The invention provides proteins and compositions for the treatment and prevention of

Streptococcus agalactiae (Group B streptococcus; GBS). BACKGROUND ART

The Gram-positive bacterium Streptococcus agalactiae (or "group B streptococcus", abbreviated to "GBS") causes serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates. There are two types of neonatal infection. The first (early onset, usually within 5 days of birth) is manifested by bacteremia and pneumonia. It is contracted vertically as a baby passes through the birth canal. GBS colonises the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonised mothers will become infected. Mortality is between 50-70%. The second is a meningitis that occurs 10 to 60 days after birth. If pregnant women are vaccinated with type III capsule so that the infants are passively immunised, the incidence of the late onset meningitis is reduced but is not entirely eliminated.

The "B" in "GBS" refers to the Lancefield classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into 10 serotypes (la, lb, II, III, IV, V, VI, VII, VII and XI) based on the structure of their polysaccharide capsule.

Investigations have been conducted into the development of protein-based and polysaccharide- based vaccines against GBS but currently, no GBS vaccine is commercially available. There therefore remains a need for effective vaccines against S. agalactiae infection. It is an object of the invention to provide proteins and immunogenic compositions which can be used in the development of vaccines against S. agalactiae infection.

DISCLOSURE OF THE INVENTION

The present invention provides polypeptides consisting of SEQ ID NO: 80 or SEQ ID NO:81, polypeptides consisting of fragments of SEQ ID NO:80 or SEQ ID NO:81, and polypeptides that have at least 70%> sequence identity to such polypeptides. These polypeptides contain the cross-protective epitopes of a GBS polypeptide termed 'GBS67', also known as ancillary protein 1 (API), that is present in GBS pilus structures. Pilus structures in GBS are considered to be interesting vaccine candidates. GBS has three pilus variants, each encoded by a distinct pathogenicity island, PI-1, PI-2a and PI-2b [1, 2]. Each pathogenicity island consists of 5 genes coding for: the pilus backbone protein (BP); 2 ancillary proteins (API and AP2); and 2 sortase proteins that are involved in the assembly of the pili.

All GBS strains carry at least one of these 3 pathogenicity islands and the sequences of the pilus structural proteins (BP, API and AP2) encoded by these pathogenicity islands are generally well conserved. The sequence of ancillary protein 1 (API) or GBS67 encoded by pathogenicity island 2a (API -2a), varies between GBS strains. At least 2 families of the GBS67 protein exist.

The original 'GBS67' (SAG 1408) sequence was annotated in reference 3 as a cell wall surface anchor family protein (see GI: 22534437). The amino acid sequence of full length GBS67 as found in the 2603 strain is given as SEQ ID NO: l herein. GBS strains CJBl 11, 515 and

NEM316 express GBS67 sequences which belong to the same family as the GBS67 sequence from the 2603 strain. The amino acid sequences of full-length GBS67 as found in the CJBl 11, 515 and NEM316 strains are given as SEQ ID NOS: 9, 13 and 17 herein.

A variant of GBS67 (SAI1512) exists in strain H36B. This variant 'GBS67' (SAG1408) sequence was annotated in reference 4 as a cell wall surface anchor family protein (see GI: 77405751). The amino acid sequence of full length GBS67 as found in the H36B strain is given as SEQ ID NO:5 herein. GBS strains DK21 and CJBl 10 express GBS67 sequences which belong to the same family as the GBS67 sequence from the H36B strain. The amino acid sequences of full- length GBS67 as found in the DK21 and CJBl 10 strains are given as SEQ ID NOS: 21 and 25 herein.

As shown herein, serum raised against the amino acid sequence of full-length GBS67 as found in the 2603 strain and related strains is active against strains of GBS that express the amino acid sequence of full-length GBS67 as found in the H36B strain and related strains, and vice versa. Full-length GBS67 thus provides cross-protection against GBS strains expressing GBS67 variants from either of the two families. GBS67 variants are thus cross-protective and provide protection against multiple strains of GBS67. The inventors have succeeded in identifying fragments of the full-length GBS67 sequences that contain the epitopes responsible for cross-protection. In particular, as described in co-pending application PCT/IB2011/000562, the inventors identified a 251 amino acid fragment located at amino acids 616-866 ofthe GBS67 sequence from the 2603 strain given in SEQ ID NO: l. The amino acid of this fragment, referred to herein as "fragment 3", is given in SEQ ID NO:5.

Equivalent fragment 3 sequences from other GBS67 strains that also contain the epitopes responsible for cross-protection are given in SEQ ID NO: 8 (H36B strain), SEQ ID NO: 12 (CJBl 11 strain), SEQ ID NO: 16 (515 strain), SEQ ID NO:20 (NEM316 strain), SEQ ID NO:24 (DK21 strain) and SEQ ID NO:28 (CJBl 10 strain).

By utilising homology modeling with RrgA (S. pneumoniae), the inventors have now identified a region of GBS67 within these fragment 3 sequences that is responsible for providing cross- protection and comprises cross-protective epitopes. This region is termed Domain 4, and in RrgA this domain comprises an isopeptide bond that is important for peptidase resistance and antisera raised against a fragment of RrgA that comprises Domain 4 has been shown to be cross-reactive and to recognise the native pili of S. pneumoniae from different clades.

Domain 4 from strains 2603 and 515 consists of SEQ ID NO:80. Domain 4 from strains CJBl 11, NEW316, H36B, DK21 and CJBl 10 consists of SEQ ID NO:81. Domain 4 from strains 2603, CJB 111, 515and NEM316 is located at amino acids 732-856 of the full-length GBS67 sequences provided in SEQ ID NOS: l, 9, 13 and 17, respectively. Domain 4 from strains H36B, DK21 and CJBl 10 is located at amino acids 727-851 of the full-length GBS67 sequences provided in SEQ ID NOS: 5, 21 and 25, respectively.

The polypeptides of the invention thus comprise cross-protective epitopes from Domain 4 and are thus important vaccine candidates but are easier to synthesise and formulate into vaccine compositions than full-length GBS67 because they are shorter.

A fragment of the GBS67 sequence as found in the 2603 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:80 herein. The amino acid sequence of SEQ ID NO:80 is a 125 amino acid fragment located at amino acids 732-856 of the GBS67 sequence from the 2603 strain given in SEQ ID NO: 1.

A fragment of the GBS67 sequence as found in the H36B strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:81 herein. The amino acid sequence of SEQ ID NO: 81 is a 125 amino acid fragment located at amino acids 727-851 of the GBS67 sequence from the H36B strain given in SEQ ID NO:5. A fragment of the GBS67 sequence as found in the 515 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO: 80 herein. The amino acid sequence of SEQ ID NO:80 is a 125 amino acid fragment located at amino acids 732-856 of the GBS67 sequence from the 515 strain given in SEQ ID NO: 13.

A fragment of the GBS67 sequence as found in the CJB111 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:81 herein. The amino acid sequence of SEQ ID NO:81 is a 125 amino acid fragment located at amino acids 732-856 of the GBS67 sequence from the CJB111 strain given in SEQ ID NO:9.

A fragment of the GBS67 sequence as found in the NEM316 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:81 herein. The amino acid sequence of SEQ ID NO:81 is a 125 amino acid fragment located at amino acids 732-856 of the GBS67 sequence from the NEM316 strain given in SEQ ID NO: 17.

A fragment of the GBS67 sequence as found in the DK21 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:81 herein. The amino acid sequence of SEQ ID NO:81 is a 125 amino acid fragment located at amino acids 727-851 of the GBS67 sequence from the DK21 strain given in SEQ ID NO:21. A fragment of the GBS67 sequence as found in the CJB110 strain that contains epitopes responsible for cross-protection is given as SEQ ID NO:81 herein. The amino acid sequence of SEQ ID NO:81 is a 125 amino acid fragment located at amino acids 727-851 of the GBS67 sequence from the CJB110 strain given in SEQ ID NO:25.

The present invention provides a GBS67 polypeptide consisting of:

(a) the amino acid sequence of SEQ ID NO: 80,

(b) the amino acid sequence of SEQ ID NO:81,

(c) a fragment of SEQ ID NO:80 comprising at least 7 amino acids,

(d) a fragment of SEQ ID NO:81 comprising at least 7 amino acids, or

(e) an amino acid sequence with at least 70% sequence identity to any one of (a), (b), (c) and (d).

In certain embodiments of the invention, fragments of SEQ ID NO: 80 or SEQ ID NO:81 consist of SEQ ID NO: 80 or SEQ ID NO:81 except for a certain number of amino acid deletions. For example, in certain embodiments, the polypeptides of the invention consist of SEQ ID NO:80 or SEQ ID NO:81 except for the deletion of 0 to 5, 0 to 10, 0 to 15, 0 to 20, 0 to 25, 0 to 30, 0 to 35, 0 to 40, 0 to 45 or 0 to 50 amino acids from the N-terminus, and/or the deletion of 0 to 5, 0 to 10, 0 to 15, 0 to 20, 0 to 25, 0 to 30, 0 to 35, 0 to 40, 0 to 45 or 0 to 50 amino acids from the C- terminus. The polypeptides of the invention may thus consist of SEQ ID NO: 80 or SEQ ID NO: 81 except for the deletion of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from the N-terminus and/or from the C-terminus. The polypeptides of this aspect of the invention are thus identical (i.e. 100% identical) to a fragment of SEQ ID NO: 80 or SEQ ID NO:81.

The polypeptides of this aspect of the invention that consist of a fragment of SEQ ID NO: 80 or SEQ ID NO:81 may consist of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 60, 70, 75, 80, 85, 90, or 100 amino acids; or consist of 20-100, 40-100, 60-100, 80-100, 20-80, 40-80, 60-80, 20-60, 40-60, or 20-40 amino acids.

In certain embodiments, the invention provides polypeptides consisting of an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 80, the amino acid sequence of SEQ ID NO:81, a fragment of SEQ ID NO:80, or a fragment of SEQ ID NO:81. In certain embodiments, the sequence identity is at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% sequence identity.

