31 ; and/or (b) comprising both a fragment of at least 'n' consecutive amino acids from amino acids 1-96 of SEQ ID NO: 31 and a fragment of at least 'n' consecutive amino acids from amino acids 103-205 of SEQ ID NO: 31 , 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). These polypeptides (e.g. SEQ ID NO: 32) can elicit antibodies (e.g. when administered to a human) which recognise both the wild-type staphylococcal protein comprising SEQ ID NO: 1 and the wild-type staphylococcal protein comprising SEQ ID NO: 2. Thus the immune response will recognise both of antigens EsxA and EsxB. Preferred fragments of (b) provide an epitope from SEQ ID NO: 1 and an epitope from SEQ ID NO: 2.

A preferred composition thus includes all four of: (i) a single polypeptide including both an EsxA antigen and an EsxB antigen e.g. comprising SEQ ID NO: 31 ; (ii) a FhuD2 antigen e.g. comprising SEQ ID NO: 6; (iii) a StaOl 1 antigen e.g. comprising SEQ ID NO: 33; and (iv) a H35L mutant form of Hla e.g. comprising SEQ ID NO: 13. In some embodiments, a composition may include one or more further polypeptides; in other embodiments the only polypeptides in a composition are these four specified polypeptides, and these polypeptides can even be the only immunogenic components in a composition.

Although SEQ ID NOs: 31 , 6, 33 and 13 are useful amino acid sequences in a combination, the invention is not limited to these precise sequences. Thus 1 , 2, 3 or all 4 of these sequences can independently be modified by up to 5 single amino changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions) provided that the modified sequence can elicit antibodies which still bind to a polypeptide consisting of the unmodified sequence.

Another useful composition includes all four of: (i) a first polypeptide having amino acid sequence SEQ ID NO: 32; (ii) a second polypeptide having amino acid sequence SEQ ID NO: 7; (iii) a third polypeptide having amino acid sequence SEQ ID NO: 8; and (iv) a fourth polypeptide having amino acid sequence SEQ ID NO: 27. In some embodiments, a composition may include one or more further polypeptides; in other embodiments the only polypeptides in the composition are these four specified polypeptides, and these polypeptides can even be the only immunogenic components in a composition. Although SEQ ID NOs: 32, 7, 8 and 27 are useful amino acid sequences in a combination, the invention is not limited to these precise sequences. Thus 1, 2, 3 or all 4 of these four sequences can independently be modified by 1, 2, 3, 4 or 5 single amino changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions) provided that the modified sequence can elicit antibodies which still bind to a polypeptide consisting of the unmodified sequence. In a preferred embodiment, a composition thus includes these four specified polypeptides with 1, 2, 3 or all 4 of SEQ ID NO: 32, 7, 8 and 27 independently modified by 1 single amino acid substitution, deletion and/or insertion.

For instance, wild-type FhuD2, StaOl l and EsxAB polypeptide sequences (e.g. SEQ ID NOs: 6, 31 and 33) each include a single cysteine residue which can lead to inter-polypeptide disulfide bridges, forming both homodimers and heterodimers. Such inter-linked polypeptides are undesirable and so Sta006, StaOl 1 and EsxB sequences can be modified to remove their natural cysteine residues, such that they do not contain free thiol groups (under reducing conditions). The wild-type cysteine can be deleted or can be substituted with a different amino acid. Thus: a FhuD2antigen can comprise SEQ ID NO: 34; a StaOl l antigen can comprise SEQ ID NO: 36; and a EsxB antigen can comprise SEQ ID NO: 35 (e.g. as an EsxAB hybrid comprising SEQ ID NO: 40). Examples of such sequences include, but are not limited to, SEQ ID NOs: 37, 39, and 38. These sequences can be used singly as substitutes for the corresponding wild-type sequences, or in combination. Thus a particularly useful composition includes all four of: (i) a first polypeptide having amino acid sequence SEQ ID NO: 38; (ii) a second polypeptide having amino acid sequence SEQ ID NO: 37; (iii) a third polypeptide having amino acid sequence SEQ ID NO: 39; and (iv) a fourth polypeptide having amino acid sequence SEQ ID NO: 27. In some embodiments a composition may include one or more further polypeptides; in other embodiments the only polypeptides in a composition are these four specified polypeptides. When more than one polypeptide is present, they may be present at substantially equal masses i.e. the mass of each of them is within +5% of the mean mass of all the polypeptides. Thus, when four polypeptides are present, they may be present at a mass ratio of a:b:c:d, where each of a-d is between 0.95 and 1.05.

Aside from EsxA, EsxB, Hla, FhuD2 and StaOl l, other S.aureus antigens exist, and a composition can optionally include one or more further S.aureus antigens. For instance, both saccharide and polypeptide antigens are known for S.aureus. Thus a composition might include a S.aureus saccharide antigen e.g. known saccharide antigens include the exopolysaccharide of S.aureus, which is a poly-N-acetylglucosamine (PNAG), and the capsular saccharides of S.aureus, which can be e.g. from type 5, type 8 or type 336. A composition might also include a ClfA antigen, an IsdA antigen, an IsdB antigen, an IsdC antigen, and/or an IsdH antigen (each as defined on pages 15-17 of reference 3). In some embodiments, a composition includes a S.aureus antigen as defined above, and also an antigen from a different organism {e.g. from a virus or from another bacterium).

S.aureus infections of mammalian bones and joints

The invention immunises a mammal such that it raises an immune response which is then useful for preventing a future S.aureus infection of its bones and joints, or can contribute to treating an existing S.aureus infection of this type.

S.aureus infects various mammals (including cows, dogs, horses, and pigs), but the preferred mammal for use with the invention is a human.

By preventing or treating S.aureus infection of bones and joints, the invention is thus suitable for preventing or treating particular disorders including, but not limited to, osteomyelitis, septic arthritis, and prosthetic joint infection (PJI). In many cases these disorders may be associated with the formation of a S.aureus biofilm.

Osteomyelitis is an infection and inflammation of a bone or bone marrow. Symptoms include pain and tenderness over the affected area of bone. S.aureus can reach a bone by two principal routes: from an infection in another part of the body via the bloodstream i. e. hematogenous osteomyelitis, which is often seen in children; or following an injury, particularly a deep cut which reaches or exposes a bone (with or without fracture). It can affect any bone, but the most commonly affected are the femur, tibia, fibula, humerus, vertebrae, the maxilla, and the mandibular bodies. Risk factors include bone fracture, bone prosthesis, bone surgery, immunosuppression, immunodeficiency, drug use, kidney dialysis, steroid use, and (less commonly for S.aureus) sickle cell disease. Osteomyelitis can be suppurative (including acute and chronic suppurative osteomyelitis) or non-suppurative (typically sclerosing, including diffuse sclerosing osteomyelitis, focal sclerosing osteomyelitis, and Garre's sclerosing osteomyelitis).

A bone infection can spread to a joint (particularly in children) and infect its synovial membrane of a joint, leading to arthrosynovitis and, eventually, to septic arthritis (often called suppurative arthritis). In children, large subperiosteal abscesses can form because the periosteum is loosely attached to the surface of the bone.

In patients who receive a prosthetic bone or joint, S.aureus can cause PJI, whose diagnosis is detailed in references 14-16. Thus a mammal which is immunised according to the invention may have a prosthetic bone or joint, or may be an intended recipient of such prostheses (e.g. a pre-operative orthopedic surgery patient). In this way the invention can be useful for preventing or treating nosocomial S.aureus infection, which is often associated with prostheses.

