WO2012072769A1

WO2012072769A1 - Pneumococcal rrgb epitopes and clade combinations

Info

Publication number: WO2012072769A1
Application number: PCT/EP2011/071566
Authority: WO
Inventors: Vega Masignani; Monica Moschioni; Werner Pansegrau
Original assignee: Novartis Ag
Priority date: 2010-12-01
Filing date: 2011-12-01
Publication date: 2012-06-07

Abstract

Epitopes that give rise to protective antibodies have been identified at residue numbers 40 to 59 (in Domain 1) and 494 to 508 (in Domain 4) of the RrgB pilus protein. Immunogenic compositions comprising these epitopes and functionally equivalent sequences are provided. Pneumococcal pilus subunit RrgB has at least three clades. Serum raised against a given clade is active against pneumococci which express that RrgB clade, but is not active against strains which express one of the other two clades i.e. there is intra-clade cross-protection, but not inter-clade cross-protection. Thus an immunogenic composition can include epitopes from more than one different clade of RrgB to improve strain coverage against pilus-containing pneumococci. These multiple clades may be present in the immunogenic composition as separate polypeptides or may be fused as a single polypeptide chain.

Description

PNEUMOCOCCAL RrgB EPITOPES AND CLADE COMBINATIONS TECHNICAL FIELD

This invention is in the field of immunising against Streptococcus pneumoniae (pneumococcus). BACKGROUND OF THE INVENTION

S.pneumoniae has a pilus known as pilus-1 encoded by a 14-kb islet (PI-1) having seven genes encoding: the RlrA transcriptional regulator, three pilus subunits with LPXTG-type cell wall sorting signals, and three sortase enzymes involved in synthesis of the pilus polymer and in the incorporation of ancillary pilus components. RrgB is the major subunit that forms the backbone of the structure, while the other two pilins (RrgA, RrgC) are ancillary structural proteins [1 -4]. RrgA is the major pilus-1 adhesin; bacteria lacking RrgA are less adherent to epithelial cells than wild-type organisms.

SUMMARY OF THE INVENTION

The present invention relates, in a first aspect, to an immunogenic composition comprising:

(a) a first amino acid sequence, where the first amino acid sequence comprises or consists of: SEQ ID NO.100, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 100, or an amino acid sequence that competes with SEQ ID NO.100 for binding to an antibody raised against SEQ ID NO.100, or a fragment of at least 7 amino acids of SEQ ID NO.100; and/or

(b) a second amino acid sequence, where the second amino acid sequence comprises or consists of: SEQ ID NO.101 , or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 101, or an amino acid sequence that competes with SEQ ID NO.101 for binding to an antibody raised against SEQ ID NO.101, or a fragment of at least 7 amino acids of SEQ ID NO.101 ; and/or

(c) a third amino acid sequence, where the third amino acid sequence comprises or consists of: SEQ ID NO.102, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 102, or an amino acid sequence that competes with SEQ ID NO.102 for binding to an antibody raised against SEQ ID NO.102, or a fragment of at least 7 amino acids of SEQ ID NO.102; and/or

(d) a fourth amino acid sequence, where the fourth amino acid sequence comprises or consists of: SEQ ID NO.103, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 103, or an amino acid sequence that competes with SEQ ID NO.103 for binding to an antibody raised against SEQ ID NO.103, or a fragment of at least 7 amino acids of SEQ ID NO.103; and/or

(e) a fifth amino acid sequence, where the fifth amino acid sequence comprises or consists of: SEQ ID NO.104, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 104, or an amino acid sequence that competes with SEQ ID NO.104 for binding to an antibody raised against SEQ ID NO.104, or a fragment of at least 7 amino acids of SEQ ID NO.104; and/or

(f) a sixth amino acid sequence, where the sixth amino acid sequence comprises or consists of: SEQ ID NO.105, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 105, or an amino acid sequence that competes with SEQ ID NO.105 for binding to an antibody raised against SEQ ID NO.105, or a fragment of at least 7 amino acids of SEQ ID NO.105.

In a second aspect, the present invention relates to a polypeptide comprising a first, second, third, fourth, fifth and/or sixth amino acid sequence as defined above.

The present invention also relates, in a third aspect, to a polypeptide comprising amino acid sequence:

A-{-X-L-}_n-B

wherein: each X is an amino acid sequence of first polypeptide, second polypeptide, third polypeptide, fourth polypeptide, fifth polypeptide or sixth polypeptide as defined in the first aspect; L is an optional linker amino acid sequence; A is an optional N terminal amino acid sequence; B is an optional C terminal amino acid sequence; n is an integer of 2 or more.

The present invention further relates to a bacterium which expresses a polypeptide according to the second or third aspect.

The present invention also relates to an antibody that binds to a polypeptide according to the second or third aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows results of a bacteremia study with five RrgB chimeras and a control. The figures are CFU/ml. Each mark shows data for a single mouse.

Figure 2 shows results of a mortality study with five RrgB chimeras and a control. The figures are survival time in days. Each mark shows data for a single mouse. Figure 3 shows a gel with four lanes. From left to right the lanes contain: MW markers; a Ι-ΙΙ-ΠΙ chimera; a I-II-III chimera; and a BSA standard. The arrow indicates a MW of 214kDa.

Figure 4A shows passive protection data using four mAbs raised against the TIGR4 RrgB, or a saline control. The y-axis shows CFU/ml for 24 hour bacteremia. Figure 4B shows results of a mortality study with the four mAbs raised against the TIGR4 RrgB.

Figures 5 and 6 show western blots using mAbs raised against the TIGR4 sequence (Figure 5) or the 6B sequence (Figure 6). Lanes are, from left to right: marker; RrgB Ι-ΙΙ-ΠΙ; RrgB ΙΙ-Ι-ΙΠ; RrgB Π- III-I; RrgB ΙΠ-Ι-ΙΙ; RrgB III-II-I; RrgB TIGR4; RrgB 6B; RrgB 23F; BSA control.

Figure 7 shows (A) bacteremia and (B) mortality data after immunisation with alum-adjuvanted Ι-Π- III chimera, III-II-I chimera, TIGR4 or alum alone. In Figure 7A the data are CFU/ml and in Figure 7B the data are survival time in days.

Figure 8 shows OPKA results against TIGR4 strain, showing % OPKA killing against serum dilution. Diamonds show positive control sera; pre-immune sera are filled boxes, visible near the x-axis; the other five lines are for sera raised against the five chimeras.

Figure 9 shows a tree based on % identity for SEQ ID NOs: 1-3 & 85-96. The label is the SEQ ID.

Figure 10 shows OPKA results against S.pneumoniae serotype 6B, showing % killing against serum dilution.

Figure 11 shows shows OPKA results against S.pneumoniae serotype 6B, showing % killing against serum dilution up to a dilution of 1/131220.

Figure 12 shows (A) bacteremia and (B) mortality data after immunisation with ΙΠ-ΙΙ-Ι chimera at different doses. In Figure 12A the data are CFU/ml and in Figure 12B the data are survival time in days.

Figure 13 shows (A) bacteremia and (B) mortality data after immunisation with 20μg RrgB ΠΙ-ΙΙ-Ι chimera. In Figure 13 A the data are CFU/ml and in Figure 13B the data are survival time in days. Figure 14 demonstrates that the ΙΠ-ΙΙ-Ι RrgB chimera is protective using the MF59 adjuvant. Diamonds show adjuvanted RrgB chimera, circles show MF59 alone.

Figure 15 shows (A) bacteremia and (B) mortality data after subcutaneous immunisation with RrgB III-II-I chimera. In Figure 15A the data are CFU/ml and in Figure 15B the data are survival time in days. Figure 16 shows that RrgB ΠΙ-ΙΙ-Ι chimera elicits production of functional antibodies in a passive protection study, compared to a Normal Rabbit Serum (NRS) control, in a 24hour bacteremia assay.

Figure 17 shows OPKA results against (A) TIGR4 and (B) ST35B, showing % killing against serum dilution. Diamonds show Anti-T4, circles show RrgB ΙΠ-ΙΙ-Ι chimera and squares show NSK.

Figure 18 shows OPKA results against TIGR4 strain, showimg that the OPA activity is specifically due to antibodies against RrgB III-II-I chimeras.

Figure 19 shows that single RrgB domains confer protection in vivo. Figure 19A shows active immunisation: Triangles show RrgB chimera, diamonds show Dl domain, squares show D4 domain and circles show Alum. Figure 19B shows that Anti-RrgB Dl and D4 sera are protective in passive serum transfer experiments. Bacteraemia (Figure 19B, left panel): circles represent the Log CFU per ml of blood for individual animals; horizontal bars represent the mean value of the Log CFU/ml ± SEM for the group; the dotted line represents the detection limit (values under the dotted line correspond to animals in which no CFU were detected). Survival (Figure 19B, right panel): the survival course for each group is represented. ** P < 0.01; * P < 0.05

Figure 20 shows western blot analysis of different RrgB domains (single domains Dl, D2, D3 and D4 and multi-domain fragments Dl -3, D2-4, D3-4) tested for binding with each of four protective mAbs raised against TIGR4 RrgB.

Figure 21 shows a western blot analysis performed with monoclonal antibody 23F8/C10 binding to trypsin-digested RrgB.

Figure 22 (A) is a model of RrgB domain Dl amino acid sequence onto the domain 1 crystal structure of S.pyogenes pilus backbone Spy0128. (B) is S.pneumoniae RrgB crystal structure (D2- D3) and modelled Dl domain. (C) is a 3D reconstruction electron density map of the S.pneumoniae pilus.