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. 5. 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. 6. Another preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [7], 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 [8].

The sequence identity is determined across the length (i.e. the full length) of the polypeptide having sequence identity to SEQ ID NO: 80, SEQ ID NO: 81, a fragment of SEQ ID NO: 80 or a fragment of SEQ ID NO:81. Thus, the polypeptides of the invention do not include any full- length GBS67 amino acid sequences because these have less than 70% sequence identity with SEQ ID NO: 80, SEQ ID NO: 81, a fragment of SEQ ID NO: 80 or a fragment of SEQ ID NO:81, as determined across the length of SEQ ID NO:80, SEQ ID NO:81, a fragment of SEQ ID NO: 80 or a fragment of SEQ ID NO:81. In certain embodiments, "percent (%) sequence identity" with respect to fragments identified herein is determined by the number of matching identical residues between the two amino acid sequences divided by the total number of residues of the longer, generally full length, sequence. Examples of polypeptides consisting of an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 80, the amino acid sequence of SEQ ID NO:81, a fragment of SEQ ID NO: 80, or a fragment of SEQ ID NO:81 are polypeptides having 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 SEQ ID NO: 80, the SEQ ID NO: 81, a fragment of SEQ ID NO: 80, or a fragment of SEQ ID NO:81, respectively.

In certain embodiments, the polypeptide of the invention consists of SEQ ID NO: 80, SEQ ID NO:81, a fragment of SEQ ID NO:80 or a fragment of SEQ ID NO:81 except for the substitution of 0 to 5, 0 to 10, 0 to 15 or 0 to 20 amino acids. These substitutions may or may not be contiguous.

In certain embodiments, the polypeptides of the invention consist of SEQ ID NO: 80, SEQ ID NO: 81 , a fragment of SEQ ID NO: 80 or a fragment of SEQ ID NO: 81 except for the deletion of 0 to 5, 0 to 10, 0 to 15, 0 to 20, 0 to 25, 0 to 30, 0 to 35, 0 to 40, 0 to 45 or 0 to 50 amino acids from the central region of SEQ ID NO: 80, SEQ ID NO: 81, a fragment of SEQ ID NO: 80 or a fragment of SEQ ID NO:81 . An example of such an internal deletion is the deletion of amino acids 66-77 of SEQ ID NO:80 or SEQ ID NO:81, or the deletion of equivalent amino acids from fragments of SEQ ID NO: 80 or SEQ ID NO:81. These internal deletions can be contiguous or can consist of numerous separate deletions. These internal deletions in polypeptides consisting of SEQ ID NO: 80 or SEQ ID NO:81 may or may not be present in combination with N- or C- terminal deletions discussed above.

Although the polypeptides of the invention do not include any full-length GBS67 polypeptides, the polypeptides of the invention can comprise limited additional GBS67 sequence at their N- terminus or C-terminus. For example, sequences with 70% sequence identity with SEQ ID NO: 80 and SEQ ID NO:81 include fragments of GBS67 having as their N-terminus (i) aspartic acid 693 of SEQ ID NO: 1, 9, 13 or 17, or aspartic acid 688 of SEQ ID NO: 5, 21 or 25; or (ii) arginine 722 of SEQ ID NO: 1, 9, 13 or 17, or arginine 717 of SEQ ID NO: 5, 21 or 25.

Similarly, sequences with 70% sequence identity with SEQ ID NO: 80 and SEQ ID NO:81 include fragments of GBS67 having as their C-terminus isoleucine 867 of SEQ ID NO: l, 9, 13 or 17, or isoleucine 862 of SEQ ID NO: 5, 21 or 25. Therefore, in certain embodiments, the polypeptides of the invention consist of amino acids 693-732, 722-732 and/or 856-867 of SEQ ID NO: 1, 9, 13 or 17 (corresponding to amino acids 688-727, 717-727 and/or 851-862 of SEQ ID NO: 5, 21 or 25), in addition to Domain 4. Therefore, in certain embodiments, the polypeptides of the present invention consist of the amino acids sequence of any one of SEQ ID NOs: 83-88, or homologues thereof.

In certain embodiments, the polypeptides of the invention do not comprise the sequence

YQLIEAVSPEDY (SEQ ID NO:82). Thus, in certain embodiments, the polypeptides of the invention consist of: a) amino acids 1-65 and/or 78-125 of SEQ ID NO:80 or SEQ ID NO:81. b) fragments of amino acids 1-65 or 78-125 of SEQ ID NO: 80 or SEQ ID NO:81; or c) polypeptides with at least 70% identity to amino acids 1-65 and/or 78-125 of SEQ ID NO:80 or SEQ ID NO:81. The polypeptides of the invention discussed above comprise epitopes that are capable of inducing cross-protection. By "epitope" is meant the part of the polypeptide that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and that elicits an immune response. They may also be referred to as "antigenic determinants". The epitopes contained in the polypeptides of the invention may be a B-cell epitopes and/or a T-cell epitopes. Such epitopes can be identified empirically (e.g. using PEPSCAN [9,10] or similar methods), or they can be predicted (e.g. using the Jameson- Wo If antigenic index [11], matrix-based approaches [12], MAPITOPE [13], TEPITOPE [14,15], neural networks [16], OptiMer & EpiMer [17, 18], ADEPT [19], Tsites [20], hydrophilicity [21], antigenic index [22] or the methods disclosed in references 23-27, etc.). The polypeptides of the invention are capable of inducing cross-protection against strains of GBS expressing variant GBS67 peptides. Thus, the polypeptides of the invention will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild- type GBS protein having amino acid sequence SEQ ID NO: l (strain 2603) and to the wild-type GBS protein having amino acid sequence SEQ ID NO:5 (strain H36B). The polypeptides of the invention are thus capable of competing with both SEQ ID NO: 1 and SEQ ID NO: 5 for binding to an antibody raised against SEQ ID NO: l or SEQ ID NO:5.

The polypeptides of the invention will typically also, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type GBS protein having amino acid sequence SEQ ID NO:9 (strain CJB111), the wild-type GBS protein having amino acid sequence SEQ ID NO: 13 (strain 515), the wild-type GBS protein having amino acid sequence SEQ ID NO: 17 (strain NEM316), the wild-type GBS protein having amino acid sequence SEQ ID NO:21 (strain DK21), and the wild-type GBS protein having amino acid sequence SEQ ID NO:25 (strain CJB110). The polypeptides of the invention are thus also capable of competing with these wild-type GBS proteins having SEQ ID NOs:9, 13, 17, 21 or 25 for binding to an antibody raised against these proteins.

Antibodies can readily be generated against the polypeptides of the invention using standard 5 immunisation methods and the ability of these antibodies to bind to the wild-type GBS proteins of SEQ ID NOs: 1, 5, 9, 13, 17, 21 and 25 can be assessed using standard assays such as ELISA assays.

Similarly, the ability of polypeptides to compete with antibodies raised against the wild-type GBS proteins can be readily determined using competition assay techniques known in the art,

10 including equilibrium methods such as ELISA, kinetic methods such as BIACORE® and by flow cytometry methods. A polypeptide that competes with wild-type GBS proteins of SEQ ID NOs: 1, 5, 9, 13, 17, 21 and 25 for binding to an antibody against one of these wild-type GBS proteins will cause a reduction in the observed total binding of the wild-type GBS protein to the antibody, compared to when the polypeptide is not present. Typically, this reduction in binding is

15 10% or greater, 20% or greater, 30%> or greater, 40%> or greater, 60%> or greater, for example a reduction in binding of 70% or more in the presence of the polypeptide of the invention compared to antibody binding observed for the GBS proteins having SEQ ID NO: l, 5, 9, 13, 17, 21 or 25.

The ability of the polypeptides of the invention to induce cross-protection against strains of GBS 0 expressing variant GBS67 proteins has been confirmed in animal models, such as the maternal immunization models described in the examples in which female mice are immunized with the polypeptides and their pups are challenged with GBS strains expressing variant GBS67 proteins.

The polypeptides of the invention may be provided in the form of a hybrid polypeptide. The hybrid polypeptide may comprise additional GBS or non-GBS polypeptide sequences, but they 5 may not be derived from GBS67. The invention thus provides a hybrid polypeptide that consists of any polypeptide of the invention discussed above and between 1 and 40 additional amino acids comprising a sequence that has less than 50%> sequence identity to SEQ ID NO: 1, 5, 9, 13, 17, 21 and 25. Additional hybrid polypeptides are discussed in more detail below.

The invention also provides a nucleic acid comprising a nucleotide sequence encoding a

30 polypeptide or a hybrid polypeptide of the invention. The invention also provides an immunogenic composition comprising a polypeptide, a hybrid polypeptide or a nucleic acid of the invention. Such an immunogenic composition may be used in methods of treating or preventing diseases or conditions associated with GBS.

The invention also provides a cell (typically a bacterium) which expresses a polypeptide or a hybrid polypeptide of the invention.

Hybrid polypeptides

The polypeptides of the invention can be expressed in combination with other polypeptides as a single polypeptide chain (a 'hybrid' polypeptide or 'chimera'). Hybrid polypeptides offer two main advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need to be employed in order to produce two polypeptides which are both antigenically useful.

Hybrid polypeptides can include sequences from other GBS antigens and/or from other non-GBS antigens. Usually, the hybrid polypeptides include sequences from other GBS sequences, such as other pilus subunits. These other GBS sequence may be to the N-terminus or to the C-terminus of the GBS67 polypeptides. Different hybrid polypeptides may be mixed together in a single formulation.

Hybrid polypeptides may be represented by the formula NH ₂ -A-{-X-L-}„-B-COOH.

X is a GBS67 Domain 4 polypeptide of the invention, including fragments and polypeptides with sequence identity to the Domain 4 polypeptide and fragments thereof as discussed above.