The invention is useful for preventing or treating any of these disorders which arise in the bones or in the joints. A patient may have a bone disorder, a joint disorder, or both. Immunogenic compositions and medicaments

Immunogenic compositions according to the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Compositions may thus be pharmaceutically acceptable. They will usually include components in addition to the antigens e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference 117.

Compositions will generally be administered to a mammal in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, although some vaccines are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other vaccines are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use. Thus a composition may be dried, such as a lyophilised formulation. Reference 4 discloses the use of lyophilisation with S.aureus immunogenic compositions.

A composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free from (i.e. less than 5μg/ml) mercurial material e.g. thiomersal-free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred.

To improve thermal stability, a composition may include a temperature protective agent (see below).

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+2mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290-310 mOsm/kg.

Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20mM range.

Compositions may include a metal ion chelator, in particular a divalent metal ion chelator such as EDTA. Reference 4 discloses that inclusion of EDTA can improve stability of the compositions disclosed herein. The final concentration of EDTA in an immunogenic composition can be about 1-50 mM, about 1-10 mM or about 1-5 mM, preferably about 2.5 mM.

The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8. The composition is preferably sterile. The composition is preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition is preferably gluten-free.

The composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material.

S. aureus infections can affect various areas of the body and so a composition may be prepared in various forms. For example, a composition 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 (e.g. a lyophilised composition or a spray-freeze dried composition). A composition may be prepared for topical administration e.g. as an ointment, cream or powder. A composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). A composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. A composition may be prepared as a suppository or pessary. A composition may be prepared for nasal, aural or ocular administration e.g. as drops. A composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.

Where a composition is to be prepared extemporaneously prior to use (e.g. where a component is presented in lyophilised form) and is presented as a kit, the kit may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection. Human vaccines are typically administered in a dosage volume of about 0.5ml, although a half volume (i.e. about 0.25ml) may also be useful e.g. for children.

Immunogenic compositions administered according to the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include one or more adjuvants (see below). The compositions may elicit both a cell mediated immune response as well as a humoral immune response. This immune response will preferably induce long lasting (e.g. neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to S.aureus.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other 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. Where more than one antigen is included in a composition then two antigens may be present at the same dose as each other or at different doses.

As mentioned above, a composition may include a temperature protective agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminium salt). As described in reference 17, 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 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').

Methods of treatment, and administration of an immunogenic composition

The invention provides a method for preventing or treating S. aureus infection of a mammal's bones and joints, by administering to the mammal at least one antigen selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1 ; and Hla.

The invention also provides the use of at least one antigen in the manufacture of a medicament for preventing or treating S.aureus infection of a mammal's bones and joints, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1 ; and Hla.

The invention also provides at least one antigen for use in immunising a mammal to prevent or treat S.aureus infection of its bones and joints, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1 ; and Hla.

The invention also provides at least one antigen for use in a method for preventing or treating S.aureus infection of a mammal's bones and joints, by administering the antigen(s) to the mammal, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1; and Hla.

These methods, uses and antigens elicit an immune response which is effective for preventing or treating S. aureus infections of bones and joints. The immune response can involve antibodies and/or cell-mediated immunity.

As mentioned above, the mammal is preferably a human. The human can be a child {e.g. a toddler or infant), a teenager, or an adult. In some embodiments the human may have a prosthetic bone or joint, or may be an intended recipient of such prostheses (e.g. a pre-operative orthopedic surgery patient). A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. The vaccines are not suitable solely for these groups, however, and may be used more generally in a human population. Other mammals which can usefully be immunised according to the invention are cows, dogs, horses, and pigs.

One way of checking efficacy of therapeutic treatment involves monitoring S. aureus infection of a joint or bone after administration of the compositions according to the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses, systemically (such as monitoring the level of IgGl and IgG2a production) and/or mucosally (such as monitoring the level of IgA production), against the antigens in the administered composition after its administration. Another way of assessing the immunogenicity of the compositions is to express the antigens recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays. A positive reaction between the protein and the patient sample indicates that the patient has mounted an immune response to the protein in question.

The efficacy of vaccine compositions can also be determined in vivo by challenging animal models of S. aureus infection, e.g., guinea pigs or mice, with the vaccine compositions. There are three generally useful animal models for the study of S. aureus infectious disease, namely: (i) the murine abscess model [18], (ii) the murine lethal infection model [18], and (iii) the murine pneumonia model [19]. In relation to evaluating efficacy for preventing/treating S. aureus infection of a mammal's bones and joints, however, different models are needed, for example as disclosed in the examples below.

Compositions 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 mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Intramuscular injection is the most typical route for administering compositions according to the invention. The invention may be used to elicit systemic and/or mucosal immunity, preferably to elicit an enhanced systemic and/or mucosal immunity. Preferably the enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. Preferably, the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA.

Dosage can be by 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. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

Immunogenic compositions may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines.

Immunogenic compositions may be administered to patients in combination with an antibiotic. For instance, they may be administered at substantially the same time as an antibiotic. Similarly, they may be administered to a subject who is receiving antibiotic therapy. Similarly, they may be administered as part of a co-therapy which involves administration of both a composition as discussed herein and an antibiotic. The antibiotic will be effective against a S.aureus bacterium, for instance a beta-lactam.

Strains and variants

Antigens are discussed above by reference to existing nomenclature (e.g. "EsxA") and exemplary sequences given as GI numbers and also in the sequence listing. The invention is not limited to these precise sequences. Genome sequences of several strains of S.aureus are available, including those of MRSA strains N315 and Mu50 [20], MW2, N315, COL, MRSA252, MSSA476, RF122, USA300 (very virulent), JH1 , JH9, NCTC 8325, and Newman. Standard search and alignment techniques can be used to identify in any of these (or other) further genome sequences the homolog of any particular sequence mentioned herein Moreover, the specific sequences disclosed herein can be used to design primers for amplification of homologous sequences from other strains. Thus the invention encompasses such variants and homologs from any strain of S.aureus, as well as non-natural variants. In general, suitable variants of a particular SEQ ID NO include its allelic variants, its polymorphic forms, its homologs, its orthologs, its paralogs, its mutants, etc. Thus, for instance, polypeptides used with the invention may, compared to the SEQ ID NO herein, include one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid substitutions, such as conservative substitutions (i.e. substitutions of one amino acid with another which has a related side chain). Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. The polypeptides may also include one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, etc.) single amino acid deletions relative to the SEQ ID NO sequences. The polypeptides may also include one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, etc.) insertions (e.g. each of 1 , 2, 3, 4 or 5 amino acids) relative to the SEQ ID NO sequences.

Similarly, a polypeptide used with the invention may comprise an amino acid sequence that:

(a) is identical (i.e. 100% identical) to a sequence disclosed in the sequence listing;

(b) shares sequence identity (e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) or more) with a sequence disclosed in the sequence listing;

(c) has 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b); or

(d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, each moving window of x amino acids from N-terminus to C-terminus (such that for an alignment that extends to p amino acids, where p>x, there are p-x+1 such windows) has at least xy identical aligned amino acids, where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and

xy is not an integer then it is rounded up to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [21], using default parameters (e.g. with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [22].