Figure 23 shows (A) 48 hour bacteremia and (B) mortality data against 6B-Finland strain (i.v. challenge) after i.p. immunisation with RrgB III-II-I chimera when combined with different combinations of further polypeptide antigens (20μg antigens). In Figure 23A the data are CFU/ml and in Figure 23B the data are survival time in days. In both (A) and (B): column 1 shows a combination of spr0057, spr0096 and spr2021 ; column 2 shows a combination of SP2216-1 , SP 1732-3 and PsaA; column 3 shows RrgB ΙΠ-ΙΙ-Ι chimera; column 4 shows RrgB ΠΙ-ΙΙ-Ι chimera combined with spr0057, spr0096 and spr2021 ; column 5 shows RrgB ΠΙ-ΙΙ-Ι chimera combined with SP2216-1, SP1732-3 and PsaA; and column 6 shows an alum control. Figure 24 shows (A) 48 hour bacteremia and (B) mortality data against 35B-SME15 strain (i.v. challenge) after i.p. immunisation with RrgB III-II-I chimera when combined with different combinations of further polypeptide antigens (2C^g antigens). In Figure 24A the data are CFU/ml and in Figure 24B the data are survival time in days. In both (A) and (B): column 1 shows a combination of spr0057, spr0096 and spr2021 ; column 2 shows a combination of SP2216-1 , SP 1732-3 and PsaA; column 3 shows RrgB ΙΠ-ΙΙ-Ι chimera; column 4 shows RrgB ΠΙ-ΙΙ-Ι chimera combined with spr0057, spr0096 and spr2021 ; column 5 shows RrgB ΠΙ-ΙΙ-Ι chimera combined with SP2216-1, SP1732-3 and PsaA; and column 6 shows an alum control.

Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data using a ΙΠ-ΙΙ-Ι chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control (i.p. immunisation, i.p. challenge with TIGR4 2.1E+02 CFU/mouse). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 26 shows a 24 hour bacteremia assay in BALB/c mice using a ΠΙ-Π-Ι chimera that contains a polyhistidine tag compared to (i) a tag-less ΙΙΙ-Π-Ι chimera, (ii) a combination of spr0057, spr0096 and spr2021 , (iii) the combination of spr0057, spr0096 and spr2021 further combined with the tag- less ΠΙ-ΙΙ-Ι chimera, and (iv) an alum control (i.p. immunisation, i.p. challenge with TIGR4 1.6E+02 CFU/mouse).

Figure 27 shows (A) a 48 hour bacteremia assay and (B) mortality data using a ΙΠ-ΙΙ-Ι chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control (i.p. immunisation, i.v. challenge with 35B-SME15 4.6E+07 CFU/mouse). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 28 shows (A) a 48 hour bacteremia assay and (B) mortality data using a ΙΠ-ΙΙ-Ι chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control (i.p. immunisation, i.v. challenge with 6BFinlandl2 9.4E+07 CFU/mouse). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 29 shows (A) a 48 hour bacteremia assay and (B) mortality data using a ΙΠ-ΙΙ-Ι chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control (i.p. immunisation, i.v. challenge with TIGR4 6.3E+05 CFU/mouse). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 30 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with 20μg ΙΠ-ΙΙ-Ι chimera, compared to an alum control (i.p. immunisation, i.v. challenge with TIGR4). The data in (A) are CFU/ml and in (B) are survival time in days. Figure 31 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 2C^g ΙΠ-ΙΙ-Ι chimera, compared to an alum control (i.p. immunisation, i.p. challenge with TIGR4). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 32 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 2C^g ΠΙ-ΙΙ-Ι chimera, compared to an alum control (i.p. immunisation, i.v. challenge with 35B- SME15). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 33 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 2C^g ΠΙ-ΙΙ-Ι chimera, compared to an alum control (i.p. immunisation, i.v. challenge with 6B Finlandl2). The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 34 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with III- II-I chimera, compared to an alum control (i.p. immunisation, i.v. challenge with TIGR4) when challenged with a TIGR4 strain overexpressing pilus (T4+) compared to a TIGR4 train expressing very low amounts of pilus (T4-) . The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 35 shows 48 hour bacteremia assays after immunisation with II-I-III and ΙΠ-ΙΙ-Ι chimeras (A) when challenged with a 6BFinll2 strain overexpressing pilus (i.p. immunisation, i.v. challenge with 6BFinlandl2 overexpressing pilus 7.0E+09 CFU/mouse) and (B) when challenged with a 6BFinll2 train expressing only very low amounts of pilus (i.p. immunisation, i.v. challenge with 6BFinlandl2 underexpressing pilus 7.3E+09 CFU/mouse). Both (A) and (B) also show data for: a combination of spr0057, spr0096 and spr2021 ; a 6BFinland-CRMl 97 conjugate; and alum. The data in (A) are CFU/ml and in (B) are survival time in days.

Figure 36 is an in silico analysis of the MLST database showing that, for a collection of 113 Acute Otitis Media S.pneumoniae isolates, pilus- 1 is more prevalent in strains that are resistant to antibiotics (erythromycin-resistance, penicillin-resistance and multiple-drug-resistance) compared to strains that are susceptible to antibiotics.

Figure 37 shows that polyclonal antibodies raised against TIGR4 Dl and TIGR4 D4 domains recognize linear epitopes within RrgB. Glass fiber membranes with arrayed peptides synthesized in situ covering residues 25 to 190 (Dl) and 444 to 628 (D4) of RrgB were incubated with anti RrgB Dl (A) or RrgB D4 (B) polyclonal antibodies (1 :3000). Secondary goat anti-mouse IgG alkaline phosphatase conjugated antibodies (1 :5000) were used. Linear epitopes corresponding to peptide sequences recognized by the antibodies are reported. Underlining marks residues present in two peptides adjacent in the PepScan. Figure 38 shows a solution structure of the TIGR4 RrgB Dl domain. A: Ribbon diagram of RrgB Dl domain. Secondary structure elements are shown: β strands are shown in cyan, helix in red. B: Topology diagram of the RrgB Dl domain. The a-helix is represented by red cylinder and the β- strands are cyan arrows.

Figure 39 shows a superimposition of the Dl domain (blue) and the C. diphtheriae SpaA N-terminal domain (red). The position of Lys 190residue, which is engaged in the formation of inter-molecular isopeptide bonds between two different SpaA subunits is shown along with Lys 183 of RrgB, which occupies a similar position.

Figure 40 shows a Western blot using anti-D4 (TIGR4) polyclonal antibodies probed against various bacterial lysates (lanes 1 to 4) and recombinant proteins (lanes 6 to 9). Lanes 1 to 10 are, respectively: TIGR4 (clade I) lysate; 6BFinl2 (clade II) lysate; 35BSME15 (clade III) lysate; T4 Δ RrgB lysate; SeeBlue® Molecular Weight Marker; RrgB clade I His; RrgB clade II His; RrgB clade III His; Sprl 875 His; BSA

Figure 41 shows a Western blot using anti-Dl (TIGR4) polyclonal antibodies probed against various bacterial lysates (lanes 1 to 4) and recombinant proteins (lanes 6 to 9). Lanes 1 to 10 are, respectively: TIGR4 (clade I) lysate; 6BFinl2 (clade II) lysate; 35BSME15 (clade III) lysate; TIGR4 Δ RrgB lysate; SeeBlue® Molecular Weight Marker; RrgB clade I His; RrgB clade Π His; RrgB clade ΠΙ His; Sprl875 His; BSA.

Figure 42 shows Western blot for comparison with Figures 40 and 41, using anti-RrgB ΠΙ-ΙΙ-Ι chimera (lanes 1 to 4) and anti-RrgB TIGR4 (lanes 6 to 10) antibodies probed against various bacterial lysates (lanes 1 to 4) and recombinant proteins (lanes 6 to 9). Lanes 1 to 10 are, respectively: TIGR4 (clade I) lysate / anti-RrgB ΙΠ-ΙΙ-Ι; 6BFinl2 (clade II) lysate / anti-RrgB ΙΙΙ-Π- I; 35BSME15 (clade III) lysate / anti-RrgB ΠΙ-ΙΙ-Ι; TIGR4 Δ RrgB lysate / anti-RrgB III-II-I; SeeBlue® Molecular Weight Marker; RrgB clade I His / anti-RrgB TIGR4; RrgB clade Π His / anti- RrgB TIGR4; RrgB clade ΠΙ His / anti-RrgB TIGR4; Sprl875 His / anti-RrgB TIGR4; BSA / anti- RrgB TIGR4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of linear epitopes in the Dl and D4 domains of the S.pneumoniae RrgB protein. As detailed below, the RrgB protein has four domains, Dl, D2, D3 and D4. Immunisation with full length RrgB or with separate domains of RrgB provides protection in active immunisation experiments (see Figure 19). The Dl and D4 domains show the most significant protective efficacy and the epitopes identified in these domains are expected to be involved in the protective mechanism; the epitopes have been identified at residue numbers 40 to 59 (in the Dl domain) and at residue numbers 494 to 508 (in the D4 domain) of RrgB. The invention therefore provides an amino acid epitope sequence in each of the Dl and D4 domains of RrgB. These amino acid sequences can be used to generate an immune response against full-length RrgB. The identification of epitopes in RrgB allows polypeptides and immunogenic compositions to be provided that do not contain the full length RrgB sequence, and instead contain fragments of RrgB comprising the identified epitopes. These smaller fragments may be easier to produce and administer for therapeutic benefit, but retain the ability to generate an immune response against the full length RrgB protein. The invention therefore provides polypeptides and immunogenic compositions containing one or more of the identified epitope sequences.