For each n instances of {-X-L-}, linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH ₂ -Xi-Li-X ₂ -L ₂ -COOH, NH ₂ -Xi-X ₂ -COOH,

NH ₂ -Xi-Li-X ₂ -COOH, NH ₂ -Xi-X ₂ -L ₂ -COOH, etc. Linker amino acid sequence(s) -L- will typically be short {e.g. 20 or fewer amino acids i.e. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers {i.e. comprising Gly„ where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags {i.e. His„ where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. Useful linkers are GSGS (SEQ ID NO:29), GSGGGG (SEQ ID NO:30) or GSGSGGGG (SEQ ID NO:31), with the Gly-Ser dipeptide being formed from a BamHl restriction site, thus aiding cloning and manipulation, and the (Gly) ₄ tetrapeptide being a typical poly-glycine linker. Other suitable linkers, particularly for use as the final L _n are a Leu-Glu dipeptide or Gly-Ser. Linkers will usually contain at least one glycine residue to facilitate structural flexibility e.g. a -L- moiety may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycine residues. Such glycines may be arranged to include at least two consecutive glycines in a Gly-Gly dipeptide sequence, or a longer oligo-Gly sequence i.e. Gly _n where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. -A- is an optional N-terminal amino acid sequence. This will typically be short {e.g. 40 or fewer amino acids i.e. 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification {e.g. histidine tags i.e. His„ where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If Xi lacks its own N-terminus methionine, -A- is preferably an oligopeptide {e.g. with 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine e.g. Met-Ala-Ser, or a single Met residue. In a nascent polypeptide the -A- moiety can provide the polypeptide's N-terminal methionine

(formyl-methionine, fMet, in bacteria). One or more amino acids may be cleaved from the N-terminus of a nascent -A- moiety, however, such that the -A- moiety in a mature polypeptide of the invention does not necessarily include a N-terminal methionine.

-B- is an optional C-terminal amino acid sequence. This will typically be short {e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification {e.g.

comprising histidine tags i.e. His„ where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art, such as a glutathione-S-transferase, thioredoxin, 14kDa fragment of S.aureus protein A, a biotinylated peptide, a maltose-binding protein, an enterokinase flag, etc. It is preferred that -A-, -B- and -L- sequences do not include a sequence that shares 10 or more contiguous amino acids in common with a human polypeptide sequence.

It is preferred that -A-, -B- and -L- sequences do not include a sequence that has 50% or more sequence identity to SEQ ID NO: 1 , 5, 9, 13, 17, 21 and 25.

Polypeptides Polypeptides used with the invention can be prepared in many ways e.g. by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture {e.g. from recombinant expression), from the organism itself (e.g. after bacterial culture, or direct from patients), etc. A preferred method for production of peptides <40 amino acids long involves in vitro chemical synthesis [28,29]. Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [30] chemistry.

Enzymatic synthesis [31] may also be used in part or in full. As an alternative to chemical synthesis, biological synthesis may be used e.g. the polypeptides may be produced by translation. This may be carried out in vitro or in vivo. Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine,

azidohomoalanine, etc.) [32]. Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides may have covalent modifications at the C-terminus and/or N- terminus.

Polypeptides can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.).

Polypeptides 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 pneumococcal 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%> (e.g. 5% or less) of a composition is made up of other expressed polypeptides.

Polypeptides may be attached to a solid support. Polypeptides may comprise a detectable label (e.g. a radioactive or fluorescent label, or a biotin label).

The term "polypeptide" refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can occur as single chains or associated chains. Polypeptides can be naturally or non-naturally glycosylated (i.e. the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring polypeptide). The invention provides a process for producing polypeptides of the invention, comprising culturing a host cell of to the invention under conditions which induce polypeptide expression. Although expression of the polypeptide may take place in a Streptococcus, the invention will usually use a heterologous host for expression. The heterologous host may be prokaryotic {e.g. a bacterium) or eukaryotic. It will usually 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.

The invention also provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised in part or in whole using chemical means. The invention also provides a composition comprising two or more polypeptides of the invention.

Nucleic acids

The invention also provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide or a hybrid polypeptide of the invention. For example, the invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 80 and SEQ ID NO:81, fragments of SEQ ID NO: 80 and SEQ ID NO: 81, and polypeptides having at least 70% identity to SEQ ID NO: 80, SEQ ID NO:81 and fragments of SEQ ID NO: 80 and 81. The invention also provides nucleic acids comprising nucleotide sequences having sequence identity to such nucleotide sequences. Such nucleic acids include those using alternative codons to encode the same amino acid. In particular, nucleic acids may contain alternative codons optimised for expression in specific microorganisms, e.g. E. coli.

The invention also provides nucleic acid which can hybridize to these nucleic acids.

Hybridization reactions can be performed under conditions of different "stringency". Conditions that increase stringency of a hybridization reaction of widely known and published in the art. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25°C, 37°C, 50°C, 55°C and 68°C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%>, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or de-ionized water. Hybridization techniques and their optimization are well known in the art [e.g. see refs 33 & 34, etc.].

The invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers). Nucleic acids according to the invention can take various forms (e.g. single-stranded, double-stranded, vectors, primers, probes, labelled etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary single-stranded forms which make up the double-stranded form. Primers and probes are generally single-stranded, as are antisense nucleic acids.

Nucleic acids of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other nucleic acids (e.g. free from naturally-occurring nucleic acids), particularly from other GBS or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure. Nucleic acids of the invention are preferably GBS nucleic acids.

Nucleic acids of the invention may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc. Nucleic acid of the invention may be attached to a solid support (e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.). Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe.

The term "nucleic acid" includes in general means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc.. Where nucleic acid of the invention takes the form of RNA, it may or may not have a 5' cap.

Nucleic acids of the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence in a host cell, "viral vectors" which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector. Preferred vectors are plasmids. A "host cell" includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention.

Where a nucleic acid is DNA, it will be appreciated that "U" in a RNA sequence will be replaced by "T" in the DNA. Similarly, where a nucleic acid is RNA, it will be appreciated that "T" in a DNA sequence will be replaced by "U" in the RNA.

The term "complement" or "complementary" when used in relation to nucleic acids refers to Watson-Crick base pairing. Thus the complement of C is G, the complement of G is C, the complement of A is T (or U), and the complement of T (or U) is A. It is also possible to use bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T).

Nucleic acids of the invention can be used, for example: to produce polypeptides in vitro or in vivo; as hybridization probes for the detection of nucleic acid in biological samples; to generate additional copies of the nucleic acids; to generate ribozymes or antisense oligonucleotides; as single- stranded DNA primers or probes; or as triple-strand forming oligonucleotides.

The invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means.

The invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.

Immunogenic compositions

The polypeptides and hybrid polypeptides of the invention are useful as active ingredients in immunogenic compositions. Such immunogenic compositions may be useful as vaccines. These vaccines may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.

It will be understood that, in the context of vaccination, the term "prophylactic" also includes the case wherein the immune system of a subject has been primed (e.g by vaccination) to trigger an immune response and repel infection. A vaccinated subject may thus get infected, but is subsequently able to completely repel or better able to repel the infection than a control subject. Hence this term includes providing protective immunity.

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 [35].

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 of the invention may be dried, such as a lyophilised formulation.

The 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 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.

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.

Human vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e. about 0.25ml) may be administered to children.

Immunogenic compositions of the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include one or more adjuvants. The adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below.

Adjuvants which may be used in compositions of the invention include, but are not limited to:

• mineral salts, such as aluminium salts and calcium salts, including hydroxides (e.g.

oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates) and sulphates, etc. [e.g. see chapters 8 & 9 of ref. 36]; · oil- in- water emulsions, such as squalene-water emulsions, including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a

microfluidizer) [Chapter 10 of ref. 36, see also ref. 37-40, chapter 10 of ref. 41 and chapter 12 of ref. 42], complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A);

• saponin formulations [chapter 22 of ref. 36], such as QS21 [43] and ISCOMs [chapter 23 of ref. 36];

• virosomes and virus-like particles (VLPs) [44-50];

• bacterial or microbial derivatives, such as non-toxic derivatives of enterobacterial

lipopolysaccharide (LPS), Lipid A derivatives [51, 52], immunostimulatory oligonucleotides [53-58], such as IC-31™ [59] (deoxynucleotide comprising 26-mer sequence 5'-(IC)i3-3' (SEQ ID NO:32) and polycationic polymer peptide comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO:33)) and ADP-ribosylating toxins and detoxified derivatives thereof [60-70];

• human immunomodulators, including cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [71, 72], interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor; • bioadhesives and mucoadhesives, such as chitosan and derivatives thereof, esterified hyaluronic acid microspheres [73] or mucoadhesives, such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulos [74]; · microparticles (i.e. a particle of -lOOnm to ~150um in diameter, more preferably ~200nm to ~30μιη in diameter, and most preferably ~500nm to ~10μιη 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.);

• liposomes [Chapters 13 & 14 of ref. 36, ref. 75-77]; · polyoxyethylene ethers and polyoxyethylene esters [78];

• PCPP formulations [79 and 80];

• muramyl peptides, including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-5/7-glycero-3-hydroxyphosphoryloxy)- ethylamine MTP-PE); and

• imidazoquinolone compounds, including Imiquamod and its homologues (e.g. "Resiquimod 3M") [81 and 82].

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [83]; (2) a saponin (e.g. QS21) + a nontoxic LPS derivative (e.g. 3dMPL) [84]; (3) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) + a cholesterol; (4) a saponin (e.g. QS21) + 3dMPL + IL-12 (optionally + a sterol) [85]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [86]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed in chapter 7 of ref. 36. The use of an aluminium hydroxide and/or aluminium phosphate adjuvant is useful, particularly in children, and antigens are generally adsorbed to these salts. Squalene-in-water emulsions are also preferred, particularly in the elderly. Useful adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & alum or resiquimod & alum. A combination of aluminium phosphate and 3dMPL may be used.

The compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response.