Where hybrid polypeptides are used, the individual antigens within the hybrid (i.e. individual -X- moieties) may be from one or more strains. Where n=2, for instance, X2 may be from the same strain as Xi or from a different strain. Where n=3, the strains might be (i)

(iii) X^X₂=X₃ (iv) or (v) X^X^, etc.

Within group (c), deletions or substitutions may be at the N-terminus and/or C-terminus, or may be between the two termini. Thus a truncation is an example of a deletion. Truncations may involve deletion of up to 40 (or more) amino acids at the N-terminus and/or C-terminus. N-terminus truncation can remove leader peptides e.g. to facilitate recombinant expression in a heterologous host. C-terminus truncation can remove anchor sequences e.g. to facilitate recombinant expression in a heterologous host.

In general, when an antigen comprises a sequence that is not identical to a complete S. aureus sequence from the sequence listing (e.g. when it comprises a sequence listing with <100%> sequence identity thereto, or when it comprises a fragment thereof) it is preferred in each individual instance that the antigen can elicit an antibody which recognises the respective complete S.aureus sequence. Polypeptides used with the invention

Polypeptides used with the invention can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, etc.). Polypeptides used with the invention can be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis, etc). Recombinantly-expressed proteins are preferred, particularly for hybrid polypeptides.

Polypeptides used with the invention are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other staphylococcal or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90%) pure i.e. less than about 50%), and more preferably less than about 10%o (e.g. 5%) of a composition is made up of other expressed polypeptides. Thus the antigens in the compositions are separated from the whole organism with which the molecule is expressed. 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 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.

Although expression of the polypeptides of the invention may take place in a Staphylococcus, the invention will usually use a heterologous host for expression (recombinant expression). The heterologous host may be prokaryotic (e.g. a bacterium) or eukaryotic. It may be E.coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g. M.tuberculosis), yeasts, etc. Compared to the wild-type S.aureus genes encoding polypeptides of the invention, it is helpful to change codons to optimise expression efficiency in such hosts without affecting the encoded amino acids. Adjuvants

As mentioned above, immunogenic compositions used according to the invention may include one or more adjuvants. Adjuvants which may be used with the invention 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 (or mixtures thereof). Calcium salts include calcium phosphate (e.g. the "CAP" particles disclosed in ref. 23). Aluminum salts include hydroxides and phosphates etc. , with the salts taking any suitable form (e.g. gel, crystalline, amorphous, etc.). Adsorption to these salts is preferred (e.g. all antigens may be adsorbed). The mineral containing compositions may also be formulated as a particle of metal salt [24].

The adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of reference 25). The invention can use any of the "hydroxide" or "phosphate" adjuvants that are in general use as adjuvants. The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. 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.

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.

Aluminium phosphate adjuvants generally have a P0₄/A1 molar ratio 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 0.5-20μηι (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.

The invention can use 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: l, >6: l, >7: l, >8: l, >9: l, efc.

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-in-water emulsions

Oil-in-water emulsion compositions suitable for use as adjuvants in the invention include squalene- in-water emulsions, such as MF59 (see Chapter 10 of ref. 25; see also ref. 26) and AS03 [27].

Various oil-in-water emulsion adjuvants 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 emulsion will include submicron oil droplets, and emulsions with droplets having a diameter less than 220nm are preferred as they can be subjected to filter sterilization.

The emulsion comprises one or more oils. Suitable oil(s) include those from, for example, an animal (such as fish) or a vegetable source. The oil is ideally biodegradable (metabolisable) and biocompatible. 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 metabolisable and so may be used. 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 metabolisable 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. Preferred emulsions comprise squalene, a shark liver oil which is a branched, unsaturated terpenoid. Squalane, the saturated analog to squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other useful oils are the tocopherols, particularly in combination with squalene. Where the oil phase of an emulsion includes a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols can be used, but a-tocopherols are preferred. D-a-tocopherol and DL-a-tocopherol can both be used. A preferred a-tocopherol is DL-a-tocopherol. An oil combination comprising squalene and a tocopherol (e.g. DL-a-tocopherol) can be used.

The oil in the emulsion may comprise a combination of oils e.g. squalene and at least one other oil.

The aqueous component of the emulsion can be plain water (e.g. w.f.i.) or can include further components e.g. solutes. For instance, it may include salts to form a buffer e.g. citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. A buffered aqueous phase is preferred, and buffers will typically be included in the 5-20mM range.

In addition to the oil and cationic lipid, an emulsion can include a non-ionic surfactant and/or a zwitterionic surfactant. Such surfactants include, but are not limited to: the polyoxyethylene 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- 1 ,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); polyoxyethylene-9-lauryl ether; 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 polysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures, or Tween 80/Triton-X 100 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol. Useful mixtures can comprise a surfactant with a HLB value in the range of 10-20 (e.g. polysorbate 80, with a HLB of 15.0) and a surfactant with a HLB value in the range of 1-10 (e.g. sorbitan trioleate, with a HLB of 1.8).

Preferred amounts of oil (% by volume) in the final emulsion are between 2-20% e.g. 5-15%, 6-14%, 7-13%), 8-12%). A squalene content of about 4-6% or about 9-1 1% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion are between 0.001%) and 8%o. For example: polyoxyethylene sorbitan esters (such as polysorbate 80) 0.2 to 4%o, in particular between 0.4-0.6%, between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%, between 1.9-2.1%, about 2%, or 0.85-0.95%), or about 1%>; sorbitan esters (such as sorbitan trioleate) 0.02 to 2%o, in particular about 0.5% or about 1%>; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%. The absolute amounts of oil and surfactant, and their ratio, can be varied within wide limits while still forming an emulsion. A skilled person can easily vary the relative proportions of the components to obtain a desired emulsion, but a weight ratio of between 4: 1 and 5: 1 for oil and surfactant is typical (excess oil).

An important parameter for ensuring immunostimulatory activity of an emulsion, particularly in large animals, is the oil droplet size (diameter). The most effective emulsions have a droplet size in the submicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the average droplet size is less than 250nm e.g. less than 200nm, less than 150nm. The average droplet size is usefully in the range of 80-180nm. Ideally, at least 80%) (by number) of the emulsion's oil droplets are less than 250 nm in diameter, and preferably at least 90%. These droplet sizes can conveniently be achieved by techniques such as micro fluidisation. Apparatuses for determining the average droplet size in an emulsion, and the size distribution, are commercially available. These typically use the techniques of dynamic light scattering and/or single-particle optical sensing e.g. the Accusizer™ and Nicomp™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), or the Zetasizer™ instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan).

Ideally, the distribution of droplet sizes (by number) has only one maximum i. e. there is a single population of droplets distributed around an average (mode), rather than having two maxima. Preferred emulsions have a polydispersity of <0.4 e.g. 0.3, 0.2, or less.

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' [28-30], as described in more detail in Chapter 10 of ref. 31 and chapter 12 of ref. 32. The MF59 emulsion advantageously includes citrate ions e.g. lOmM sodium citrate buffer.

• An emulsion comprising squalene, a tocopherol, and polysorbate 80. The emulsion may include phosphate buffered saline. These emulsions may have by volume from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight ratio of squalene tocopherol is preferably <1 (e.g. 0.90) as this can provide a more stable emulsion. Squalene and polysorbate 80 may be present volume ratio of about 5:2 or at a weight ratio of about 11 :5. Thus the three components (squalene, tocopherol, polysorbate 80) may be present at a weight ratio of 1068: 1 186:485 or around 55:61 :25. One such emulsion ('AS03 ') 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 microfiuidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm. The emulsion may also include a 3-de-O- acylated monophosphoryl lipid A (3d MPL). Another useful emulsion of this type may comprise, per human dose, 0.5-10 mg squalene, 0.5-1 1 mg tocopherol, and 0.1-4 mg polysorbate 80 [33] e.g. in the ratios discussed above.