The RrgB pilus subunit has at least three clades. Reference amino acid sequences for the three full length clades are SEQ ID NOs: 1, 2 and 3 herein. The clades are well conserved at their N- and C- termini but deviate in between. SEQ ID NOs: 1 and 2 are 46% identical; SEQ ID NOs: 1 and 3 are 51% identical; SEQ ID NOs: 2 and 3 are 65% identical. Epitopes have been identified at residue numbers 40 to 59 (in the Dl domain) and at residue numbers 494 to 508 (in the D4 domain) of RrgB. The epitopes in each of the three clades are identified in the following table:

Thus, in a first aspect the invention provides an immunogenic composition comprising:

(a) a first amino acid sequence, where the first amino acid sequence comprises or consists of: SEQ ID NO.100, or an amino acid sequence having at least a% sequence identity to SEQ ID

NO: 100, or an amino acid sequence that competes with SEQ ID NO.100 for binding to an antibody raised against SEQ ID NO.100, or a fragment of at least u contiguous amino acids from SEQ ID NO.100; and/or (b) a second amino acid sequence, where the second amino acid sequence comprises or consists of: SEQ ID NO.101, or an amino acid sequence having at least b% sequence identity to SEQ ID NO: 101 , or an amino acid sequence that competes with SEQ ID NO.101 for binding to an antibody raised against SEQ ID NO.101 , or an a fragment of at least v contiguous amino acids from SEQ ID NO.101 ; and/or

(c) a third amino acid sequence, where the third amino acid sequence comprises or consists of: SEQ ID NO.102, or an amino acid sequence having at least c% sequence identity to SEQ ID NO: 102, or an amino acid sequence that competes with SEQ ID NO.102 for binding to an antibody raised against SEQ ID NO.102, or a fragment of at least w contiguous amino acids from SEQ ID NO.102; and/or

(d) a fourth amino acid sequence, where the fourth amino acid sequence comprises or consists of: SEQ ID NO.103, or an amino acid sequence having at least d% sequence identity to SEQ ID NO: 103, or an amino acid sequence that competes with SEQ ID NO.103 for binding to an antibody raised against SEQ ID NO.103, or a fragment of at least x contiguous amino acids from SEQ ID NO.103; and/or

(e) a fifth amino acid sequence, where the fifth amino acid sequence comprises or consists of: SEQ ID NO.104, or an amino acid sequence having at least e% sequence identity to SEQ ID NO: 104, or an amino acid sequence that competes with SEQ ID NO.104 for binding to an antibody raised against SEQ ID NO.104, or a fragment of at least y contiguous amino acids from SEQ ID NO.104; and/or

(f) a sixth amino acid sequence, where the sixth amino acid sequence comprises or consists of: SEQ ID NO.105, or an amino acid sequence having at least f/o sequence identity to SEQ ID NO: 105, or an amino acid sequence that competes with SEQ ID NO.105 for binding to an antibody raised against SEQ ID NO.105, or a fragment of at least z contiguous amino acids from SEQ ID NO.105.

Serum raised against a given RrgB clade is active against pneumococci which express that clade, but is not active against strains which express one of the other two clades i.e. there is intra-clade cross-protection, but not inter-clade cross-protection. According to one embodiment of the invention, therefore, an immunogenic composition includes epitopes from at least two different clades of RrgB. As detailed in the Table above, SEQ ID NOs. 100 and 101 are from a first clade, SEQ ID NOs. 102 and 103 are from a second clade and SEQ K) NOs. 104 and 105 are from a third clade. Therefore, the first and second amino acid sequences of the invention, relating to SEQ ID NOs. 100 and 101 , are from a first clade. The third and fourth amino acid sequences of the invention, relating to SEQ ID NOs. 102 and 103, are from a second clade. The fifth and sixth amino acid sequences of the invention, relating to SEQ ID NOs. 104 and 105, are from a third clade.

An epitope according to the invention may be combined with an epitope, or a longer sequence containing multiple epitopes, from a different clade. The different clades may be present in the immunogenic composition as separate polypeptides or may be fused as a single polypeptide chain. The inclusion of multiple RrgB clades as vaccine components improves the strain coverage of the immunogenic composition against pilus-containing pneumococci. Furthermore, it has been observed that there is a significant association between pilus-1 presence and antibiotic resistance; this observation suggests that immunising against pilus-1 using an immunogenic composition including multiple RrgB clades will have the additional advantage of protecting against pneumococci that are resistant to antibiotic treatment.

Thus the invention provides a polypeptide comprising a first, second, third, fourth, fifth and/or sixth amino acid sequence as defined above in the first aspect.

The invention also provides a polypeptide comprising amino acid sequence:

-A-{-X-L-}„-B- wherein: X is an amino acid sequence of first amino acid sequence, second amino acid sequence, third amino acid sequence, fourth amino acid sequence, fifth amino acid sequence or sixth amino acid sequence as defined above; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; n is an integer of 2 or more {e.g. 2, 3, 4, 5, 6, etc.). Optionally, the polypeptide comprises at least two of a first, second third, fourth, fifth and sixth amino acid sequence as defined above. Usually n is 2 or 3, and X moieties are selected from the following:

N Xl X₂ X₃

2 First or Second amino acid Third or Fourth amino acid - sequence sequence

2 Third or Fourth amino acid First or Second amino acid - sequence sequence

3 First or Second amino acid Third or Fourth amino acid Fifth or Sixth amino acid

sequence sequence sequence

3 First or Second amino acid Fifth or Sixth amino acid Third or Fourth amino acid

sequence sequence sequence

3 Third or Fourth amino acid Fifth or Sixth amino acid First or Second amino acid

sequence sequence sequence

3 Third or Fourth amino acid First or Second amino acid Fifth or Sixth amino acid

sequence sequence sequence

3 Fifth or Sixth amino acid Third or Fourth amino acid First or Second amino acid

sequence sequence sequence

3 Fifth or Sixth amino acid First or Second amino acid Third or Fourth amino acid

sequence sequence sequence

In each of the combinations exemplified in the table above, the two alternatives for each instance of X_1; X₂ and X3 can optionally be combined, so that both of the recited alternative amino acid sequences are administered. For example, in the first line of the Table, Xi could contain the first and second amino acid sequence, and/or X₂ could contain the third and fourth amino acid sequence.

The invention also provides a cell (typically a bacterium, such as a pneumococcus) which expresses a first, second, third, fourth, fifth and/or sixth amino acid sequence as defined above in the first aspect.

The invention further provides an antibody that binds to: a polypeptide comprising a first, second, third, fourth, fifth and/or sixth amino acid sequence as defined above in the first aspect; or a polypeptide comprising amino acid sequence -A-{-X-L-}„-B- .

The first, second, third, fourth, fifth and sixth amino acid sequences

The value of a is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of b is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of d is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of e is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of/is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The values of a, b, c, d, e and / may be the same or different. In some embodiments, a, b, c, d, e and / are identical. Typically, a, b, c, d, e and / are at least 90 e.g. at least 95.

The value of u is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The value of v is at least 7 e.g. 8, 9, 10, 11, 12, 13 or 14. The value of w is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, or 14. The value of y is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, or 14. The values of u,v,w,x, y and z may be the same or different. In some embodiments, u,v,w,x, y and z are identical.

Fragments preferably comprise an epitope from the respective SEQ ID NO: sequence. Other useful fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, or more) from the N- terminus of the respective SEQ ID NO: while retaining at least one epitope thereof. Truncation by 1 - 5 amino acids at the N-terminus is convenient e.g. removal of aa 1-5 of any of SEQ ID NOs: 100 to 105.

Amino acid sequences within the scope of the invention typically compete with an identified epitope for binding to an antibody raised against the identified epitope; an amino acid sequence that competes with SEQ ID NO.100, 101, 102, 103, 104 or 105 for binding to an antibody raised against SEQ ID NO.100, 101, 102, 103, 104 or 105, respectively, is within the scope of the invention. Antibodies can readily be generated that bind to the SEQ ID NOs. identified herein, as detailed below. Determining whether a test amino acid sequence competes with SEQ ID NO. 100, 101, 102, 103, 104 or 105 for binding to the antibody can be readily determined using competition assay techniques known in the art for determining competition, including by equilibrium methods such as ELISA, kinetic methods such as BIACORE® and by flow cytometry methods. An amino acid sequence that competes with SEQ K) NO. 100, 101 , 102, 103, 104 or 105 for binding to an antibody will cause a reduction in the observed total binding of the SEQ ID NO. to the antibody when the test sequence is present, compared to when the test sequence is not present. Typically, this reduction in binding is 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, for example a reduction in binding of 70% or more in the presence of the test sequence compared to antibody binding observed for the SEQ ID NO. in the absence of the test sequence. First, second, third, fourth, fifth and sixth polypeptides comprise or consist of the first, second, third, fourth, fifth and/or sixth amino acid sequences, respectively. These polypeptides can consist of, i.e. contain only, the respective amino acid sequence or can contain additional amino acid residues or sequences. Typically, each of the first, second, third, fourth, fifth and sixth polypeptides consists of 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 30 or fewer, or 25 or fewer amino acid residues.

The RrgB protein can be split into four domains (Dl to D4) between its leader peptide and its LPXTG anchor. These four domains are as follows in SEQ ID NOs: 1 to 3, and the positions in further RrgB sequences which correspond to these residues can readily be identified by alignment:

Based on passive protection studies, useful fragments of RrgB may retain epitopes from at least domains Dl and/or D4. As shown in Figure 20, antibodies have been raised that bind to domain Dl , domain D4 and a fragment containing domains D2 to D4. Further, Figure 19A shows that active immunisation with individual Dl and D4 domains confers protection in vivo and Figure 19B shows that sera raised against Dl and D4 are also protective in both bacteraemia and survival experiments.