Two types of T cells, CD4 and CD8 cells, are generally thought necessary to initiate and/or enhance cell mediated immunity and humoral immunity. CD8 T cells can express a CD8 co-receptor and are commonly referred to as Cytotoxic T lymphocytes (CTLs). CD8 T cells are able to recognized or interact with antigens displayed on MHC Class I molecules.

CD4 T cells can express a CD4 co-receptor and are commonly referred to as T helper cells. CD4 T cells are able to recognize antigenic peptides bound to MHC class II molecules. Upon interaction with a MHC class II molecule, the CD4 cells can secrete factors such as cytokines. These secreted cytokines can activate B cells, cytotoxic T cells, macrophages, and other cells that participate in an immune response. Helper T cells or CD4+ cells can be further divided into two functionally distinct subsets: THl phenotype and TH2 phenotypes which differ in their cytokine and effector function.

Activated THl cells enhance cellular immunity (including an increase in antigen- specific CTL production) and are therefore of particular value in responding to intracellular infections.

Activated THl cells may secrete one or more of IL-2, IFN-γ, and TNF-β. A THl immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs). A THl immune response may also act to expand the immune response by stimulating growth of B and T cells with IL-12. THl stimulated B cells may secrete IgG2a.

Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection. An enhanced immune response may include one or more of an enhanced THl immune response and a TH2 immune response. A THl immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a THl immune response (such as IL-2, IFN-γ, and TNF- β), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced THl immune response will include an increase in IgG2a production.

A THl immune response may be elicited using a THl adjuvant. A THl adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant. THl adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides. Immunostimulatory

oligonucleotides, such as oligonucleotides containing a CpG motif, are preferred THl adjuvants for use in the invention.

A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells. Preferably, the enhanced TH2 immune response will include an increase in IgGl production.

A TH2 immune response may be elicited using a TH2 adjuvant. A TH2 adjuvant will generally elicit increased levels of IgGl production relative to immunization of the antigen without adjuvant. TH2 adjuvants suitable for use in the invention include, for example, mineral containing compositions, oil-emulsions, and ADP-ribosylating toxins and detoxified derivatives thereof. Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.

A composition may include a combination of a THl adjuvant and a TH2 adjuvant. Preferably, such a composition elicits an enhanced THl and an enhanced TH2 response, i.e., an increase in the production of both IgGl and IgG2a production relative to immunization without an adjuvant. Still more preferably, the composition comprising a combination of a THl and a TH2 adjuvant elicits an increased THl and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a THl adjuvant alone or immunization with a TH2 adjuvant alone). The immune response may be one or both of a THl immune response and a TH2 response. Preferably, immune response provides for one or both of an enhanced THl response and an enhanced TH2 response. The enhanced immune response may be one or both of a systemic and a mucosal immune response. Preferably, the immune response provides for one or both of an enhanced systemic and an enhanced mucosal immune response. Preferably the mucosal immune response is a TH2 immune response. Preferably, the mucosal immune response includes an increase in the production of IgA.

Streptococcal infections can affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray- freeze dried composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The 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. Alternatively such kits may comprise one or more antigens in lyophilised form and a physiologically acceptable carrier in liquid form, such as saline, to reconstitute the antigen or antigens prior to administration to a patient.

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. 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.

Methods of treatment, and administration of the vaccine

The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a polypeptide, hybrid polypeptide, nucleic acid or an immunogenic composition as described above. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method may raise a booster response.

The invention also provides a polypeptide, hybrid polypeptide, nucleic acid or an immunogenic composition described above for use as a medicament e.g. for use in raising an immune response in a mammal.

The invention also provides the use of a polypeptide, hybrid polypeptide, nucleic acid or an immunogenic composition described above in the manufacture of a medicament for raising an immune response in a mammal. By raising an immune response in the mammal by these uses and methods, the mammal can be protected against disease and/or infection caused by GBS e.g. against meningitis.

The invention also provides a delivery device pre-filled with an immunogenic composition of the invention.

Particularly the mammal is a female mammal, more particularly a female mammal of reproductive age, yet more particularly a female mammal of reproductive age that is pregnant or is trying to conceive. The mammal is preferably a human. The human may be a teenager or an adult.

One way of checking efficacy of therapeutic treatment involves monitoring GBS infection after administration of the compositions of the invention. One way of checking efficacy of prophylactic treatment involves testing post-immunisation sera in standard tests; for example, sera can be tested in an opsonophagocytic killing assay (OPKA), with the ability to opsonise bacteria indicating protective efficacy. Another way of checking efficacy of prophylactic treatment involves post-immunisation challenge in an animal model of GBS infection, e.g., guinea pigs or mice. One such model is described in reference 87. Another way of assessing the immunogenicity of the compositions of the present invention is to express the polypeptides recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays. A positive reaction between the polypeptide and the patient sample indicates that the patient has mounted an immune response to the polypeptide in question. This method may also be used to identify immunodominant antigens and/or epitopes within antigens.

Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration.

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.).

Vaccines prepared according to the invention may be used to treat both children and adults. Thus a human patient may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients for receiving the vaccines are adolescents (e.g. 13-20 years old), pregnant women, and the elderly (e.g. >50 years old, >60 years old, and preferably >65 years. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population. Vaccines produced by the invention 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 e.g. at substantially the same time as a rubella vaccine, a varicella vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, an inactivated polio virus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Y vaccine), a respiratory syncytial virus vaccine, an human papillomavirus vaccine, an influenza virus vaccines (including a pandemic influenza virus vaccine) etc. Vaccines of the invention may also be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional) an antiviral compound, and in particular an antiviral compound active against influenza virus (e.g.

oseltamivir and/or zanamivir). These antivirals include neuraminidase inhibitors, such as a (3R,4R,5S)-4-acetylamino-5-amino-3(l-ethylpropoxy)-l-cyclohe xene-l-carboxylic acid or 5- (acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5 -trideoxy-D-glycero-D- galactonon-2-enonic acid, including esters thereof (e.g. the ethyl esters) and salts thereof (e.g. the phosphate salts). A preferred antiviral is (3R,4R,5S)-4-acetylamino-5-amino-3(l-ethylpropoxy)- 1-cyclohexene-l-carboxylic acid, ethyl ester, phosphate (1 : 1), also known as oseltamivir phosphate (TAMIFLU™).

Combinations

In addition to a GBS67 polypeptide described above, a composition may include: (i) one or more further polypeptides that elicit antibody responses against GBS proteins, particularly against GBS proteins other than GBS67; (ii) a capsular saccharide from GBS; and/or (iii) one or more further immunogens that elicit antibody responses that recognise epitopes on non-GBS organisms.

Combinations with further polypeptide antigens

GBS67 polypeptides described above may be combined with one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10) polypeptide antigens selected from the group consisting of: (1) a (GBS80) antigen; (2) a GBS59 antigen; (3) a GBS 1523 antigen; (4) a GBS 104 antigen; (5) a GBS 1524 antigen; (6) a GBS3 antigen; (7) a SAN1485 antigen; (8) a GBS147 antigen; (9) a GBS328 antigen; and/or (10) a GBS84 antigen.

These further antigens may be added as separate polypeptides. As an alternative, they may be added as hybrids e.g. a GBS80-GBS1523 hybrid. As a further alternative, they may be fused to a GBS67 polypeptide of the invention to provide a hybrid polypeptide.

Any of these combinations may also include one or more GBS capsular saccharide(s), which will typically be conjugated to carrier protein(s). Further information about such saccharides and conjugation is provided below.

GBS80 The original 'GBS80' (SAG0645) sequence was annotated in reference 3 as a cell wall surface anchor family protein (see GI: 22533660). For reference purposes, the amino acid sequence of full length GBS80 as found in the 2603 strain is given as SEQ ID NO:34 herein. Preferred GBS80 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:34; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:34, 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 GBS80 proteins include variants of SEQ ID NO: 34.

Preferred fragments of (b) comprise an epitope from SEQ ID NO:34. 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:34 while retaining at least one epitope of SEQ ID NO:34. Other fragments omit one or more protein domains.

Wild-type GBS-80 contains a N-terminal leader or signal sequence region at amino acids 1-37 of SEQ ID NO:34. One or more amino acids from the leader or signal sequence region of GBS80 can be removed, e.g. SEQ ID NO:35. The wild-type sequence also contains a C-terminal transmembrane region at amino acids 526-543 of SEQ ID NO:34. One or more amino acids from the transmembrane region and/or a cytoplasmic region may be removed, e.g. SEQ ID NO:36. Wild-type GBS80 contains an amino acid motif indicative of a cell wall anchor at amino acids 521-525 of SEQ ID NO:34. In some recombinant host cell systems it may be useful to remove this motif to facilitate secretion of a recombinant GBS80 polypeptide from the host cell. Thus the transmembrane and/or cytoplasmic regions and the cell wall anchor motif may be removed from GBS80, e.g. SEQ ID NO:37. Alternatively, in some recombinant host cell systems it may be useful to use the cell wall anchor motif to anchor the recombinantly expressed polypeptide to the cell wall. The extracellular domain of the expressed polypeptide may be cleaved during purification or the recombinant polypeptide may be left attached to either inactivated host cells or cell membranes in the final composition, e.g. SEQ ID NO:38. A particularly immunogenic fragment of wild-type GBS80 is located towards the N-terminus of the polypeptide, and is SEQ ID NO:39.

GBS59

GBS59 is the pilus backbone protein encoded by pathogenicity island 2a (BP-2a). For reference purposes, the amino acid sequence of full length GBS59 as found in the 2603 strain is given as SEQ ID NO:40 herein. Preferred GBS59 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60%> 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:40; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:40, wherein 'η' 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 GBS59 proteins include variants of SEQ ID NO:40. Preferred fragments of (b) comprise an epitope from SEQ ID NO:40. 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:40 while retaining at least one epitope of SEQ ID NO:40. Other fragments omit one or more protein domains.