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 10μg/ml 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™ L121"). 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 [34] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [35] (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 [36]. 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. The emulsion may include a TLR4 agonist [37]. Such emulsions may be lyophilized.

An emulsion of squalene, poloxamer 105 and Abil-Care [38]. The final concentration (weight) of these components in adjuvanted vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and 2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone; caprylic/capric triglyceride). • 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 39, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, 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 40, 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 in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [41].

• 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) [42].

• 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) [42].

In some embodiments an emulsion may be mixed with antigen(s) extemporaneously, at the time of delivery, and thus the adjuvant and antigen(s) may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. In other embodiments an emulsion is mixed with antigen during manufacture, and thus the composition is packaged in a liquid adjuvanted form. 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: 1 and 1 :5) but is generally about 1 : 1. Where concentrations of components are given in the above descriptions of specific emulsions, these concentrations are typically for an undiluted composition, and the concentration after mixing with an antigen solution will thus decrease.

C. Saponin formulations [chapter 22 of ref. 25]

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 officianalis (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, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in ref. 43. Saponin formulations may also comprise a sterol, such as cholesterol [44]. Combinations of saponins and cholesterols can be used to form particles called ISCOMs (chapter 23 of ref. 25). 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, QUA & QHC. ISCOMs are further described in refs. 44-46. Optionally, the ISCOMS may be devoid of additional detergent [47]. A review of the development of saponin based adjuvants can be found in refs. 48 & 49.

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. 50. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22μηι membrane [50]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 (see below).

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in refs. 51 & 52.

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 53, 54 and 55 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. 56-61.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [62]. 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. 63-65. 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. 62 & 66-68.

A useful CpG adjuvant is CpG7909, also known as ProMune™ (Coley Pharmaceutical Group, Inc.). Another is CpG1826. As an alternative, or in addition, to using CpG sequences, TpG sequences can be used [69], and these oligonucleotides may be free from unmethylated CpG motifs. The immunostimulatory oligonucleotide may be pyrimidine-rich. For example, it may comprise more than one consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref. 69), and/or it may have a nucleotide composition with >25% thymidine (e.g. >35%, >40%, >50%, >60%, >80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (e.g. CCCC, as disclosed in ref. 69), and/or it may have a nucleotide composition with >25% cytosine (e.g. >35%, >40%, >50%, >60%, >80%, etc.). These oligonucleotides may be free from unmethylated CpG motifs. Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides. A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-31™ [70]. 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: 41). The polycationic polymer may be a peptide comprising 1 1-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 42). The oligonucleotide and polymer can form complexes e.g. as disclosed in references 71 & 72.

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. 73 and as parenteral adjuvants in ref. 74. 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-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 75-82. A useful CT mutant is or CT-E29H [83]. 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. 84, specifically incorporated herein by reference in its entirety. E. TLR agonists

Compositions can include a TLR agonist i.e. a compound which can agonise a Toll-like receptor. Most preferably, a TLR agonist is an agonist of a human TLR. The TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11; preferably it can activate human TLR4 or human TLR7.

Agonist activity of a compound against any particular Toll-like receptor can be determined by standard assays. Companies such as Imgenex and Invivogen supply cell lines which are stably co-transfected with human TLR genes and NFKB, plus suitable reporter genes, for measuring TLR activation pathways. They are designed for sensitivity, broad working range dynamics and can be used for high-throughput screening. Constitutive expression of one or two specific TLRs is typical in such cell lines. See also reference 85. Many TLR agonists are known in the art e.g. reference 86 describes certain lipopeptide molecules that are TLR2 agonists, references 87 to 90 each describe classes of small molecule agonists of TLR7, and references 91 & 92 describe TLR7 and TLR8 agonists for treatment of diseases.

A TLR agonist used with the invention ideally includes at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour [93]. Phosphorus-containing adsorptive moieties are particularly useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc. Preferably the TLR agonist includes at least one phosphonate group.

Thus, in preferred embodiments, a composition includes a TLR agonist (more preferably a TLR7 agonist) which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt [93].

TLR agonists useful with the invention may include a single adsorptive moiety, or may include more than one e.g. between 2 and 15 adsorptive moieties. Typically a compound will include 1, 2 or 3 adsorptive moieties.

Useful phosphorus-containing TLR agonists can be represented by formula (Al):

wherein:

R^x and R^Y are independently selected from H and C₁-C6 alkyl; selected from a covalent bond, O and NH; selected from a covalent bond, O, C(O), S and NH; L is a linker e.g. selected from, Ci-C₆alkylene, Ci-C₆alkenylene, arylene, heteroarylene, Ci-C₆alkyleneoxy and -((CH₂)_pO)_q(CH₂)_p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, Ci-C₄alkyl, -OP(0)(OH)₂ and -P(0)(OH)₂; each p is independently selected from 1 , 2, 3, 4, 5 and 6; q is selected from 1 , 2, 3 and 4; n is selected from 1 , 2 and 3; and

A is a TLR agonist moiety.

In one embodiment, the TLR agonist according to formula (Al) is as follows: R^x and R^Y are H; X is O; L is selected from C₁-C6 alkylene and -((CH₂)_pO)_q(CH₂)_p- each optionally substituted with 1 to 2 halogen atoms; p is selected from 1 , 2 and 3; q is selected from 1 and 2; and n is 1. Thus in these embodiments the adsorptive moiety comprises a phosphate group.

Other useful TLR agonists of formula (Al) are disclosed on pages 6-13 of reference 94.

Compositions can include an imidazoquinolone compound, such as Imiquimod ("R-837") [95,96], Resiquimod ("R-848") [97], and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in references 98 to 102.

Compositions can include a TLR4 agonist, and most preferably an agonist of human TLR4. TLR4 is expressed by cells of the innate immune system, including conventional dendritic cells and macrophages [103]. Triggering via TLR4 induces a signalling cascade that utilizes both the MyD88- and TRIF-dependent pathways, leading to NF-κΒ and IRF3/7 activation, respectively. TLR4 activation typically induces robust IL-12p70 production and strongly enhances Thl-type cellular and humoral immune responses.

Various useful TLR4 agonists are known in the art, many of which are analogs of endotoxin or lipopolysaccharide (LPS). For instance, the TLR4 agonist can be: 3d-MPL (i.e. 3-O-deacylated monophosphoryl lipid A; present in GSK's 'AS04' adjuvant, with further details in references 104 to 107glucopyranosyl lipid A (GLA) [108] or its ammonium salt; an aminoalkyl glucosaminide phosphate, such as RC-529 or CRX-524 [ 109-1 1 1]; E5564 [112,1 13]; or a compound of formula I, II or III as defined in reference 1 14, or a salt thereof, such as compounds 'ER 803058', 'ER 803732', 'ER 804053', 'ER 804058', 'ER 804059', 'ER 804442', 'ER 804680', 'ER 803022', 'ER 804764' or 'ER 804057' (also known as E6020).