A polypeptide comprising the first or second amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 1 (strain TIGR4). In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 2 or to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 3.

A polypeptide comprising the third or fourth amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 2 (strain Finland^6B-12). In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 1 or to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 3.

A polypeptide comprising the fifth or sixth amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 3 (strain Taiwan 23F -15). In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 1 or to the wild-type pneumococcus protein having amino acid sequence SEQ ID NO: 2.

Although the first, third and fifth amino acid sequences may share some sequences in common, overall they have different amino acid sequences. Similarly, although the second, fourth and sixth amino acid sequences may share some sequences in common, overall they have different amino acid sequences.

Where the invention uses epitopes from only two RrgB clades a composition or polypeptide can include both: (a) a first and/or second amino acid sequence as defined above; and (b) a third and/or fourth amino acid sequence as defined above. In an alternative embodiment the composition includes both: (a) a first and/or second amino acid sequence as defined above; and (b) a fifth and/or sixth amino acid sequence as defined above. In an alternative embodiment the composition includes both: (a) a third and/or fourth amino acid sequence as defined above; and (b) a fifth and/or sixth amino acid sequence as defined above.

The invention therefore provides an immunogenic composition or polypeptide comprising at least one amino acid sequence selected from two or three of the following groups:

(a) the first and second amino acid sequence;

(b) the third and fourth amino acid sequence; and

(c) the fifth and sixth amino acid sequence.

An immunogenic composition or polypeptide according to the invention will therefore typically contain

two, three, four, five or six different amino acid sequences. Typically, amino acid sequences from two or three clades are present.

Amino acid sequences used with the invention, may, compared to SEQ ID NOs: 100, 101, 102, 103, 104 or 105 include one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e. replacements of one amino acid with another which has a related side chain. Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. The polypeptides may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to a reference sequence. The polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to a reference sequence.

A polypeptide used with the invention may comprise an amino acid sequence that:

(a) is identical (i.e. 100% identical) to SEQ ID NO: 100, 101, 102, 103, 104 or 105;

(b) shares sequence identity SEQ K) NO: 100, 101, 102, 103, 104 or 105;

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

(d) when aligned SEQ ID 100, 101 , 102, 103, 104 or 105 using a pairwise alignment algorithm, each moving window of x amino acids from N-terminus to C-terminus (such that for an alignment that extends to p amino acids, where p>x, there are p-x+1 such windows) has at least xy identical aligned amino acids, where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and if xy is not an integer then it is rounded up to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [5], 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 [6].

Within group (c), deletions or substitutions may be at the N-terminus and/or C-terminus, or may be between the two termini. Thus a truncation is an example of a deletion. Truncations may involve deletion of up to 5 (or more) amino acids at the N-terminus and/or C-terminus.

In general, when a polypeptide of the invention comprises a sequence that is not identical to a complete pneumococcal epitope sequence from SEQ ID NOs: 100, 101, 102, 103, 104 or 105 (e.g. when it comprises a sequence listing with <100% sequence identity thereto, or when it comprises a fragment thereof), it is preferred in each individual instance that the polypeptide can elicit an antibody that recognises the complete pneumococcal sequence.

For reference, SEQ ID NOs: 1 to 3 and 85 to 96 are 15 unique RrgB sequences which have been identified in 45 different strains. Any of these sequences can be used for implementing the invention.

Hybrid polypeptides

Different RrgB epitopes used in the invention do not have to be present as separate polypeptides but can instead be expressed 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 contain epitopes from a single RrgB clade or from multiple RrgB clades.

Hybrid polypeptides can include sequences from only RrgB antigens but in other embodiments can include non-RrgB antigens (usually pneumococcal non-RrgB antigens), such as other pilus subunits. If non-RrgB antigens are present these may be to the N-terminus of any two RrgB sequences, to the C-terminus of any two RrgB sequences, or may be between two RrgB sequences.

In one embodiment, a hybrid polypeptide according to the invention consists of 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 34 or fewer, or 33 or fewer amino acid residues.

Different hybrid polypeptides may be mixed together in a single formulation. Hybrids may be combined with non-hybrid RrgB antigens or other non-RrgB antigens.

Hybrid polypeptides may be represented by the formula NH₂-A-{-X-L-}„-B-COOH.

If a -X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of Xi will be retained, but the leader peptides of X₂ ... X_n will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-.

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 NH₂-Xi-Li-X₂-L₂-COOH, NH₂-Xi-X₂-COOH, NH₂-Xi-Li-X₂-COOH, NH₂-Xi-X₂-L₂-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. A useful linker is GSGGGG (SEQ ID NO: 7) or GSGSGGGG (SEQ ID NO: 8), with the Gly-Ser dipeptide being formed from a BamH 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, 11, 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, fJVIet, 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, 11 , 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, such as SEQ ID NO: 9), 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-transf erase, 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.

In some embodiments, a -L- moiety comprises a non-RrgB antigen. In some embodiments, the -A- moiety comprises a non-RrgB antigen, and in some the -B- moiety comprises a non-RrgB antigen.

The invention also provides nucleic acid which encodes a hybrid polypeptide of the invention. 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 [7,8]. Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [9] chemistry. Enzymatic synthesis [10] 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.) [1 1] . 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 hybrid polypeptide of the invention. The invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences. Such nucleic acids include those using alternative codons to encode the same amino acid.

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 12 & 239, etc.].

The invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers).

Nucleic acid 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 pneumococcal 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 pneumococcal 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

Mixtures 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.

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 234.

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, for example two, three, four 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: A. Mineral-containing compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides {e.g. oxyhydroxides), phosphates {e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 13], or mixtures of different mineral compounds, with the compounds taking any suitable form {e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt.

The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm ¹ and a strong shoulder at 3090-3100cm ¹ [chapter 9 of ref. 13]. The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology {e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pi of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate {i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO₄/AI molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm^"1 {e.g. when heated to 200°C) indicates the presence of structural hydroxyls [ch. 9 of ref. 13].

The PCVAl^3"1" molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO₄/AI molar ratio between 0.84 and 0.92, included at

3 +

0.6mg Al /ml. The aluminium phosphate will generally be particulate {e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20μηι (e.g. about 5-10μηι) after any antigen adsorption. Adsorptive capacities of between 0.7- 1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

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

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

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

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

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 13; see also ref. 14] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund' s adjuvant (CFA) and incomplete Freund' s adjuvant (IF A) may also be used.

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

220nm are preferred as they can be subjected to filter sterilization. The invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6, 10,15, 19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Other preferred oils are the tocopherols (see below). Oil in water emulsions comprising sqlauene are particularly preferred. Mixtures of oils can be used.

Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as tri ethyl eneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. As mentioned above, detergents such as Tween 80 may contribute to the thermal stability seen in the examples below. Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.

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

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

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

• An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer.

• An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an a-tocopherol succinate). The emulsion may include these three components at a mass ratio of about 75: 1 1 : 10 (e.g. 750μg/ml polysorbate 80, 110μg/ml Triton X-100 and 100μg/ml α-tocopherol succinate), and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

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

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

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

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

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

• An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [26].

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

C. Saponin formulations [chapter 22 of ref. 13]

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

Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21 , QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in ref. 27. Saponin formulations may also comprise a sterol, such as cholesterol [28].

Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexs (ISCOMs) [chapter 23 of ref. 13]. ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA & QHC. ISCOMs are further described in refs. 28-30. Optionally, the ISCOMS may be devoid of additional detergent [31].

A review of the development of saponin based adjuvants can be found in refs. 32 & 33. D. Virosomes and virus-like particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HTV, RNA-phages, QB-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi). VLPs are discussed further in refs. 34-39. Virosomes are discussed further in, for example, ref. 40

E. Bacterial or microbial derivatives

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

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 41. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22μηι membrane [41]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [42,43].

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in refs. 44 & 45.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. References 46, 47 and 48 disclose possible analog substitutions e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in refs. 49-54. The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [55]. The CpG sequence may be specific for inducing a Thl immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs. 56-58. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, refs. 55 & 59-61.

A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-3 1™ [62] . Thus an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g. between 15-40 nucleotides) including at least one (and preferably multiple) Cpl motifs (i.e. a cytosine linked to an inosine to form a dinucleotide), and (ii) a polycationic polymer, such as an oligopeptide (e.g. between 5-20 amino acids) including at least one (and preferably multiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC)i3-3' (SEQ ID NO: 80). The polycationic polymer may be a peptide comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 81).

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E.coli (E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 63 and as parenteral adjuvants in ref. 64. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 65-72. A useful CT mutant is or CT-E29H [73]. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 74, specifically incorporated herein by reference in its entirety.

F. Human immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1 , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [75], etc.) [76], interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor. A preferred immuno modulator is IL-12. G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [77] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [78].

H. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles {i.e. a particle of -lOOnm to ~150μηι in diameter, more preferably ~200nm to ~30μηι in diameter, and most preferably ~500nm to -ΙΟμιτι in diameter) formed from materials that are biodegradable and non-toxic {e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface {e.g. with SDS) or a positively-charged surface {e.g. with a cationic detergent, such as CTAB). L Liposomes (Chapters 13 & 14 of ref. 13)

Examples of liposome formulations suitable for use as adjuvants are described in refs. 79-81.