Variants of GBS59 exist in strains H36B, 515, CJB1 1 1 , DK21 and CJB1 10. For reference purposes, the amino acid sequence of full length GBS59 as found in the 515, CJB1 1 1 , H36B, CJB1 10 and DK21 strains are given as SEQ ID NOs: 41 , 42, 43, 44, and 45. Preferred GBS59 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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 NOs: 41 , 42, 43, 44, or 45; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NOs: 41 , 42, 43, 44, or 45, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NOs: 41 , 42, 43, 44, or 45. 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 NOs: 41 , 42, 43, 44, or 45 while retaining at least one epitope of SEQ ID NOs: 41 , 42, 43, 44, or 45. Other fragments omit one or more protein domains.

GBS 1523

The original 'GBS 1523' (SAN1518; Spbl) sequence was annotated in reference 4 as a cell wall surface anchor family protein (see GI: 77408651). For reference purposes, the amino acid sequence of full length GBS 1523 as found in the COH1 strain is given as SEQ ID NO:46 herein. Preferred GBS 1523 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:46; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:46, 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 GBS 1523 proteins include variants of SEQ ID NO:46. Preferred fragments of (b) comprise an epitope from SEQ ID NO:46. 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:46 while retaining at least one epitope of SEQ ID NO:46. Other fragments omit one or more protein domains.

Wild-type GBS 1523 contains a N-terminal leader or signal sequence region at amino acids 1 to 29 of SEQ ID NO:46 which may be removed in fragments, e.g. SEQ ID NO:47. The wild-type sequence contains an amino acid motif indicative of a cell wall anchor (LPSTG) at amino acids 468-472 of SEQ ID NO:46. In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant polypeptide from the cell.

Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed polypeptide to the cell wall. The extracellular domain of the expressed polypeptide may be cleaved during purification or the recombinant polypeptide may be left attached to either inactivated host cells or cell membranes in the final composition. An E box containing a conserved glutamic residue has also been identified at amino acids 419-429 of SEQ ID NO:46, with a conserved glutamic acid at residue 423. The E box motif may be important for the formation of oligomeric pilus-like structures, and so useful fragments of GBS1523 may include the conserved glutamic acid residue. A mutant of GBS 1523 has been identified in which the glutamine (Q) at position 41 of SEQ ID NO:46 is substituted for a lysine (K), as a result of a mutation of a codon in the encoding nucleotide sequence from CAA to AAA. This substitution may be present in the GBS 1523 sequences and GBS1523 fragments (e.g. SEQ ID NO:48). Where the compositions include both GBS80 and GBS 1523, a hybrid polypeptide may be used. Examples of GBS80-GBS 1523 hybrids are found in reference 88 and include the polypeptides of SEQ ID NOS: 49-52.

GBS 104

The original 'GBS 104' (SAG0649) sequence was annotated in reference 3 as 'a cell wall surface anchor family protein' (see GI: 22533664). For reference purposes, the amino acid sequence of full length GBS 104 as found in the 2603 strain is given as SEQ ID NO:53 herein. Preferred GBS 104 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:53; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:53, 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 GBS 104 proteins include variants of SEQ ID NO:53. Preferred fragments of (b) comprise an epitope from SEQ ID NO:40. 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:53 while retaining at least one epitope of SEQ ID NO:53. Other fragments omit one or more protein domains.

GBS 1524 For reference purposes, the amino acid sequence of full length GBS 1524 (SAN1519) as found in the COHl strain is given as SEQ ID NO:54 herein. Preferred GBS 1524 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:54; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:54, 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 GBS 1524 proteins include variants of SEQ ID NO:54. Preferred fragments of (b) comprise an epitope from SEQ ID NO:54. 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:54 while retaining at least one epitope of SEQ ID NO: 54. Other fragments omit one or more protein domains.

GBS3

The original 'GBS3' (SAG2603; BibA) sequence was annotated in reference 3 as 'a pathogenicity protein' (see GL22535109). For reference purposes, the amino acid sequence of full length GBS3 as found in the 2603 strain is given as SEQ ID NO:55 herein. Preferred GBS3 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:55; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:55, 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 GBS3 proteins include variants of SEQ ID NO:55. Preferred fragments of (b) comprise an epitope from SEQ ID NO:35. 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:55 while retaining at least one epitope of SEQ ID NO:55. Other fragments omit one or more protein domains.

Wild-type GBS3 contains a N-terminal leader or signal sequence region at amino acids 1 to 36 of SEQ ID NO:55 which may be removed in fragments, e.g. SEQ ID N0563. GBS3 also contains an amino acid motif indicative of a cell wall anchor (LPXTG), a transmembrane region and cytoplasmic domains (see reference 89). The leader or signal sequence region., the transmembrane and cytoplasmic domains, and the ceil wal l anchor motif may ail be removed from GBS3 to leave a fragment comprising the eoiied-coii and praline-rich segments as set forth below (SEQ ID NO:57). Alternative fragments of GBS3 may comprise: the signal sequence region and coiled coil segment (SEQ ID NO:58); the coiled coil segment (SEQ ID NO:59); or the signal sequence region, coiled coil segment, and proime-rieb segment (SEQ ID NO:60),

Variants of GBS3 exist in the 5.15 strain (SAL21 18), CJB 1 .1 .1 strain (SAM 1974) and COH i strain (SAN2207). Reference amino acid sequences for full-le gth GBS3 in the 515 strain, the C.IBl 1 1 strain and the ( C )i i i strain are given herein as SEQ ID O:61, SEQ ID NC):62 and SEQ ID O:63 respectively. Thus, GBS3 polypeptides for use with the invention may also comprise an amino acid sequence: (a) having 60% 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:61 , SEQ I D NO:62 or SEQ ID NO:63; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:6 1 , SEQ I D NO:62 or SEQ ID NO:63, 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 GBS3 proteins include variants of SEQ ID NO:61 , SEQ ID NO:62 or SEQ ID N():63. Preferred fragments of (b) comprise an epitope from SEQ ID NO:6L SEQ ID NO:62 or SEQ ID NO:63. 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:61 , SEQ ID O:62 or SEQ ID NO:63 while retaining at least one epitope of SEQ ID NO:6E SEQ ID NO:62 or SEQ ID NO:63. Other fragments omit one or more protein domains.

The invention includes the use of fragments of GBS3 from the 515, cjbl 1 1 and cohl strains that are analogous to fragments of GBS3 from the 2603 strain discussed in detail above, e.g. lacking the N-terminal leader or signal sequence region; comprising the coil ed-coi S and pro line-rich segments: comprising the signal sequence region and coiled coil segment; comprising the coiled coil segment; or comprising the signal sequence region., coiled coil segment, and proline-rich segment.

SAN 1485

The original 'SAN1485' sequence was annotated in reference 4 as 'cell wall surface anchor family protein ' (see GI: 77408233). For reference purposes, the amino acid sequence of full length SAN1485 as found in the COHI strain is given as SEQ ID NO:64 herein. Preferred SAN 1485 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:64; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:64, 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 SA 1485 5 proteins include variants of SEQ ID NO: 64. Preferred fragments of (b) comprise an epitope from SEQ ID NO:64. 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:64 while retaining at least one epitope of SEQ ID NO:64. Other fragments omit one or more protein domains.

10 GBS 147

The original 'GBS 147' (SAG0416) sequence was annotated in reference 3 as 'a putative protease' (see GI: GL22533435). For reference purposes, the amino acid sequence of full length GBS 147 as found in the 2603 strain is given as SEQ ID NO:65 herein. Preferred GBS 147 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% or more identity (e.g.

15 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO:65 and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:65, 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 GBS 147 proteins include variants of SEQ ID NO: 65. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 65. Other

20 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:65 while retaining at least one epitope of SEQ ID NO:65.

GBS328

25 The original 'GBS328' (SAG1333) sequence was annotated in reference 3 as ' 5 '-nucleotidase family protein ' (see GI: 22534359). For reference purposes, the amino acid sequence of full length GBS328 as found in the 2603 strain is given as SEQ ID NO:66 herein. Preferred GBS328 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60%> or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

30 97%, 98%, 99%, 99.5% or more) to SEQ ID NO:66; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:66, 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 GBS328 proteins include variants of SEQ ID NO:66. Preferred fragments of (b) comprise an epitope from SEQ ID NO:66. 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:66 while retaining at least one epitope of SEQ ID NO:66. Other fragments omit one or more protein domains. GBS84

The original 'GBS84' (SAG0907) sequence was annotated in reference 3 as 'a putative lipoprotein' (see GI: 22533929). For reference purposes, the amino acid sequence of full length GBS84 as found in the 2603 strain is given as SEQ ID NO:67 herein. Preferred GBS84 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% 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:67; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO:67, 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 GBS84 proteins include variants of SEQ ID NO:67. Preferred fragments of (b) comprise an epitope from SEQ ID NO:67. 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:67 while retaining at least one epitope of SEQ ID NO:67. Other fragments omit one or more protein domains.

Combinations with GBS saccharides

GBS67 polypeptides may be combined with one or more GBS capsular saccharide(s), which will typically be conjugated to carrier protein(s). Thus the invention provides an immunogenic composition comprising a combination of:

(1) a GBS67 polypeptide as discussed above; and

(2) one or more GBS capsular saccharides. A saccharide used in component (2) of this combination is ideally present as a conjugate comprising a saccharide moiety and a carrier protein moiety. The carrier moiety in the conjugate may be a single GBS67 polypeptide, a hybrid GBS67 polypeptide, a non-GBS67 GBS polypeptide, or a non-GBS polypeptide.

The saccharide is from the capsular saccharide of GBS. The saccharide may be a polysaccharide having the size that arises during purification of the saccharide from bacteria, or it may be an oligosaccharide achieved by fragmentation of such a polysaccharide. A composition may include a capsular saccharide from one or more of the following streptococcal serotypes: la, lb, Ia/c, II, III, IV, V, VI, VII and VIII. A composition may include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, or 8 serotypes. Including a saccharide from one or more of serotypes la, lb, II, III & V is useful. The capsular saccharides of each of these five serotypes include: (a) a terminal N-acetyl-neuraminic acid (NeuNAc) residue (commonly referred to as sialic acid), which in all cases is linked 2→3 to a galactose residue; and (b) a N-acetyl- glucosamine residue (GlcNAc) within the trisaccharide core.