The invention is particularly useful when using human TLR7 agonists, such as a compound of formula (K). These agonists are discussed in detail in reference 1 15:

( )

wherein:

R¹ is H, Ci-Cealkyl, -C(R⁵)₂OH, -L'R⁵, -I^R⁶, -L²R⁵, -L²R⁶, -OL²R⁵, or -OL²R⁶;

L¹ is -C(O)- or -0-;

L² is Ci-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or -((CR⁴R⁴)_pO)_q(CH₂)_p-, wherein the Ci-C₆alkylene and C2-C₆alkenylene of L² are optionally substituted with 1 to 4 fluoro groups;

each L³ is independently selected from Ci-C₆alkylene and -((CR⁴R⁴)_pO)_q(CH₂)_p-, wherein the Ci-C₆alkylene of L³ is optionally substituted with 1 to 4 fluoro groups;

L⁴ is arylene or heteroarylene;

R² is H or Ci-C₆alkyl;

R³ is selected from Ci-C₄alkyl, -L³R⁵, -L'R⁵, -L³R⁷, -L³L⁴L³R⁷, -L³L⁴R⁵, -L³L⁴L³R⁵, -OL³R⁵, -OL³R⁷, -OL³L⁴R⁷, -OL³L⁴L³R⁷, -OR⁸, -OL³L⁴R⁵, -OL³L⁴L³R⁵ and -C(R⁵)₂OH ;

each R⁴ is independently selected from H and fluoro;

R⁵ is -P(0)(OR⁹)₂,

R⁶is -CF₂P(0)(OR⁹)₂ or -C(0)OR¹⁰;

R⁷ is -CF₂P(0)(OR⁹)₂ or -C(0)OR¹⁰;

R⁸ is H or C C₄alkyl;

each R⁹ is independently selected from H and Ci-C₆alkyl;

R¹⁰ is H or Ci-C₄aIkyl;

each p is independently selected from 1, 2, 3, 4, 5 and 6, and

q is 1, 2, 3 or 4.

The compound of formula (K) is preferably of formula (Κ'):

( ')

wherein:

Ρ¹ is selected from H, Ci-C₆alkyl optionally substituted with COOH and -Y-L-X- P(0)(OR^x)(OR^Y);

P² is selected from H, C C₆alkyl, C C₆alkoxy and -Y-L-X-P(0)(OR^x)(OR^Y);

with the proviso that at least one of P¹ and P² is -Y-L-X-P(0)(OR^x)(OR^Y);

R^B is selected from H and Ci-C₆alkyl;

R^x and R^Y are independently selected from H and Ci-C₆alkyl;

X is selected from a covalent bond, O and NH;

Y is selected from a covalent bond, O, C(O), S and NH;

L is selected from, a covalent bond Ci-C₆alkylene, Ci-C₆alkenylene, arylene, heteroarylene, Ci-C₆alkyleneoxy and -((CH₂)_pO)_q(CH₂)_p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, Ci-C₄alkyl, -OP(0)(OH)₂ and -P(0)(OH)₂;

each p is independently selected from 1, 2, 3, 4, 5 and 6; and

q is selected from 1, 2, 3 and 4.

In some embodiments of formula (Κ'): P¹ is selected from Ci-C₆alkyl optionally substituted with COOH and -Y-L-X-P(0)(OR^x)(OR^Y); P² is selected from C C₆alkoxy and -Y-L-X- P(0)(OR^x)(OR^Y); R^B is C C₆alkyl; X is a covalent bond; L is selected from C C₆alkylene and - ((CH₂)_pO)_q(CH₂)_p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C C₄alkyl, -OP(0)(OH)₂ and -P(0)(OH)₂; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.

A preferred compound of formula (K) for use with the invention is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy) ethoxy)ethoxy)phenethyl)benzo[f] [ 1 ,7] naphthyridin-8-yl)propanoic acid, or compound 'Kl ':

This compound can be used as free base or in the form of a pharmaceutically acceptable salt e.g. an arginine salt [1 16].

F. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of -lOOnm to ~150μηι in diameter, more preferably ~200nm to ~30μηι in diameter, and most preferably ~500nm to -ΙΟμηι 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).

Adjuvant combinations

The individual adjuvants listed above may also be included in combinations. For instance, a combination of an aluminium hydroxide and an aluminium phosphate adjuvant can be used. Similarly, a combination of aluminium phosphate and 3dMPL may be used.

A particularly preferred adjuvant combination is an insoluble metal salt (e.g. an aluminium salt, such as an aluminium hydroxide) and a TLR agonist (e.g. a human TLR7 agonist, such as compound 'K2' identified above), as disclosed in references 3 and 93. The TLR agonist is preferably adsorbed to the metal salt, and the S. aureus antigen(s) can also be adsorbed to the metal salt. A composition including a TLR agonist of the invention adsorbed to a metal salt can also include a buffer (e.g. a phosphate or a histidine or a Tris buffer). When such a composition includes a phosphate buffer, however, it is preferred that the concentration of phosphate ions in the buffer should be less than 50mM e.g. <40mM, <30mM, <20mM, <10mM, or <5mM, or between l-15mM. A histidine buffer is preferred e.g. between l-50mM, between 5-25mM, or about lOmM. A composition can include a mixture of both an aluminium oxyhydroxide and an aluminium hydroxyphosphate, and a TLR agonist may be adsorbed to one or both of these salts.

As mentioned above, a maximum of 0.85mg/dose Al⁺⁺⁺ is preferred. Because the inclusion of a TLR agonist can improve the adjuvant effect of aluminium salts then the invention advantageously permits lower amounts of Al⁺⁺⁺ per dose, and so a composition can usefully include between 10 and 250μg of Al⁺⁺⁺ per unit dose. Current pediatric vaccines typically include at least 300μg Al⁺⁺⁺. In concentration terms, a composition may have an Al⁺⁺⁺ concentration between 10 and 500 μ^ηιΐ e.g. between 10-300μ^ηι1, between 10-200μ^ηι1, or between 10-100μ^ηι1.

In general, when a composition includes both a TLR agonist and an aluminium salt, the weight ratio of agonist to Al⁺⁺⁺ will be less than 5: 1 e.g. less than 4: 1, less than 3: 1, less than 2: 1, or less than 1 : 1. Thus, for example, with an Al⁺⁺⁺ concentration of 0.5mg/ml the maximum concentration of TLR agonist would be 1.5mg/ml. But higher or lower levels can be used.

Where a composition includes a TLR agonist and an insoluble metal salt, it is preferred that at least 50% (by mass) of the agonist in the composition is adsorbed to the metal salt e.g. >60%, >70%, >80%, >85%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, or even 100%. Thus, in one embodiment, the invention uses an immunogenic composition comprising:

^■ an aluminium hydroxide adjuvant;

^■ a TLR7 agonist of formula (K), such as compound K2;

^■ a first polypeptide comprising SEQ ID NO: 6, or a modified amino acid sequence which differs from SEQ ID NO: 6 by up to 5 single amino changes provided that the modified sequence can elicit antibodies which bind to a polypeptide consisting of SEQ ID NO: 6;

^■ a second polypeptide comprising SEQ ID NO: 13, or a modified amino acid sequence which differs from SEQ ID NO: 13 by up to 5 single amino changes provided that the modified sequence can elicit antibodies which bind to a polypeptide consisting of SEQ ID NO: 13;

^■ a third polypeptide comprising SEQ ID NO: 31, or a modified amino acid sequence which differs from SEQ ID NO: 31 by up to 5 single amino changes provided that the modified sequence can elicit antibodies which bind to a polypeptide consisting of SEQ ID NO: 31;

^■ a fourth polypeptide comprising SEQ ID NO: 33, or a modified amino acid sequence which differs from SEQ ID NO: 33 by up to 5 single amino changes provided that the modified sequence can elicit antibodies which bind to a polypeptide consisting of SEQ ID NO: 33, in which the TLR7 agonist and/or at least one of the polypeptides is/are adsorbed to the aluminium hydroxide adjuvant.