J Polyoxyethylene ether and polyoxyethylene ester formulations

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [82]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [83] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [84]. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4- lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23 -lauryl ether. K. Polwhosvhazene (PCPP)

PCPP formulations are described, for example, in refs. 85 and 86.

L. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-M D P ) , a n d N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-i^,«- glycero-3 -hydroxyphosphoryloxy)-ethylamine MTP-PE). M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues {e.g. "Resiquimod 3M"), described further in refs. 87 and 88.

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 [89]; (2) a saponin {e.g. QS21) + a non-toxic LPS derivative {e.g. 3dMPL) [90] ; (3) a saponin {e.g. QS21 ) + a non-toxic LPS derivative {e.g. 3dMPL) + a cholesterol; (4) a saponin {e.g. QS21 ) + 3 dMPL + IL-12 (optionally + a sterol) [91] ; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [92]; (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. 13.

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 Π 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 TH1 adjuvant and a TH2 adjuvant. Preferably, such a composition elicits an enhanced TH1 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 TH1 and a TH2 adjuvant elicits an increased TH1 and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a TH1 adjuvant alone or immunization with a TH2 adjuvant alone).

The immune response may be one or both of a TH1 immune response and a TH2 response. Preferably, immune response provides for one or both of an enhanced TH1 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.

Pneumococcal 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. 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.

Nucleic acid immunisation

The immunogenic compositions described above include polypeptide antigens from S. pneumoniae . In all cases, however, the polypeptide antigens can be replaced by nucleic acids (typically DNA) encoding those polypeptides, to give compositions, methods and uses based on nucleic acid immunisation [93 to 100].

The nucleic acid encoding the immunogen is expressed in vivo after delivery to a patient and the expressed immunogen then stimulates the immune system. The active ingredient will typically take the form of a nucleic acid vector comprising: (i) a promoter; (ii) a sequence encoding the immunogen, operably linked to the promoter; and optionally (iii) a selectable marker. Preferred vectors may further comprise (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). In general, (i) & (v) will be eukaryotic and (iii) & (iv) will be prokaryotic.

Preferred promoters are viral promoters e.g. from cytomegalovirus (CMV). The vector may also include transcriptional regulatory sequences (e.g. enhancers) in addition to the promoter and which interact functionally with the promoter. Preferred vectors include the immediate-early CMV enhancer/promoter, and more preferred vectors also include CMV intron A. The promoter is operably linked to a downstream sequence encoding an immunogen, such that expression of the immunogen-encoding sequence is under the promoter's control. Where a marker is used, it preferably functions in a microbial host (e.g. in a prokaryote, in a bacteria, in a yeast). The marker is preferably a prokaryotic selectable marker (e.g. transcribed under the control of a prokaryotic promoter). For convenience, typical markers are antibiotic resistance genes.

The vector is preferably an autonomously replicating episomal or extrachromosomal vector, such as a plasmid.

The vector preferably comprises an origin of replication. It is preferred that the origin of replication is active in prokaryotes but not in eukaryotes.

Preferred vectors thus include a prokaryotic marker for selection of the vector, a prokaryotic origin of replication, but a eukaryotic promoter for driving transcription of the immunogen-encoding sequence. The vectors will therefore (a) be amplified and selected in prokaryotic hosts without polypeptide expression, but (b) be expressed in eukaryotic hosts without being amplified. This arrangement is ideal for nucleic acid immunization vectors.

The vector may comprise a eukaryotic transcriptional terminator sequence downstream of the coding sequence. This can enhance transcription levels. Where the coding sequence does not have its own, the vector preferably comprises a polyadenylation sequence. A preferred polyadenylation sequence is from bovine growth hormone.

The vector may comprise a multiple cloning site. In addition to sequences encoding the immunogen and a marker, the vector may comprise a second eukaryotic coding sequence. The vector may also comprise an IRES upstream of said second sequence in order to permit translation of a second eukaryotic polypeptide from the same transcript as the immunogen. Alternatively, the immunogen- coding sequence may be downstream of an IRES.

The vector may comprise unmethylated CpG motifs e.g. unmethylated DNA sequences which have in common a cytosine preceding a guanosine, flanked by two 5' purines and two 3' pyrimidines. In their unmethylated form these DNA motifs have been demonstrated to be potent stimulators of several types of immune cell.

Vectors may be delivered in a targeted way. Receptor-mediated DNA delivery techniques are described in, for example, references 101 to 106. Therapeutic compositions containing a nucleic acid are administered in a range of about lOOng to about 200mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about ^g to about 2 mg, about 5μg to about 500μg, and about 20μg to about 100μg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g. for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy. Where greater expression is desired over a larger area of tissue, larger amounts of vector or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.

Vectors can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally references 107 to 1 10).

Viral-based vectors for delivery of a desired nucleic acid and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e.g. references 1 11 to 121), alphavirus-based vectors (e.g. Sindbis virus vectors, Sem ki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532); hybrids or chimeras of these viruses may also be used), poxvirus vectors (e.g. vaccinia, fowlpox, canarypox, modified vaccinia Ankara, etc.), adenovirus vectors, and adeno- associated virus (AAV) vectors (e.g. see refs. 122 to 127). Administration of DNA linked to killed adenovirus [128] can also be employed.

Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone [e.g. 128], ligand-linked DNA [129], eukaryotic cell delivery vehicles cells [e.g. refs. 130 to 134] and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in refs. 135 and 136. Liposomes (e.g. immunoliposomes) that can act as gene delivery vehicles are described in refs. 137 to 141. Additional approaches are described in references 142 & 143.

Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in ref. 143. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation [e.g. refs. 144 & 145]. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun [146] or use of ionizing radiation for activating transferred genes [144 & 145]. Delivery of DNA using PLG {poly(lactide-co-glycolide)} microparticles is a particularly preferred method e.g. by adsorption to the microparticles, which are optionally treated to have a negatively- charged surface (e.g. treated with SDS) or a positively-charged surface (e.g. treated with a cationic detergent, such as CTAB). 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 an immunogenic composition of the invention. 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 epitopes from at least two different RrgB clades for combined use as a medicament e.g. for use in raising an immune response in a mammal. Typically, epitopes from three different RrgB clades are combined for use as a medicament. As explained above, the first and second amino acid sequences of the invention, relating to SEQ ID NOs. 100 and 101, are from a first clade. The third and fourth amino acid sequences of the invention, relating to SEQ ID NOs. 102 and 103, are from a second clade. The fifth and sixth amino acid sequences of the invention, relating to SEQ ID NOs. 104 and 105, are from a third clade.

The invention also provides the use of epitopes from at least two different RrgB clades in the manufacture of a medicament for raising an immune response in a mammal. Typically, epitopes from three different RrgB clades are used 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 pneumococcal disease and/or infection e.g. against pneumococcal meningitis.

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

The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involves monitoring pneumococcal 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 pneumococcal infection, e.g., guinea pigs or mice. One such model is described in reference 147. 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 THl 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.). In one embodiment, multiple doses may be administered approximately 6 weeks, 10 weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and 14 weeks, as often used in the World Health Organisation's Expanded Program on Immunisation ("EPI"). In an altemative embodiment, two primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g. about 6, 8, 10 or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third primary dose, e.g. about 6, 8, 10, or 12 months after the third primary dose.

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 the elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g. <5 years old), hospitalised patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients. 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 measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.influenzae type b vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Y vaccine), a respiratory syncytial virus vaccine, etc.

Combinations

A composition useful for immunisation comprises an RrgB epitope identified herein. In a typical embodiment, a composition useful for immunisation comprises epitopes from at least two RrgB clades, typically three RrgB clades, either as a hybrid polypeptide or as separate polypeptides. In addition, a composition may include: (i) one or more further polypeptides that elicit antibody responses against pneumococcal proteins, particularly against pneumococcal proteins other than RrgB; (ii) a capsular saccharide from pneumococcus; and/or (iii) one or more further immunogens that elicit antibody responses that recognise epitopes on non-pneumococcal organisms. As detailed above, compositions of the invention comprising combinations such as these can optionally comprise one or more adjuvants, for example two or more adjuvants. Suitable adjuvants include mineral salts such as aluminium salts, and squalene-water emulsions such as MF59.

Combinations with further polypeptide antigens [148]

RrgB epitopes from one or more clades may be combined with one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13) polypeptide antigens selected from the group consisting of: (1) a spr0057 antigen; (2) a spr0565 antigen; (3) a sprl098 antigen; (4) a sprl416 antigen; (5) a sprl418 antigen; (6) a spr0867 antigen; (7) a sprl431 antigen; (8) a sprl739 antigen; (9) a spr2021 antigen; (10) a spr0096 antigen; (11) a sprl707 antigen; (12) a sprl875 antigen; and/or (13) a spr0884 antigen.

Similarly, RrgB epitopes from one or more clades may be combined with one or more (i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or all 20) polypeptide antigens selected from the group consisting of: (1) ClpP; (2) LytA; (3) PhtA; (4) PhtB; (5) PhtD; (6) PhtE; (7) ZmpB; (8) CbpD; (9) CbpG; (10) PvaA; (11) CPL1 ; (12) PspC; (13) PspA; (14) PsaA; (15) PrtA; (16) Spl33; (17) PiaA; (18) PiuA; (19) CbiO; and/or (20) 30S nbosomal protein S8.

These further antigens may be added as separate polypeptides. As an alternative, they may be added as hybrids e.g. a spr0057-spr0096 hybrid or a spr0096-spr2021 hybrid, a spr0565-PhtD hybrid, etc. As a further alternative, they may be fused to a RrgB epitope sequence to provide a hybrid polypeptide e.g. a RrgB-spr0057 hybrid.