Saccharides used according to the invention may be in their native form, or may have been modified. For example, the saccharide may be shorter than the native capsular saccharide, or may be chemically modified. For instance, the saccharide may be de-O-acetylated (partially or fully), de-N-acetylated (partially or fully) or N-propionated (partially or fully), etc. De- acetylation may occur before, during or after conjugation, but preferably occurs before conjugation. Depending on the particular saccharide, de-acetylation may or may not affect immunogenicity. The relevance of O-acetylation on GBS saccharides in various serotypes is discussed in ref. 90, and in some embodiments O-acetylation of sialic acid residues at positions 7, 8 and/or 9 is retained before, during and after conjugation e.g. by protection/de-protection, by re-acetylation, etc. However, typically the GBS saccharide used in the present invention has substantially no O-acetylation of sialic acid residues at positions 7, 8 and/or 9. The effect of de-acetylation etc. can be assessed by routine assays. Another possible modification is the removal of sialic acid residues from the saccharide, such as side-chain terminal sialic acids [91]. In particular, when a serotype V capsular saccharide is used in the invention, it may be modified by desialylation as described in ref. [91]. Desialylated GBS serotype V capsular saccharide may be prepared by treating purified GBS serotype V capsular saccharide under mildly acidic conditions {e.g. 0.1M sulphuric acid at 80°C for 60 minutes) or by treatment with neuraminidase, as described in ref. [91]. In another example, full-length polysaccharides may be depolymerised to give shorter fragments for use with the invention e.g. by hydrolysis in mild acid, by heating, by sizing chromatography, etc. Chain length has been reported to affect immunogenicity of GBS saccharides in rabbits [92]. In particular, when a serotype II and/or III capsular saccharide is used in the invention, it may be depolymerised as described in ref. 93. This document describes the partial depolymerization of type II and type III capsular saccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-D-mannose residues.

Capsular saccharides can be purified by known techniques, as described in the references herein such as ref. 94. A typical process involves base extraction, centrifugation, filtration,

R ase/DNase treatment, protease treatment, concentration, size exclusion chromatography, ultrafiltration, anion exchange chromatography, and further ultrafiltration. As an alternative, the purification process described in ref. 95 can be used. This process involves base extraction, ethanol/CaCl ₂ treatment, CTAB precipitation, and re-solubilisation.

The invention is not limited to saccharides purified from natural sources, however, and the saccharides may be obtained by other methods, such as total or partial synthesis.

Saccharides will typically be conjugated to a carrier protein. In general, covalent conjugation of saccharides to carriers enhances the immunogenicity of saccharides as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory.

Conjugation of GBS saccharides has been widely reported e.g. see refs. 96 to 103. The typical prior art process for GBS saccharide conjugation involves reductive amination of a purified saccharide to a carrier protein such as tetanus toxoid (TT) or CRM 197 [97]. The reductive amination involves an amine group on the side chain of an amino acid in the carrier and an aldehyde group in the saccharide. As GBS capsular saccharides do not include an aldehyde group in their natural form then this is typically generated before conjugation by oxidation {e.g. periodate oxidation) of a portion of the saccharide's sialic acid residues [97, 104]. Conjugate vaccines prepared in this manner have been shown to be safe and immunogenic in humans for each of GBS serotypes la, lb, II, III, and V [105].

Preferred carrier proteins are bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. These are commonly used in conjugate vaccines. A carrier protein in a conjugate may or may not be one of the GBS59 antigens of (1). If it is not a GBS59 antigen it may instead be a different GBS antigen. In some embodiments, though, the carrier is not a GBS antigen, and may be e.g. a bacterial toxin or toxoid.

Typical carrier proteins are diphtheria or tetanus toxoids or mutants thereof. Fragments of toxins or toxoids can also be used e.g. fragment C of tetanus toxoid [106]. The CRM 197 mutant of diphtheria toxin [107-109] is a particularly useful with the invention. Other suitable carrier proteins include N. meningitidis outer membrane protein complex [110], synthetic peptides [111,112], heat shock proteins [113,114], pertussis proteins [115,116], cytokines [117], lymphokines [118], hormones [118], growth factors, artificial proteins comprising multiple human CD4 ⁺ T cell epitopes from various pathogen-derived antigens [119] such as N19 [120], protein D from H. influenzae [121-123], iron-uptake proteins [124], toxin A or B from C. difficile [125], recombinant P. aeruginosa exoprotein A (rEPA) [126], etc. Where a composition includes more than one conjugate, each conjugate may use the same carrier protein or a different carrier protein.

In some embodiments, a single conjugate may carry saccharides from multiple serotypes [127]. Usually, however, each conjugate will include saccharide from a single serotype. Conjugates may have excess carrier (w/w) or excess saccharide (w/w). In some embodiments, a conjugate may include equal weights of each. For example, conjugates with a saccharide :protein ratio (w/w) of between 1 :5 and 5 : 1 may be used, in particular ratios between 1 :5 and 2: 1.

The carrier molecule may be covalently conjugated to the carrier directly or via a linker. Direct linkages to the protein may be achieved by, for instance, reductive amination between the saccharide and the carrier, as described in, for example, references 118 and 128. The saccharide may first need to be activated e.g. by oxidation. Linkages via a linker group may be made using any known procedure, for example, the procedures described in references 129 and 130. A preferred type of linkage is an adipic acid linker, which may be formed by coupling a free -NH ₂ group (e.g. introduced to a glucan by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate [131, 132]. Another preferred type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a saccharide CDI [133, 134] followed by reaction with a protein to form a carbamate linkage. Other linkers include β-propionamido [135], nitrophenyl-ethylamine [136], haloacyl halides [137], glycosidic linkages [138], 6-aminocaproic acid [139], ADH [140], C ₄ to C ₁₂ moieties [141], etc. Carbodiimide condensation can also be used [142].

Combinations with non-GBS antigens

The GBS67 polypeptides of the invention may be used in combination with non-GBS antigens. Thus the invention provides an immunogenic composition comprising a combination of:

(1) a GBS67 polypeptide of the invention as discussed above; and (2) one or more antigen(s) selected from the group consisting of: diphtheria toxoid; tetanus toxoid; one or more pertussis antigens; hepatitis B virus surface antigen; an inactivated polio virus antigen;; a conjugate of the capsular saccharide antigen from serogroup C of Neisseria meningitidis; a conjugate of the capsular saccharide antigen from serogroup Y of Neisseria meningitidis; a conjugate of the capsular saccharide antigen from serogroup W135 of Neisseria meningitidis; a conjugate of the capsular saccharide antigen from serogroup A of Neisseria meningitides; one or more influenza antigens; and one or more human papillomavirus antigens. Diphtheria toxoid can be obtained by treating (e.g. using formaldehyde) diphtheria toxin from Corynebacterium diphtheriae. Diphtheria toxoids are disclosed in more detail in, for example, chapter 13 of reference 143.

Tetanus toxoid can be obtained by treating (e.g. using formaldehyde) tetanus toxin from

Clostridium tetani. Tetanus toxoids are disclosed in more detail in chapter 27 of reference 143.

Pertussis antigens in vaccines are either cellular (whole cell, Pw) or acellular (Pa). The invention can use either sort of pertussis antigen. Preparation of cellular pertussis antigens is well documented (e.g. see chapter 21 of reference 143) e.g. it may be obtained by heat inactivation of phase I culture of B.pertussis. Acellular pertussis antigen(s) comprise specific purified

B.pertussis antigens, either purified from the native bacterium or purified after expression in a recombinant host. It is usual to use more than one acellular antigen, and so a composition may include one, two or three of the following well-known and well-characterized B.pertussis antigens: (1) detoxified pertussis toxin (pertussis toxoid, or 'PT'); (2) filamentous hemagglutinin ('FHA'); (3) pertactin (also known as the '69 kiloDalton outer membrane protein'). FHA and pertactin may be treated with formaldehyde prior to use according to the invention. PT may be detoxified by treatment with formaldehyde and/or glutaraldehyde but, as an alternative to this chemical detoxification procedure, it may be a mutant PT in which enzymatic activity has been reduced by mutagenesis [144]. Further acellular pertussis antigens that can be used include fimbriae (e.g. agglutinogens 2 and 3). Hepatitis B virus surface antigen (HBsAg) is the major component of the capsid of hepatitis B virus. It is conveniently produced by recombinant expression in a yeast, such as a

Saccharomyces cerevisiae.

Inactivated poliovirus (IPV) antigens are prepared from viruses grown on cell culture and then inactivated (e.g. using formaldehyde). Because poliomyelitis can be caused by one of three types of poliovirus, as explained in chapter 24 of reference 143, a composition may include three poliovirus antigens: poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain).

When a composition includes one of diphtheria toxoid, tetanus toxoid or an acellular pertussis antigen in component (2) then it will usually include all three of them i.e. component (2) will include a D-T-Pa combination. When a composition includes one of diphtheria toxoid, tetanus toxoid or a cellular pertussis antigen in component (2) then it will usually include all three of them i.e. component (2) will include a D-T-Pw combination.

Human papillomavirus antigens include LI capsid proteins, which can assemble to form structures known as virus-like particles (VLPs). The VLPs can be produced by recombinant expression of LI in yeast cells {e.g. in S.cerevisiae) or in insect cells {e.g. in Spodoptera cells, such as S.frugiperda, or in Drosophila cells). For yeast cells, plasmid vectors can carry the LI gene(s); for insect cells, baculo virus vectors can carry the LI gene(s). More preferably, the composition includes LI VLPs from both HPV- 16 and HPV- 18 strains. This bivalent combination has been shown to be highly effective [145]. In addition to HPV- 16 and HPV- 18 strains, it is also possible to include LI VLPs from HPV-6 and HPV-11 strains to give a tetravalent combination.