For example, as explained in more detail elsewhere herein: the first polypeptide can comprise SEQ ID NO: 34; the second polypeptide can comprise SEQ ID NO: 13; the third polypeptide can comprise SEQ ID NO: 40; and the fourth polypeptide can comprise SEQ ID NO: 36. Thus the composition can use a mixture of four polypeptides having SEQ ID NOs: 37, 27, 38 and 39.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 117-124, etc. "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 [125,126] or similar methods), or they can be predicted (e.g. using the Jameson- Wolf antigenic index [127], matrix-based approaches [128], MAPITOPE [129], TEPITOPE [130,131], neural networks [132], OptiMer & EpiMer [133, 134], ADEPT [135], Tsites [136], hydrophilicity [137], antigenic index [138] or the methods disclosed in references 139-143, 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".

Where an antigen "domain" is omitted, this may involve omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, of an extracellular domain, etc.

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, x+10%.

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 ref. 144. 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 disclosed in ref. 145.

Phosphorous-containing adjuvants used with the invention may exist in a number of protonated and deprotonated forms depending on the pH of the surrounding environment, for example the pH of the solvent in which they are dissolved. Therefore, although a particular form may be illustrated, it is intended that these illustrations are merely representative and not limiting to a specific protonated or deprotonated form. For example, in the case of a phosphate group, this has been illustrated as - OP(0)(OH)₂ but the definition includes the protonated forms [OP(0)(OH₂)(OH)]⁺ and -[OP(0)(OH)₂]²⁺ that may exist in acidic conditions and the deprotonated forms -[OP(0)(OH)(0)]^~ and [OP(0)(0)2]^2" that may exist in basic conditions.

Compounds can exist as pharmaceutically acceptable salts. Thus, compounds (e.g. adjuvants) may be used in the form of their pharmaceutically acceptable salts i.e. physiologically or toxicologically tolerable salt (which includes, when appropriate, pharmaceutically acceptable base addition salts and pharmaceutically acceptable acid addition salts). 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.

MODES FOR CARRYING OUT THE INVENTION

Model system

Bioluminescent S. aureus strains were used to infect mice in the lateral tail vein (i.v.) and infection progression was followed for at least 1 week after the injection using an IVIS 100™ machine (Perkin Elmer). The i.v. route of infection was chosen trying to mimic a hematogenous source of infection. One day after injection S. aureus reached the knee joints and was able to establish a local infection that persisted for at least 7 days. Bioluminescence values showed a very good correlation when compared to colony forming units (CFUs) counted after knee joint washes on the last day of the experiment. Various different strains had the ability to reach the knee joint. Finally the presence of bacteria in knee joints after i.v. inoculation was confirmed using confocal microscopy.

The in vivo imaging approach was used to better understand whether bacteria which had reached the joint could invade the bone tissue (thus potentially causing osteomyelitis). Mice were again i.v. infected with a bioluminescent strain and then followed for 1 week post infection. The IVIS spectrum-CT system™, which combines a camera able to acquire bioluminescent signals and a tomography system that can scan sections of the animal body, revealed that bioluminescent bacteria were not only present in the knee joint area but also in the tibia. These findings suggested that S.awrews-mediated hematogenous infection could cause arthrosynovitis and osteomyelitis. Histopathological analysis of tissues obtained from infected mice showed that the normal architecture of bones and joints was effaced by the presence of mixed inflammatory cells (with prevalence of neutrophils and macrophages) as compared with control animals. In particular, the tibia was destroyed and granulomas/abscesses were observed. Periostal inflammation was severe, causing thickening of the periostal tissue. Inflammation was evident in the synovia, which appeared severely thickened. Elongated cells could be seen which formed a wall around foci of inflammation composed by neutrophils and macrophages. Within the inflammatory focus, clusters of bright pick eosinophylic slightly hyaline material consistent with the presence of bacteria were observed. Indeed, immune-histochemical staining demonstrated that S.aureus was present in bone abscesses and a wall of neutrophils was built up all around these structures.

To set up an arthrosynovitis mouse model of infection suitable for long lasting in vivo studies, the Newman strain was chosen as it has been largely used in research worldwide being well adapted for animal studies. The first objective was to find a dose high enough to homogeneously infect a group of mice both systemically and in the knee joints, but without inducing severe disease and/or death. Mice were intravenously infected with doses ranging from lxl 0⁷ to lxlO⁴ CFU/mouse and followed up to 3 weeks post infection. Kidneys of infected animals were collected and knee joint washes were performed. Mice treated with the dose of lxl 0⁷ showed evident signs of illness and, from four days after the infection, mice began to die, while doses as low Ixl0⁵-lxl0⁴ resulted in poor or even absent infection. For these reasons a dose of lxl 0⁶ was chosen for our purposes since no animal died during the observation time and all of them were significantly infected both systemically and locally.

Animals were inoculated with 10⁶ CFUs and the progression of S. aureus dissemination was followed for up to 90 days after the infection. CFU counts in kidneys were used as marker of systemic infection and CFU counts in joint washes as marker of local infection in the knee joints. CFUs in the blood was used as a possible second marker of systemic infection since it had been reported that during the chronicization of the pathology S. aureus could escape from the organs ad exploit the bloodstream for further spreading. The peak of infection seemed to be reached between 1 and 2 weeks after the inoculum when CFUs recovered in joints and kidneys were maximal. Starting from 1 month after infection, the CFUs started to decrease both in kidneys and in knee joints as the infection was being controlled by the host to a certain extent. On the other hand, CFUs in the blood seemed to increase during time reaching the highest level at the latest time point 90 days after infection. To better understand whether infection was being controlled or was becoming chronic, H&E staining of slices from knee joints was used. These analyses demonstrated that after intravenous infection with S. aureus mice developed arthrosynovitis and osteomyelitis and that both an acute (7-14 days) and a sub-acute/chronic (90 days) phase could be clearly detected.

The humoral response during the time both in situ and systemically was also analyzed. IgM and IgG against Hla were titrated both in sera and in the joint washes of infected mice at each time point as marker of infection progression. Anti-Hla IgM peaked seven days after the bacterial injection both in the knee joints and in the serum, while IgG level reached the maximum level at 14 and 30 days after inocula in joint and serum respectively, decreasing then in knee washes along with CFU decrease, while remaining almost stable during the time in sera. As cytokine secretion can be considered a reliable indicator of immune activation, cytokine levels at different time points after infection were assessed. In order to understand if the local inflammatory response was specific and if differences existed with the systemic response, cytokine levels were measured both in the serum and directly in situ. In the knee joints all the cytokines that increased (IL- la, IL-Ιβ, IL-6, IL-10, IL-12(p40), IL-17, eotaxin, G-CSF, GM-CSF, IFNy, KC, MCP-1, MIP-la, ΜΙΡ-Ιβ, RANTES) in comparison with time 0 showed a profile of expression that somehow correlated with the observed trend of CFU number variation during infection. Some of the cytokines found in the knee joint washes well correlated with the CFU recovered in that site at different time points. In particular, for most of them this correlation could be detected 14 days after infection (IL-6, IL-12(p40), IL-13, IL-17, G-CSF, KC, MCP-1, ΜΙΡ-Ιβ, RANTES), while for IL-12(p40) and MIP- la a good correlation was also found at 30 and 90 days post infection respectively. Immune cell populations recruited in the knee joint space and present in the blood of infected animals were also monitored. As early as 1 week post infection the immune cells counted in the knee joints of infected animals were 100 fold higher than those recovered in uninfected mice. The next step was staining those cells with specific antibodies directed against different cellular markers to better characterize the multiple cell populations. Neutrophils, macrophages (only in knee joint washes), monocytes, dendritic cells, eosinophils (only in blood), B cells, CD4+ and CD8+ T cells, NK cells were all detected.