For example, a chimeric RrgB polypeptide including epitopes from two or three RrgB clades may be combined with: (a) a mixture of spr0057, spr0096 and spr2021 ; (b) a mixture of spr0057, spr0565 and spr2021 ; (c) a mixture of spr0057, spr0096 and spr0565; (d) a mixture of spr0057, spr0096, spr0565 and spr2021 ; (e) a mixture of sprl418, spr0884 and spr0096; (f) a mixture of sprl418, spr0884 and spr2021 ; (g) a mixture of sprl418, spr0884, spr0096 and spr2021 ; (h) a mixture of spr0884, sprl416 and spr0057; (h) a mixture of spr0884, sprl416 and spr0096; (h) a mixture of spr0884, sprl416, spr0057 and spr0096; or (i) a mixture of sprl418, sprl431 and spr0565. Where these mixtures include both spr0057 and spr0096, a hybrid protein can be used e.g. comprising SEQ ID NO: 82 (see SEQ K) NO: 200 of ref. 148) or comprising SEQ K) NO: 83. Where these mixtures include both spr0096 and spr2021, a hybrid protein can be used e.g. comprising SEQ ID NO: 84 (see SEQ ID NO: 205 of ref. 148).

In a further example, a chimeric RrgB polypeptide including epitopes from two or three RrgB clades may be combined with a pneumococcal immunogen comprising an spr2021 (also referred to as SP2216) antigen, an SP1732 antigen and optionally a PsaA antigen. A suitable pneumococcal immunogen of this sort is the immunogen disclosed in reference 159 that comprises the antigens "SP2216-1" (SEQ ID NO: 1 in reference 159; SEQ ID NO: 97 herein), "SP 1732-3" (SEQ ID NO: 2 in reference 159; SEQ ID NO: 98 herein) and, optionally, PsaA (SEQ ID NO: 3 in reference 159; SEQ ID NO: 99 herein). Polypeptides comprising immunogenic fragments of these SEQ ID NOs can be used in place of the actual disclosed SEQ ID NOs e.g. comprising at least one immunogenic fragment from each of SEQ ID NOs 97 & 98. Polypeptides comprising variants of spr2021 (SP2216), SP1732 and optionally PsaA can also be used in place of the actual disclosed SEQ ID NOs e.g. comprising at least one variant from each of SEQ ID NOs 97 and 98. Examples of this combination include the combination of a pneumococcal immunogen as disclosed in reference 159 with a chimeric RrgB polypeptide comprising chimera ΙΙ-Ι-ΙΠ (e.g. SEQ ID NO: 21) or chimera ΙΠ- II-I (e.g. SEQ ID NO:15) as detailed below. The further antigens may be added as separate polypeptides. As an alternative, they may be added as hybrids e.g. a spr2021 -SP1732 hybrid or a spr2021 -SP1732-PsaA hybrid. As a further alternative, they may be fused to a RrgB polypeptide sequence, e.g. a chimeric RrgB polypeptide, to provide a hybrid polypeptide e.g. a RrgB-spr2021 -SP1732 hybrid. As detailed above, compositions of the invention comprising combinations such as these can optionally comprise one or more adjuvants. Suitable adjuvants include mineral salts such as aluminium salts, and squalene-water emulsions such as MF59.

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

The original 'spr0057' sequence was annotated in reference 149 as 'Beta-N-acetyl-hexosaminidase precursor' (see GI: 15902101). For reference purposes, the amino acid sequence of full length spr0057 as found in the R6 strain is given as SEQ ID NO: 23 herein. Preferred spr0057 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: 23; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 23, 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 spr0057 proteins include variants of SEQ K⁾ NO: 23. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 23. 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: 23 while retaining at least one epitope of SEQ ID NO: 23. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 38, which omits the natural leader peptide and sortase recognition sequences. Another suitable fragment is SEQ ID NO: 24, which has N-terminal and C-terminal truncations. SEQ ID NO: 27 is a variant of SEQ ID NO: 24 based on a different wild-type strain and is a useful spr0057 sequence for use with the invention.

The original 'spr0565' sequence was annotated in reference 149 as 'beta-galactosidase precursor' (see GI:15902609). For reference purposes, the amino acid sequence of full length spr0565 as found in the R6 strain is given as SEQ ID NO: 25 herein. Preferred spr0565 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: 25; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 25, 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 spr0565 proteins include variants of SEQ ID NO: 25 (e.g. SEQ K⁾ NO: 45; see below). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 25. 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: 25 while retaining at least one epitope of SEQ ID NO: 25. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 42, which omits the natural leader peptide and sortase recognition sequences. Other suitable fragments are SEQ ID NOs: 43 and 44. These shortened versions of spr0565 are particularly useful because the natural polypeptide is very long (>2000 aa).

A variant form of spr0565 is SEQ ID NO: 45 herein. The use of this variant form for immunisation is reported in reference 150 (SEQ ID NO: 178 therein). Useful spr0565 polypeptides may thus 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: 45; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 45, wherein V is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These polypeptides include variants of SEQ ID NO: 45. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 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 NO: 45 while retaining at least one epitope of SEQ ID NO: 45. Other fragments omit one or more protein domains. Immunogenic fragments of SEQ ID NO: 45 are identified in table 1 of reference 150.

The original 'sprl 098' sequence was annotated in reference 149 as 'Sortase' (see GL 15903141). For reference purposes, the amino acid sequence of full length sprl098 as found in the R6 strain is given as SEQ ID NO: 26 herein. Preferred sprl098 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: 26; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 26, 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 sprl098 proteins include variants of SEQ ID NO: 26. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 26. 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 K) NO: 26 while retaining at least one epitope of SEQ ID NO: 26. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 46, which omits the natural leader peptide sequence.

The original 'sprl416' sequence was annotated in reference 149 as 'hypothetical protein' (see GI:15903459). For reference purposes, the amino acid sequence of full length sprl416 as found in the R6 strain is given as SEQ ID NO: 28 herein. Preferred sprl416 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: 28; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 28, 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 sprl416 proteins include variants of SEQ ID NO: 28. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 28. 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: 28 while retaining at least one epitope of SEQ ID NO: 28. Other fragments omit one or more protein domains.

The original 'sprl418' sequence was annotated in reference 149 as 'hypothetical protein' (see GI:15903461). For reference purposes, the amino acid sequence of full length sprl418 as found in the R6 strain is given as SEQ ID NO: 29 herein. Preferred sprl418 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: 29; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 29, 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 sprl418 proteins include variants of SEQ ID NO: 29. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 29. 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: 29 while retaining at least one epitope of SEQ ID NO: 29. Other fragments omit one or more protein domains.

The original 'spr0867' sequence was annotated in reference 149 as 'Endo-beta-N- acetylglucosaminidase' (see GI:15902911 ). For reference purposes, the amino acid sequence of full length spr0867 as found in the R6 strain is given as SEQ ID NO: 30 herein. Preferred spr0867 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: 30; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 30, wherein V 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 spr0867 proteins include variants of SEQ ID NO: 30. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 30. 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: 30 while retaining at least one epitope of SEQ ID NO: 30. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 48, which omits the natural leader peptide sequence.

The original 'sprl431 ' sequence was annotated in reference 149 as Ί ,4-beta-N-acetylmuramidase' (see GI: 15903474). It is also known as 'LytC, and its use for immunisation is reported in reference 171. For reference purposes, the amino acid sequence of full length sprl431 as found in the R6 strain is given as SEQ ID NO: 31 herein. Preferred sprl431 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: 31 ; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 31 , wherein V 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 sprl431 proteins include variants of SEQ ID NO: 31. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 31. 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: 31 while retaining at least one epitope of SEQ ID NO: 31. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 49, which omits the natural leader peptide sequence.

The 'sprl739' polypeptide is pneumolysin (e.g. see GL 15903781 ). For reference purposes, the amino acid sequence of full length sprl739 as found in the R6 strain is given as SEQ ID NO: 32 herein. Preferred sprl739 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: 32; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 32, 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 sprl739 proteins include variants of SEQ ID NO: 32. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 32. 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: 32 while retaining at least one epitope of SEQ ID NO: 32. Other fragments omit one or more protein domains. Mutant forms of pneumolysin for vaccination use are known in the art [183, 151 -156], and these mutant forms may be used with the invention. Detoxification can be achieved by C-terminal truncation (e.g. see ref. 157) e.g. deleting 34 amino acids, 45 amino acids, 7 amino acids [158], etc. Further mutations, numbered according to SEQ ID NO: 32, include Pro325→Leu (e.g. SEQ ID NO: 50) and/or Trp433→Phe (e.g. SEQ ID NO: 51). These mutations may be combined with C-terminal truncations e.g. to combine a Pro325→Leu mutation with a 7-mer truncation (e.g. SEQ ID NO: 52).

The original 'spr2021 ' sequence was annotated in reference 149 as 'General stress protein GSP-781 ' (see GI: 15904062). For reference purposes, the amino acid sequence of full length spr2021 as found in the R6 strain is given as SEQ ID NO: 33 herein. Preferred spr2021 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: 33; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 33, 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 spr2021 proteins include variants of SEQ ID NO: 33. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 33. 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: 33 while retaining at least one epitope of SEQ ID NO: 33. Other fragments omit one or more protein domains. One suitable fragment is SEQ ID NO: 53, which omits the natural leader peptide sequence. Reference 150 annotates spr2021 as a secreted 45kDa protein with homology to GbpB and discloses its use as an immunogen (SEQ ID NO: 243 therein; SP2216). Immunogenic fragments of spr2021 are identified in table 1 of reference 150 (page 73). Another useful fragment of spr2021 is disclosed as SEQ ID NO: 1 of reference 159 (amino acids 28-278 of SEQ ID NO: 33 herein; this useful fragment of spr2021 is provided as SEQ ID NO:97 herein; SP2216-1).