Influenza antigens may be in the form of currently an influenza virus vaccine. Various forms of influenza virus vaccine are currently available {e.g. see chapters 17 & 18 of reference [143]). Vaccines are generally based either on live virus, inactivated virus, recombinant hemagglutinin or virosomes. Inactivated vaccines may be based on whole virions, split virions, or on purified surface antigens. The antigen in vaccines of the invention may take the form of a live virus or, more preferably, an inactivated virus. The vaccine can be, for instance, a trivalent vaccine {e.g. including hemagglutinin from a A/H1N1 strain, a A/H3N2 strain and a B strain). In other embodiments the vaccine is a monovalent vaccine {e.g. including hemagglutinin from a A/H1N1 strain or a A/H5N1 strain). The vaccine can be adjuvanted {e.g. with an oil-in-water emulsion) or unadjuvanted.

Human papillomavirus antigens are in the form of hollow virus-like particles (VLPs) assembled from recombinant HPV coat proteins, typically from HPV types 16 and 18, and optionally also from HPV types 6 and 1 1.

Antibodies

Antibodies against GBS antigens can be used for passive immunisation [146]. Thus the invention provides a combination of antibodies for simultaneous, separate or sequential administration, wherein the combination includes at least two of: (a) an antibody which recognises a first amino acid sequence as defined above; (b) an antibody which recognises a second amino acid sequence as defined above; and/or (c) an antibody which recognises a third amino acid sequence as defined above; The invention also provides the use of such antibody combinations in therapy. The invention also provides the use of such antibody combinations in the manufacture of a medicament. The invention also provides a method for treating a mammal comprising the step of administering to the mammal an effective amount of such a combination. As described above for immunogenic compositions, these methods and uses allow a mammal to be protected against GBS infection.

The term "antibody" includes intact immunoglobulin molecules, as well as fragments thereof which are capable of binding an antigen. These include hybrid (chimeric) antibody molecules [147, 148]; F(ab')2 and F(ab) fragments and Fv molecules; non-covalent heterodimers [149, 150]; single-chain Fv molecules (sFv) [151]; dimeric and trimeric antibody fragment constructs; minibodies [152, 153]; humanized antibody molecules [154-156]; and any functional fragments obtained from such molecules, as well as antibodies obtained through non-conventional processes such as phage display. Preferably, the antibodies are monoclonal antibodies. Methods of obtaining monoclonal antibodies are well known in the art. Humanised or fully-human antibodies are preferred. 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 34, 35,157-162, 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.

The term "consisting of means "consisting only of. A composition "consisting of X" may not include any other components. A composition "consisting essentially of X" may not include any other active components. The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure. 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 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.

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

The term "fragment" may be used to refer to a nucleic acid or amino acid sequence which has been separated from sequences which flank it in a naturally occurring state e.g. a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, for example, the sequences adjacent to the fragment in a genome in which it naturally occurs or e.g. a portion of an amino acid sequence which has been removed from the full length polypeptide sequence and lacks sequences normally adjacent to the N and/or C terminus.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

Antibodies will generally be specific for their target. Thus they will have a higher affinity for the target than for an irrelevant control protein, such as bovine serum albumin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Alignment of GBS67 from 2603 (SAG1408) and H37B (SAI1512) showing location of fragment 1 (Frl), fragment 2 (Fr2) and fragment 3 (Fr3).

Figure 2: Purification of fragment 1 (Frl), fragment 2 (Fr2) and fragment 3 (Fr3) from 2603 (Figure 2 A) and H36B (Figure 2B)

Figure 3: Polyclonal antibodies raised against GBS67 2603 (Figure 3A) and GBS67 H36B (Figure 3B) recognise fragment 3 (Fr 3) from both variants in a Western blot analysis but not fragment 2 (Fr 2) or fragment 1 (Fr 1).

Figure 4: Antibodies against Fragment 1 from 2603 only recognize recombinant Fragment 1 from 2603 variant (1 2603) and full-length GBS67 from 2603 variant (FL 2603) in a Western blot analysis. Full-length GBS67 from H36B (FL H36B), fragments 1, 2 and 3 from H36B and fragments 2 and 3 from 2603 not recognized. Figure 5A: Antibodies against Fragment 2 from 2603 recognize recombinant Fragment 2 from 2603 variant (2 2603) and H36B variant (2 2603), as well as full-length GBS 67 from 2603 variant (FL 2603) in a Western blot analysis.

Figure 5B: Antibodies against Fragment 3 from 2603 recognize recombinant Fragment 3 from 5 2603 variant (3 2603) and H36B variant (3 2603), as well as full-length GBS 67 from 2603 variant (FL 2603) and full-length GBS67 from H36B (FL H36B) in a Western blot analysis.

Figure 6: Structural organisation and sequence variability of RrgA. (A) Protein sequence domain organization of RrgA clade I (aa 1 to 893). The leader peptide (LP), the CWSS, and the two intramolecular isopeptide bonds (K191-N695, K742-N854) are indicated. (B) The

0 organisation of the domains is displayed on the crystal structure of TIGR4 RrgA clade I.

Figure 7: The location of the domains identified on GBS67 following RrgA homology modeling. Dl IgG-like fold, D2 vWB domain and D3 IgG-like fold are Fragment 1, Fragment 2 and Fragment 3 from Figure 1.

MODES FOR CARRYING OUT THE INVENTION 5 GBS67 variants are cross-protective

Two allelic variants of GBS67 (API -2a) have been identified, one in GBS strain 2603 and one in GBS strain H36B. The GBS67 strain identified in GBS strain 2603 is predominant, being the variant that is present in 87% of GBS strains.

Either of these two GBS67 variants is capable of conferring cross-protection against GBS strains0 expressing the other GBS67 variant. For example, as shown in Table 1 below, the pups of female mice immunized with GBS67 (API -2a) from the 2603 strain are protected against challenge with GBS strains expressing either the 2603 or the H36B variant of GBS67.

Table 1: GBS67 confers cross-protection in GBS mouse maternal immunization/pup challenge model

An investigation was conducted to identify the portions of the GBS67 variants that are responsible for cross-protection. Identification of three fragments of GBS67

No crystal structure is available for API -2a (GBS67). An in silico analysis of the secondary structure of the GBS67 variants from 2603 and H36B identified three putative conserved fragments that might be responsible for cross-protective activity of GBS67 (see Table 2a). The sequences of equivalent fragments in other GBS67 strains that might be responsible for cross- protective activity is shown in Table 2b.

Table 2a: Conserved fragments of GBS67 variants

Fragment Amino SEQ ID NO Number of Theoretical MW

acid amino acid (kDa)

residues residues

Fragment 1 2603 24-217 2 194 23.5

Fragment 2 2603 218-615 3 398 47.3

Fragment 3 2603 616-866 4 251 30.4

Fragment 1 H36B 24-217 6 194 24 Fragment 2 H36B 218-610 7 393 46.8

Fragment 3 H36B 611-861 8 251 30.5

Table 2b: Conserved fragments of GBS67 variants

An alignment of the GBS67 variants from Table 2a showing the location of the 3 fragments is shown in Figure 1.

These six fragments were cloned and expressed as His-tagged proteins in HK100 and

BL21(DE3) strains. All of the fragments were over-expressed and soluble. The fragments yields obtained are shown in Table 3 below and Figure 2 shows the purified fragments isolated on gels. Table 3a: Purification of GBS672603 fragments

Table 3b: Purification of GBS67 H36B fragments

Assessment of cross-protective activity of GBS67 fragments Polyclonal antibodies raised against the 2603 and H36B GBS67 variants were capable of recognizing fragment 3 from both variants in a Western Blot analysis (Figure 3), suggesting that fragment 3 contains the epitopes responsible for inducing cross-protection.

In a subsequent Western Blot analysis, antibodies raised against fragment 1 of the 2603 GBS67 variant only recognized recombinant fragment 1 from 2603 GBS67 and full-length GBS67 from 2603 (Figure 4). These antibodies did not recognize fragment 1 from the H36B GBS67 variant or full-length GBS67 from H36B. In contrast, antibodies raised against fragment 2 from 2603 GBS67 recognized fragment 2 from H36B GBS67 and full-length H36B GBS67 (Figure 5A) and antibodies raised against fragment 3 from 2603 GBS67 recognized fragment 3 from H36B GBS67 and full-length H36 GBS67 (Figure 5B). FACs analysis demonstrates that both fragments 2 and 3 of GBS67 are highly exposed surface of the GBS bacterium (see Table 4 below).

Table 4:Surface exposure of fragments 2 and 3

Group Strain/serotype FACS Strain/serotype FACS

Exposure exposure

Fragment 1 2603 515(Ia) - 5401(11) - Fragment 2 2603 515(Ia) ++ 5401(11) +++

Fragment 3 2603 515(Ia) ++ 5401(11) +++

GBS67 2603 515(Ia) ++ 5401(11) +++

GBS67 H36B 515(Ia) ++ 5401(11) +++

The ability of fragments 1, 2 and 3 to induce cross-protection was then tested in vivo in a maternal immunization model. Female mice were immunized with fragment 1, 2 or 3 from GBS67 2603, with full-length GBS67 from 2603 or H36B, or with PBS. Pups were then challenged with the 5401 GBS strain expressing the GBS67 H36B variant. The results are shown in Table 5 below.