Locally, in the knee joints, cells belonging to the myeloid lineage increased starting from three days after the infection with an evident peak at 1 - 2 weeks post inoculum. Neutrophils were the most abundant population increasing up to 1000-fold as compared to the basal situation, but also monocytes and macrophages showed a sharp increase (10-50 fold). A similar situation, although less evident, was observed when myeloid cells were stained and counted in the blood. In this case a decrement in eosinophils, especially immediately after infection, was observed. While lymphoid cells decreased in the blood starting 3 days after the injection with S. aureus, they increased in the joints. This was true for all the lineages but not for B cells that significantly decreased in number also in the joints. During the chronic phase the cellular composition both in blood and in the knee joint came back to the baseline level.

Antibiotic treatment (for comparison)

In humans antibiotics are the first and most used treatment for S. aureus septic arthritis and osteomyelitis. Nevertheless, sometimes they are not able to eradicate bacteria from these sites because of the low vascularization of bones and joints and of the increase of S. aureus antibiotic resistant forms. In order to investigate whether a classical antibiotic treatment would be able to reduce bacterial burden in the mouse model, mice infected i.v. with about 2xl0⁶ bacteria of Newman strain were treated i.p. with 100μg/g weight of ampicillin 24 and 48 hours after infection [146). Five days later they were sacrificed, kidneys were collected and knee joint washes were performed.

A significant reduction in CFU counts was observed in kidneys, indicating that the antibiotic treatment had some positive effect on the containment of the systemic infection. In contrast, a CFUs reduction in knee joints was not observed, which could be expected on the basis of the reported difficulties to successfully treat with antibiotics such localized infections. When the cell composition in knee joint washes was analyzed no substantial differences were observed, with the exception B cells, which appeared to be more abundant in the animals treated with ampicillin.

Vaccination

A 4-valent vaccine with antigens consisting of SEQ ID NOs: 7, 8, 27 and 32 was used to immunise mice. Different groups of mice received two immunizations at a two-week interval, either with the 4-valent vaccine (10μg per antigen per dose) adjuvanted with aluminum hydroxide, or the adjuvant alone as a negative control. Ten days after the second dose, the animals were infected with Newman strain, about 2x10⁶ CFU/mouse intravenously. They were sacrificed 7 days later and then kidneys, blood, serum were collected, together with knee joint washes for microbiological and immunological analysis. Immunization resulted in a significantly lower bacterial burden both in the knee joints (about 2 logs lower; p<0.05, Mann- Whitney t test) and in the kidneys (about 1.5 logs lower; p<0.05), suggesting that immunisation is more efficacious than a classical therapeutic approach (i. e. antibiotic treatment) in reducing S. aureus mediated infection in joints.

A similar experiment was performed using S aureus Xen36 strain. Immunization with the 4-valent vaccine substantially reduced knee joint bacterial burden (again about 2 longs lower). The results were confirmed with bioluminescence assays.

Further analyses were then performed in the attempt to understand the possible mechanism(s) of action of the vaccine. To this purpose, the humoral and cellular immune components were both analysed. Antibody titres against each single antigen of the vaccine in the knee joint washes were measured, and IgG titres against all four antigens were seen (p<0.0001, Mann- Whitney U test), demonstrating that all antigens induced seroconversion in immunized animals and that measurable antibody levels could be detected in the knee joints.

Production of functional antibodies in response to the 4-valent vaccine was confirmed by use of rabbit sera in passive immunization of mice. Mice were passively immunized with sera from rabbits vaccinated with either the 4-valent vaccine/adjuvant or adjuvant alone as a negative control, and infected intravenously with S aureus. LogioCFU counts from knee joint washes showed that bacterial burden in mice immunized with sera from rabbits vaccinated with the 4-valent vaccine was lower than in mice immunized with control sera (p<0.05, Mann- Whitney U test).

Moreover, immune cells were analyzed comparing the results obtained for the actively vaccinated animals to those obtained for the negative controls. Fewer dead immune cells were found in knee joint washes of combo-immunized mice and, even if the number of total cells recovered was more or less the same in both samples, the number of neutrophils recruited was lower in the immunized group indicating the lower state of general inflammation. B cells were preserved in knee joints after immunization, implying that the vaccination could increase the number of cells recruited and/or shield them from S.aureus-mediated toxicity.

Cytokine content in knee joint washes from vaccinated mice was also analysed. Levels of IL-1 alpha, IL-lbeta, IL-17, G-CSF and MIP-1 alpha were all reduced in mice vaccinated with 4-valent vaccine/adjuvant as compared to mice vaccinated with alum adjuvant alone.

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SEQUENCE LISTING

SEQID NO: 1 (EsxA)

MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLN STADAVQEQDQQLSNNFGLQ

SEQID NO: 2 (EsxB)

MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQ KAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 3 (FhuD2)

MKKLLLPLI IMLLVLAACGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGA IVAVNQQVDQ SKVLKDKFKGVTKIGDGDVEKVAKEKPDLI IVYS DKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKA WKKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEVKQE EIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLIKAAK

SEQ ID NO: 4 (StaOll)

MMKRLNKLVLGI IFLFLVISI AGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEM VIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKIKKEIENFK FFVQYGDFKNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIE FTFVEKKEENIYFSDSLDYKKSGDV

SEQID NO: 5 (Hla)

MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKK LLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDD TGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMK AADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERY KIDWEKEEMTN

SEQ ID NO: 6 (N -terminally truncated FhuD2 of SEQ ID NO: 3)

CGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGVTKIGDG DVEKVAKEKPDLI IVYS DKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKKDWEETTAKDGKEIK KAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTSEGK PTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLIKAAK

SEQ ID NO: 7 (Example FhuD2 sequence)

MASCGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGVTKI GDGDVEKVAKEKPDLI IVYS DKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKKDWEETTAKDGK EIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTS EGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLIKAAK

SEQ ID NO: 8 (Example StaOll sequence)

MGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEMVIQPNNEDMVAKGMVLYMNRNT KTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYN PEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKS GDV

SEQ ID NO: 9 (Example StaOll sequence)

MMKRLNKLVLGI IFLFLVI SI AGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEM VIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKLKKEIENFK FFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIE FTFVEKKEENIYFSDSLDYKKSGDV

SEQ ID NO: 10 (Example StaOll sequence)

MMKRLNKLVLGI IFLFLVI SI AGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEM VIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKVKKEIENFK FFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIE FTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 11 (Example StaOll sequence)