The original 'spr0096' sequence was annotated in reference 149 as 'hypothetical protein' (see GI:15902140). For reference purposes, the amino acid sequence of full length spr0096 as found in the R6 strain is given as SEQ ID NO: 34 herein. Preferred spr0096 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 spr0096 proteins include variants of SEQ ID NO: 34 (e.g. SEQ ID NO: 54; see below). 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.

A variant form of spr0096, with an insert near its C-terminus relative to SEQ ID NO: 34, is SEQ ID NO: 54 herein. The use of this variant for immunisation is reported in reference 150 (SEQ ID NO: 150 therein), where it is annotated as a LysM domain protein. Thus a spr0096 for use with the invention may 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 V is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These polypeptides 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. Immunogenic fragments of SEQK⁾ NO: 54 are identified in table 1 of reference 150.

A spr0096 polypeptide may be used in the form of a dimer e.g. a homodimer.

The original 'sprl 707' sequence was annotated in reference 149 as 'ABC transporter substrate- binding protein - oligopeptide transport' (see GI: 15903749). For reference purposes, the amino acid sequence of full length sprl 707 as found in the R6 strain is given as SEQ ID NO: 36 herein. Preferred sprl 707 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: 36; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 36, 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 sprl 707 proteins include variants of SEQ ID NO: 36 (e.g. SEQ ID NO: 55; see below). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 36. 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: 36 while retaining at least one epitope of SEQ ID NO: 36. Other fragments omit one or more protein domains.

A variant form of sprl 707, differing from SEQ ID NO: 14 by 4 amino acids, is SEQ ID NO: 55 herein. The use of SEQ ID NO: 55 for immunisation is reported in reference 150 (SEQ ID NO: 220 therein). Thus a sprl707 polypeptide for use with the invention may 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, 1 8, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These polypeptides include variants of SEQ ID NO: 55. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 55. 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. Immunogenic fragments of SEQ ID NO: 55 are identified in table 1 of reference 150.

The original 'sprl 875' sequence was annotated in reference 149 as 'hypothetical protein' (see GI:15903916). For reference purposes, the amino acid sequence of full length sprl 875 as found in the R6 strain is given as SEQ ID NO: 35 herein. Preferred sprl 875 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: 35; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 35, 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 sprl 875 proteins include variants of SEQ ID NO: 35. 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: 35 while retaining at least one epitope of SEQ ID NO: 35. Other fragments omit one or more protein domains.

The 'spr0884' protein is a peptidylprolyl isomerase, also known as protease maturation protein. For reference purposes, the amino acid sequence of full length spr0884 is SEQ ID NO: 37 herein. Preferred spr0884 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: 37; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 37, 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 spr0884 proteins include variants of SEQ ID NO: 37. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 37. 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: 37 while retaining at least one epitope of SEQ ID NO: 37. Other fragments omit one or more protein domains. One suitable fragment is SEQ

ID NO: 56, which omits the natural leader peptide sequence. The use of spr0884 for immunisation is reported in reference 160.

ClpP is the ATP-dependent Clp protease proteolytic subunit. For reference purposes, the amino acid sequence of full length ClpP is SEQ K⁾ NO: 58 herein. In the R6 genome ClpP is spr0656 [149]. Preferred ClpP 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: 58; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 58, 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 ClpP proteins include variants of SEQ ID NO: 58. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 58. 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: 58 while retaining at least one epitope of SEQ ID NO: 58. Other fragments omit one or more protein domains. The use of ClpP for immunisation is reported in references 161 and 162. It may advantageously be used in combination with PspA and PsaA and/or PspC [161 ].

LytA is the N-acetylmuramoyl-L-alanine amidase (autolysin). For reference purposes, the amino acid sequence of full length LytA is SEQ ID NO: 59 herein. In the R6 genome LytA is sprl754 [149]. Preferred LytA 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: 59; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 59, 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 LytA proteins include variants of SEQ ID NO: 59 (e.g. GI: 18568354). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 59. 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 K) NO: 59 while retaining at least one epitope of SEQ ID NO: 59. Other fragments omit one or more protein domains. The use of LytA for immunisation is reported in reference 163, particularly in a form comprising the LytA choline binding domain fused to a heterologous promiscuous T helper epitope.

PhtA is the Pneumococcal histidine triad protein A. For reference purposes, the amino acid sequence of full length PhtA precursor is SEQ K) NO: 60 herein. In the R6 genome PhtA is sprl061 [149]. Preferred PhtA 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: 60; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 60, 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 PhtA proteins include variants of SEQ ID NO: 60. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 60. 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: 60 while retaining at least one epitope of SEQ ID NO: 60. Other fragments omit one or more protein domains. The use of PhtA for immunisation is reported in references 164 and 165.

PhtB is the pneumococcal histidine triad protein B. For reference purposes, the amino acid sequence of full length PhtB precursor is SEQ ID NO: 61 herein. Xaa at residue 578 can be Lysine. Preferred PhtB 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 K) NO: 61 ; and/or (b) comprising a fragment of at least V consecutive amino acids of SEQ ID NO: 61 , 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 PhtB proteins include variants of SEQ ID NO: 61. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 61. 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 while retaining at least one epitope of SEQ ID NO: 61. Other fragments omit one or more protein domains. The use of PhtB for immunisation is reported in references 164, 165 and 166. PhtD is the Pneumococcal histidine triad protein D. For reference purposes, the amino acid sequence of full length PhtD precursor is SEQ K) NO: 62 herein. In the R6 genome PhtD is spr0907 [149]. Preferred PhtD 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: 62; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 62, 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 PhtD proteins include variants of SEQ ID NO: 62. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 62. 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: 62 while retaining at least one epitope of SEQ ID NO: 62. Other fragments omit one or more protein domains. The use of PhtD for immunisation is reported in references 164, 165 and 167.

PhtE is the Pneumococcal histidine triad protein E. For reference purposes, the amino acid sequence of full length PhtE precursor is SEQ K) NO: 63 herein. In the R6 genome PhtE is spr0908 [149]. Preferred PhtE 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: 63; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of 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 PhtE proteins include variants of SEQ ID NO: 63. Preferred fragments of (b) comprise an epitope from 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: 63 while retaining at least one epitope of SEQ ID NO: 63. Other fragments omit one or more protein domains. The use of PhtE for immunisation is reported in references 164 and 165.

ZmpB is the zinc metalloprotease. For reference purposes, the amino acid sequence of full length ZmpB is SEQ K) NO: 64 herein. In the R6 genome ZmpB is spr0581 [149]. Preferred ZmpB 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 K) NO: 64, wherein V 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 ZmpB 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.

CbpD is the Choline binding protein D. For reference purposes, the amino acid sequence of full length CbpD is SEQ K) NO: 65 herein. In the R6 genome CbpD is spr2006 [149]. Preferred CbpD 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: 65; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 65, wherein V 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 CbpD proteins include variants of SEQ ID NO: 65 (e.g. SEQ ID NO: 66; see below). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 65. 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: 65 while retaining at least one epitope of SEQ ID NO: 65. Other fragments omit one or more protein domains. The use of CbpD for immunisation is reported in reference 171.

A variant of SEQ ID NO: 65 is SEQ ID NO: 66 herein. The use of SEQ ID NO: 66 for immunisation is reported in reference 150 (SEQ ID NO: 241 therein). Thus a CbpD polypeptide for use with the invention may 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: 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 CbpD proteins include variants of SEQ K⁾ 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. Immunogenic fragments of SEQ ID NO: 66 are identified in table 1 of ref.150.

CbpG is the Choline binding protein G. For reference purposes, the amino acid sequence of full length CbpG is SEQ K⁾ NO: 67 herein. In the R6 genome CbpG is spr0350 [149]. Preferred CbpG 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 V 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 CbpG proteins include variants of SEQ K) 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. The use of CbpG for immunisation is reported in reference 171.

PvaA (Streptococcus pneumoniae pneumococcal vaccine antigen A) is also known as splOl . For reference purposes, the amino acid sequence of full length PvaA is SEQ ID NO: 68 herein. In the R6 genome PvaA is spr0930 [149]. Preferred PvaA 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: 68; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 68, 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 PvaA proteins include variants of SEQ ID NO: 68. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 68. 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 K) NO: 68 while retaining at least one epitope of SEQ ID NO: 68. Other fragments omit one or more protein domains. The use of PvaA for immunisation is reported in references 168 and 169.

CPL1 is the pneumococcal phage CP1 lysozyme. For reference purposes, the amino acid sequence of full length CPL1 is SEQ K⁾ NO: 69 herein. Preferred CPL1 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: 69; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 69, 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 CPL1 proteins include variants of SEQ ID NO: 69. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 69. 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: 69 while retaining at least one epitope of SEQ ID NO: 69. Other fragments omit one or more protein domains. The use of CPL1 for immunisation is reported in reference 163, particularly in a form comprising the CPL1 choline binding domain fused to a heterologous promiscuous T helper epitope.

PspC is the pneumococcal surface protein C [170] and is also known as choline-binding protein A (CbpA). Its use for immunisation is reported in references 168 and 171. In the R6 strain it is sprl995 and, for reference, the amino acid sequence of full length sprl995 is SEQ ID NO: 57 herein. Preferred PspC 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: 57; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 57, 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 sprl995 proteins include variants of SEQ K⁾ NO: 57 (e.g. SEQ ID NO: 71 ; see below). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 57. 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: 57 while retaining at least one epitope of SEQ ID NO: 57. Other fragments omit one or more protein domains.