Table 5: Maternal immunization model results

These results show that fragment 3 of the GBS67 2603 variant is able to confer the same cross- protection against 5401 GBS strain expressing the GBS67 H36B variant as full-length GBS67 2603 or full-length GBS67 H36B. The ability of fragments 1, 2 and 3 of GBS67 2603 to induce protection against challenge with the 515 GBS strain expressing the GBS 67 2603 variant was confirmed by repeating the experiment described above except that pups were challenged with the 515 GBS strain, instead of the 5401 GBS strain. The results are shown in Table 6 below:

Table 6: Maternal immunization model results

Group Antigen Dead/treated % survival

1 Fragment 1 (2603) 34/39 12

2 Fragment 2 (2603) 31/64 52 3 Fragment 3 (2603) 28/40 30

4 GBS67 (2603) 16/58 72

6 PBS 51/57 10

In a further experiment, female mice were immunized with fragment 1, 2 or 3 from GBS67 H36B, with full-length GBS67 from H36B, or with PBS. Pups were challenged with the 5401 GBS strain expressing the GBS67 H36B variant. The results are shown in Table 7 below. Table 7: Maternal immunization model results

Identification of cross-protective domains of GBS67

In order to determine more precisely the location of domains responsible for cross-protection and domains that comprise epitopes responsible for cross-protection, homology modeling of GBS67 was performed. As discussed above, no crystal structure is available for GBS67. Therefore, GBS67 was aligned to RrgA (S. pneumoniae) in order to determine the location of different domains through homology modeling.

RrgA is the major determinant of in vitro adhesion associated with pilus 1 of S. pneumoniae, is protective in vivo in mouse models, and exists in two variants (clades I and II). The high- resolution crystal structure of the recombinant form of RrgA clade I has been published (163), and it describes a flexible molecule organized into four nested structural domains (Domain 1 to Domain 4). Domain 1 and Domain 2 comprise elements from both N- and C- terminal regions. Domain 1 comprises 5 β-strands and is an IgG domain. Domain 2 comprises 11 β- strands and is a CnaB-like domain. It also comprises an isopeptide bond spanning its N- and C- terminal regions. Domain 3 is vWA factor domain and it comprises 2 extensions. Domain 4 comprises 8 β-strands and is an IgG domain. Domain 4 also comprises an isopeptide bond (164) (see Figure 6).

Analysis of these domains has revealed that the sequence variations between the two variants of RrgA cluster in the distal "head" domain - Domain 3, whereas Domain 1 , Domain 2 and Domain 4, which form the stalk of the molecule, are well conserved. The isopeptide bond within Domain 4 has been shown to be important for resistance to proteolytic cleavage. Antisera raised against a C-terminal fragment of RrgA from TIGR4 (clade I) comprising Domain 4 and part of Domain 2 (amino acids 638-862) has been shown to be cross-reactive and to recognise the native pili of both TIGR4 (clade I) and SPEC-6B (clade II) in FACS analysis, whereas antisera raised against a N-terminal fragment from TIGR4 comprising most of Domain 1 and part of Domain 2 (amino acids 39-214) were always FACS negative. This demonstrates that the N-terminal of RrgA is not accessible once RrgA is incorporated into the native pilus fiber, because antisera raised against the N-terminal fragment is reactive in western blots (164).

RrgA has 38.9% sequence identity with GBS67 and 57.1% sequence similarity. Despite this relatively low identity, it was possible to perform homology modeling of the GBS67 515 variant. The location of the four domains (Domain 1 to Domain 4) are shown in Figure 7 in relation to the three fragments characterised above (Fragment 1 , Fragment 2 and Fragment 3 are labeled as Dl IgG-like fold, D2 vWB domain and D3 IgG- like fold). Modeling reveals that the isopeptide bond in Domain 4 of RrgA is present in Domain 4 of GBS67 between Lys742 and Asn850. This reveals that that Domain 4 resides within Fragment 3, which has been shown above to be capable of providing cross-protection against GBS67 challenge of different strains. As a fragment comprising Domain 4 of RrgA has been shown to be accessible in the native pilus fiber and to elicit cross-reactive antisera (as discussed above), Domain 4 of GBS67 is able to provide cross-protection against GBS67 challenge of different strains and Domain 4 contains epitopes that are responsible for cross-protection.

Domain 4 of GBS67 2603 and epitopes within this fragment may therefore be used in

immunogenic compositions instead of full-length GBS67 2603 or full-length GBS67 H36B. Similarly, Domain 4 of GBS67 H36B and epitopes within this fragment may be used in immunogenic compositions instead of full-length GBS67 2603 or full-length GBS67 H36B. Similarly, Domain 4 of GBS67 515 and epitopes within this fragment may be used in

immunogenic compositions instead of full-length GBS67 2603, full-length GBS67 H36B or full length GBS67 515. Immunization studies with GBS67 Domain 4 constructs

A range of Domain 4 polypeptides consisting of SEQ ID NO: 80 or SEQ ID NO:81, consisting of fragments of SEQ ID NO:80 or SEQ ID NO:81, or having at least 70% sequence identity to such polypeptides are tested in immunization studies such as the maternal immunization protocol described above. The polypeptides are found to provide protection against lethal challenge with a number of different GBS strains.

Materials and Methods

Bioinformatics

The complete genome sequences of Streptococcus agalactiae strains 2603 V/R (V) and H36B (lb) are available under Accession Numbers AE009948 and AAJSOOOOOOOO. Pairwise sequence alignment was obtained by ClustalW algorithm.

In order to identify the putative architecture, we used Pfam program connected to NCBI-BLAST database. Secondary structure prediction was performed using PsiPred (Protein Structure Prediction Server; UCL Bioinformatic Group) software. Bacterial strains and growth conditions

The GBS strains used in this work were 2603 V/R (serotype V), 515 (la), H36B (serotype lb) and 5401 (II). Bacteria were grown at 37°C in Todd Hewitt Broth (THB; Difco Laboratories) or in trypticase soy agar supplemented with 5% sheep blood.

Cloning, expression, purification of recombinant proteins and antisera GBS strains 2603 and H36B were used as source of DNA for cloning the sequences coding for the single fragments (fragments 1, 2 and 3) of GBS67 2603 and H36B allelic variants. Genomic DNA was isolated by a standard protocol for gram-positive bacteria using a NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer's instructions. Genes corresponding to each domain were cloned in the SpeedET or pET15-TEV vectors (N-terminal 6xHIS tag) by PIPE cloning method in E. coli HK100 strain (165). The oligos used are listed in Table 6. The resulting construct in pET15-TEV was checked for sequencing and then transformed into E. coli BL21(DE3) (Novagen). For the recombinant protein expression, the cultures were maintained at 25°C for 5h after induction with ImM IPTG for the pET clone or with 0.2% arabinose for the SpeedET clones. All recombinant proteins were purified by affinity chromatography. Briefly, cells were harvested by centrifugation and lysed in "lysis buffer", containing lOmM imidazole, lmg\ml lysozyme, 0.5 mg\ml DNAse and COMPLETE inhibitors cocktail (Roche) in PBS. The lysate was clarified by centrifugation and applied onto His-Trap HP column (Armesham

Biosciences) pre-equilibrated in PBS containing lOmM imidazole. Protein elution was performed using the same buffer containing 250mM imidazole, after two wash steps using 20mM and 50mM imidazole buffers. Protein concentration of the pure fractions was estimated using BCA assay (PIERCE).

Antisera specific for each protein were produced by immunizing CD1 mice with the purified recombinant proteins as previously described [166]. Protein-specific immune responses (total Ig) in pooled sera were monitored by ELISA.

The full length recombinant GBS67 proteins, corresponding to 2603 and H36B allelic variants (TIGR annotation SAG 1408 and SAI 1512, respectively), were produced as previously reported [2, 166].

Immunoblotting

10 ng of each purified protein were separated by 4-12% NuPage No vex pre-cast gels

(Invitrogen) and electroblotted onto nitrocellulose membranes using the iBlot™ Dry Blotting System (Invitrogen). After blocking in IX phosphate-buffered saline (PBS: 140 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04 and 1.8 mm KH2P04, pH 7.3) containing 0.05% Tween 20 and 10% skim milk for 1 h at room temperature, membranes were incubated for 1 h at room temperature (RT) with primary antibodies diluted 1 :500. After washing three times in PBS containing 0.05% Tween 20 (PBST), the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Dako). Positive bands were visualized with the Opti-4CN Substrate Kit (Bio-Rad).

ELISA

Antigen-specific antibody responses were detected by ELISA, by using of lOOng of purified recombinant antigens per well. IgG antibody titers were calculated by comparing the response curve of test serum samples with that of reference serum samples by using a reference line calculation program. The reference serum samples were a pool of serum samples obtained from mice immunized with the purified recombinant antigen, to which an arbitrary titer of 150,000 EU/mL was assigned.

FACS Mouse sera raised against purified recombinant proteins were analyzed on whole bacteria by flow cytometry to evaluate the surface-exposure of the single domains. Exponential phase bacterial cells were fixed in the presence of 0.08% (wt/vol) paraformaldehyde and incubated for 1 h at 37 °C. Fixed bacteria were then washed once with PBS, resuspended in Newborn Calf Serum (Sigma) and incubated for 20 min. at 25°C. The cells were then incubated for 1 hour at 4 °C in pre-immune or immune sera, diluted 1 :200 in dilution buffer (PBS, 20% Newborn Calf Serum, 0.1% BSA). Cells were washed in PBS-01% BSA and incubated for a further 1 h at 4 °C with a 1 : 100 dilution of R-Phycoerythrin conjugated F(ab)2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; Inc.),. After washing, cells were resuspended in PBS and analyzed with a FACS Calibur apparatus (Becton Dickinson, Franklin Lakes, NJ) using Flow Jo Software (Tree Star, Ashland, OR). Data are expressed as the difference in fluorescence between cells stained with immune sera versus pre-immune sera. Mouse active maternal immunization model

A maternal immunization/neonatal pup challenge model of GBS infection was used to verify the protective efficacy of the produced proteins in mice, as previously described [166]. Briefly, CD- 1 female mice (6-8 weeks old) were immunized on days 1 (in CFA), 21 and 35 (IFA) with either PBS or 20 mg of recombinant protein and were then bred 3 days after the last immunization. Within 48 h of birth, pups were injected intraperitoneally with a dose of different GBS strains calculated to cause 90%> lethality. Survival of pups was monitored for 2 days after challenge. Statistical analysis was performed using Fisher's exact test. All animal studies were performed according to guidelines of the Istituto Superiore di Sanita (Italy). Table 6: Primers used to clone GBS67 fragments

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