MMKRLNKLVLGI IFLFLVISI AGCGIGKEAEVKKS EKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEM VIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKLKKEIENFK FFVQYGDFKNVKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIE FTFVEKKEENIYFSDSLDYKKSGDV

SEQ ID NO: 12 (N -terminally truncated Hla SEQ ID NO: 5)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 13 (Mature Hla-H35L)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 14 (tetramer)

PSGS

SEQ ID NO: 15 (Hla-H35L with PSGS substitution)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNP VYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWK GTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 16 (Hla with PSGS substitution)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNP VYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWK GTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 17 (Hla with Y101 mutation)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 18 (Hla with D152 mutation)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPLFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 19 (Hla with H35 and Y101 mutations)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 20 (Hla with H53 andD152 mutations)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSG LAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPLFK TILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 21 (Hla fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNK SEQ ID NO: 22 (Hla fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG

SEQ ID NO: 23 (Hla fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLL

SEQ ID NO: 24 (Hla-H35L fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNK

SEQ ID NO: 25 (Hla-H35L fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAG

SEQ ID NO: 26 (Hla-H35L fragment)

ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLL

SEQ ID NO: 27 (Useful HLA sequence with H35L mutation)

MASADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGAN KSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQP DFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDF ATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

SEQ ID NO: 28 (EsxAB example)

MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLN STADAVQEQDQQLSNNFGLQASGGGSMGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQF ANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 29 (EsxBA example)

MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQ KAIDNLSQNLAKYDTLSIKQGLDRVNPASGGGSMAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWE GQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNSTADAVQEQDQQLSNNFGLQ

SEQ ID NO: 30 (linker)

ASGGGS

SEQ ID NO: 31 (EsxAB example)

AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNS TADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFAN KVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 32 (EsxAB example)

MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLN STADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFA NKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 33 (N-terminally truncated SEQ ID NO: 4)

GCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEMVIQPNNEDMVAKGMVLYMNRNTK TTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYNP EVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSG DV

SEQ ID NO: 34 (FhuD2)

GNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGVTKIGDGD VEKVAKEKPDLI IVYS DKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKKDWEETTAKDGKEIKK AIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTSEGKP TPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLIKAAK

SEQ ID NO: 35 (Cys-free EsxB sequence)

GGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQK AIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 36 (Cys-free version ofStaOll SEQ ID NO: 4)

GIGKEAEVKKS EKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEMVIQPNNEDMVAKGMVLYMNRNTKTT NGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYNPEV PSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV

SEQ ID NO: 37 (Cys-free version ofFhuD2 SEQ ID NO: 7)

MASGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGVTKIG DGDVEKVAKEKPDLI IVYS DKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKKDWEETTAKDGKE IKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTSE GKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLIKAAK

SEQ ID NO: 38 (EsxAB Cys-Ala mutant)

MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLN STADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAAQKQTQQLAEYIEGSDWEGQFA NKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 39 (useful StaOll sequence)

MGSGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWI INSEMVIQPNNEDMVAKGMVLYMNRNT KTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKI IPTKEIKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYN PEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKS GDV

SEQ ID NO: 40 (Cys-free EsxAB example)

AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNS TADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLAEYIEGSDWEGQFAN KVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP

SEQ ID NO: 41

icicicicicicicicicicicicic

SEQ ID NO: 42

KLKLLLLLKLK

Claims

1. A method for preventing or treating S.aureus infection of a mammal's bones and joints, by administering to the mammal at least one antigen selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1; and Hla.

2. The use of one or more antigens for immunising a mammal to prevent or treat S.aureus infection of its bones and joints, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1; and Hla.

3. Use of at least one antigen in the manufacture of a medicament for preventing or treating S.aureus infection of a mammal's bones and joints, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1; and Hla.

4. One or more antigen(s) for use in a method for preventing or treating S.aureus infection of a mammal's bones and joints, by administering the antigen(s) to the mammal, wherein the antigen(s) is/are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOl 1; and Hla.

5. The method, use, or antigen(s) of any preceding claim, wherein the mammal receives a composition including 2, 3, 4, or all 5 of EsxA, EsxB, FhuD2, StaOl 1, and/or Hla; for example, comprising all five of EsxA, EsxB, FhuD2, StaOl 1, and Hla.

6. The method, use, or antigen(s) of any preceding claim, wherein: the EsxA antigen elicits antibodies in the mammal that recognise SEQ ID NO: 1; the EsxB antigen elicits antibodies in the mammal that recognise SEQ ID NO: 2; the FhuD2 antigen elicits antibodies in the mammal that recognise SEQ ID NO: 3; the StaOl 1 antigen elicits antibodies in the mammal that recognise SEQ ID NO: 4; and/or the Hla antigen elicits antibodies in the mammal that recognise SEQ ID NO: 5.

7. The method, use, or antigen(s) of any preceding claim, wherein the mammal receives a composition comprising (i) a detoxified Hla and/or (ii) a fusion polypeptide of EsxA and EsxB.

8. The method, use, or antigen(s) of any preceding claim, wherein the mammal receives a composition comprising

• all four of: (i) a single polypeptide including both an EsxA antigen and an EsxB antigen e.g. comprising SEQ ID NO: 31; (ii) a FhuD2 antigen e.g. comprising SEQ ID NO: 6; (iii) a StaOl 1 antigen e.g. comprising SEQ ID NO: 33; and (iv) a H35L mutant form of Hla e.g. comprising SEQ ID NO: 13;

• all four of: (i) a first polypeptide having amino acid sequence SEQ ID NO: 32; (ii) a second polypeptide having amino acid sequence SEQ ID NO: 7; (iii) a third polypeptide having amino acid sequence SEQ ID NO: 8; and (iv) a fourth polypeptide having amino acid sequence SEQ ID NO: 27; or • all four of (i) a first polypeptide having amino acid sequence SEQ ID NO: 38; (ii) a second polypeptide having amino acid sequence SEQ ID NO: 37; (iii) a third polypeptide having amino acid sequence SEQ ID NO: 39; and (iv) a fourth polypeptide having amino acid sequence SEQ ID NO: 27.

9. The method, use, or antigen(s) of any preceding claim, wherein the mammal receives a composition comprising: (i) EsxA, EsxB, FhuD2, StaOl l, and/or Hla; and (ii) an adjuvant, such as an aluminium salt e.g. an aluminium hydroxide and/or an aluminium phosphate.

10. The method, use, or antigen(s) of claim 9, wherein the adjuvant comprises a human TLR agonist, such as a TLR7 agonist, for example a compound of formula (K) such as 3-(5-amino-2- (2-methyl-4-(2-(2-(2-phosphonoethoxy) ethoxy)ethoxy)phenethyl) benzo[f][l,7] naphthyridin-8- yl)propanoic acid.

11. The method, use, or antigen(s) of any one of claims 9 and 10, wherein the adjuvant comprises a human TLR agonist adsorbed to an aluminium salt.

12. The method, use, or antigen(s) of any preceding claim, wherein the mammal is a human.

13. The method, use, or antigen(s) of any preceding claim, for treating or preventing (i) osteomyelitis; (ii) septic arthritis; or (iii) prosthetic joint infection.

14. The method, use, or antigen(s) of any preceding claim, wherein the mammal has a prosthetic bone or joint, or is an intended recipient of a prosthetic bone or joint.

15. The method, use, or antigen(s) of any preceding claim, wherein the mammal receives the antigen(s) by injection and/or the mammal receives the antigen(s) in conjunction with an antibiotic.