A variant of PspC is known as 'Hie' . It is similar to PspC, as shown in Figure 1 of reference 172, where it is reported to bind to factor H (fH). For reference purposes, the amino acid sequence of full length Hie is SEQ ID NO: 71 herein. A Hie protein may be used with the invention in addition to or in place of a PspC polypeptide. Preferred Hie 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: 71 ; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 71 , 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 Hie proteins include variants of SEQ ID NO: 71. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 71. 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 K) NO: 71 while retaining at least one epitope of SEQ ID NO: 71. Other fragments omit one or more protein domains. PspC and/or Hie can advantageously be used in combination with PspA and/or PsaA. PspA is the Pneumococcal surface protein A. For reference purposes, the amino acid sequence of full length PspA is SEQ ID NO: 72 herein. In the R6 genome PspA is spr0121 [149]. Preferred PspA 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 K) NO: 72; and/or (b) comprising a fragment of at least V consecutive amino acids of SEQ ID NO: 72, 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 PspA proteins include variants of SEQ ID NO: 72. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 72. 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: 72 while retaining at least one epitope of SEQ ID NO: 72. Other fragments omit one or more protein domains. The use of PspA for immunisation is reported inter alia in reference 173. It can advantageously be administered in combination with PspC.

PsaA is the Pneumococcal surface adhesin. For reference purposes, the amino acid sequence of full length PsaA is SEQ ID NO: 73 herein. Preferred PsaA 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: 73; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 73, wherein V 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 PsaA proteins include variants of SEQ ID NO: 73. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 73. 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: 73 while retaining at least one epitope of SEQ ID NO: 73. Other fragments omit one or more protein domains. A useful fragment of PsaA is disclosed as SEQ ID NO: 3 in reference 159 (corresponding to amino acids 21-309 of SEQ ID NO: 73 herein; this useful fragment of PsaA is provided as SEQ ID No.99 herein). The use of PsaA for immunisation is reported in reference 174. It can be used in combination with PspA and/or PspC.

PrtA is the cell wall-associated serine proteinase. It has also been known as spl28 and spl30, and is in a subtilisin-like serine protease. For reference purposes, the amino acid sequence of full length PrtA precursor is SEQ K) NO: 74 herein. In the R6 genome PrtA is spr0561 [149]. Preferred PrtA 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: 74; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 74, wherein V 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 PrtA proteins include variants of SEQ K) NO: 74. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 74. 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: 74 while retaining at least one epitope of SEQ ID NO: 74. Other fragments omit one or more protein domains. The use of PrtA for immunisation is reported in references 175 & 176, and also in reference 168.

Spl33 is a conserved pneumococcal antigen. For reference purposes, the amino acid sequence of full length Spl33 is SEQ K) NO: 75 herein. In the R6 genome Spl33 is spr0931 [149]. Preferred Spl 33 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: 75; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 75, wherein V 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 Spl33 proteins include variants of SEQ ID NO: 75. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 75. 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: 75 while retaining at least one epitope of SEQ ID NO: 75. Other fragments omit one or more protein domains. The use of Spl 33 for immunisation is reported in reference 177.

PiaA is the membrane permease involved in iron acquisition by pneumococcus. For reference purposes, the amino acid sequence of full length PiaA is SEQ ID NO: 76 herein. In the R6 genome PiaA is spr0935 [149]. Preferred PiaA 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: 76; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 76, 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 PiaA proteins include variants of SEQ ID NO: 76. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 76. 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 K) NO: 76 while retaining at least one epitope of SEQ ID NO: 76. Other fragments omit one or more protein domains. The use of PiaA for immunisation is reported in references 178, 179 and 180, particularly in combination with PiuA.

PiuA is the ABC transporter substrate-binding protein for ferric iron transport. It is also known as FatB. For reference purposes, the amino acid sequence of full length PiuA is SEQ ID NO: 77 herein. In the R6 genome PiuA is sprl687 [149]. Preferred PiuA 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: 77; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 77, wherein V 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 PiuA proteins include variants of SEQ ID NO: 77. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 77. 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: 77 while retaining at least one epitope of SEQ ID NO: 77. Other fragments omit one or more protein domains. The use of PiuA for immunisation is reported in refs 178 to 180, particularly in combination with PiaA.

CbiO is annotated as a cobalt transporter ATP -binding subunit. For reference purposes, the amino acid sequence of full length CbiO is SEQ K⁾ NO: 78 herein. In the R6 genome CbiO is spr2025 [149] . The use of CbiO for immunisation is reported in reference 181 ('ID2' therein). Preferred CbiO 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 K) NO: 78; and/or (b) comprising a fragment of at least V consecutive amino acids of SEQ ID NO: 78, 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 CbiO proteins include variants of SEQ ID NO: 78. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 78. 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: 78 while retaining at least one epitope of SEQ ID NO: 78. Other fragments omit one or more protein domains.

For reference purposes, the amino acid sequence of 3 OS ribosomal protein S8 is SEQ ID NO: 79 herein. In the R6 genome the S8 subunit is spr0203 [149]. Preferred S8 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: 79; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 79, 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 S8 proteins include variants of SEQ ID NO: 79. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 79. 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: 79 while retaining at least one epitope of SEQ ID NO: 79. Other fragments omit one or more protein domains.

SP1732 is a membrane-associated serine/threonine kinase, StkP. The sequence of SP1732, comprising 659 amino acids, is identified in reference 150 as SEQ ID NO: 214. An exemplary fragment of this sequence, referred to as "SP 1732-3", is identified in reference 159 as SEQ ID NO: 2. For reference purposes, the amino acid sequence of SP 1732-3 is provided as SEQ ID NO: 98 herein. Preferred SP1732 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: 98; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 98, 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 SP1732 proteins include variants of SEQ ID NO: 98. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 98. 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: 98 while retaining at least one epitope of SEQ ID NO: 98. Other fragments omit one or more protein domains. Combinations with pneumococcal saccharides

RrgB epitopes from one or more clades may be combined with one or more pneumococcal 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 combination of at least two RrgB clade epitopes as discussed above, as a mixture or hybrid; and

(2) one or more pneumococcal 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 RrgB polypeptide, a hybrid RrgB polypeptide, a non-RrgB pneumococcal polypeptide, or a non-pneumococcal polypeptide.

The saccharide is from the capsular saccharide of a pneumococcus. 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. In the 7-valent PREVNAR™ product, for instance, 6 of the saccharides are presented as intact polysaccharides while one (the 18C serotype) is presented as an oligosaccharide.

A composition may include a capsular saccharide from one or more of the following pneumococcal serotypes: 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. A composition may include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or more serotypes. 7-valent, 9-valent, 10-valent, 11 - valent and 13-valent conjugate combinations are already known in the art, as is a 23-valent unconjugated combination.

For example, a 10-valent combination may include saccharide from serotypes 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent combination may further include saccharide from serotype 3. A 12-valent combination may add to the 10-valent mixture: serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B; 19A and 15B; r 22F and 15B; A 13-valent combination may add to the 11 - valent mixture: serotypes 19A and 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F; 15B and 19A; 15B and 22F. etc. One useful 13-valent combination includes capsular saccharide from serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19, 19F and 23F. If saccharides are enclosed then it is preferred to include 1, 2 or 3 of serotypes 1 , 5 and 14.

A carrier protein in a conjugate may or may not be one of the RrgB antigens of (1). If it is not a RrgB antigen it may instead be a different pneumococcal antigen, such as spr0057, spr0096 and spr2021, etc., or pneumolysin [182] or its non-toxic derivatives [183], or pneumococcal surface protein PspA [184], In some embodiments, though, the carrier is not a pneumococcal antigen, and may be e.g. a bacterial toxin or toxoid. Typical carrier proteins are diphtheria or tetanus toxoids or mutants thereof. The CRM₁₉₇ diphtheria toxin mutant [185] is useful, and is the carrier in the PREVNAR™ product. Other suitable carrier proteins include N.meningitidis outer membrane protein complex [186], synthetic peptides [187,188], heat shock proteins [189,190], pertussis proteins [191 ,192], cytokines [193], lymphokines [193], hormones [193], growth factors [193], artificial proteins comprising multiple human CD4⁺ T cell epitopes from various pathogen-derived antigens [194] such as N19 [195], protein D from H.influenzae [196-198], iron-uptake proteins [199], toxin A or B from C.difficile [200], recombinant P. aeruginosa exoprotein A (rEPA) [201], etc.

Where a composition includes more than one conjugate, each conjugate may use the same carrier protein or a different carrier protein. Reference 202 describes potential advantages when using different carrier proteins in multivalent pneumococcal conjugate vaccines

In some embodiments, a single conjugate may carry saccharides from multiple serotypes [203]. 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.

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 204 and 205. 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 206 and 207. A preferred type of linkage is an adipic acid linker, which may be formed by coupling a free -NH₂ 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 [208,209]. Another preferred type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a saccharide CDI [210, 211] followed by reaction with a protein to form a carbamate linkage. Other linkers include β-propionamido [212], nitrophenyl-ethylamine [213], haloacyl halides [214], glycosidic linkages [215], 6-aminocaproic acid [216], ADH [217], C₄ to C₁₂ moieties [218], etc. Carbodiimide condensation can also be used [219].

Combinations with non-pneumococcal antigens

The RrgB clade epitope combinations may be used in combination with non-pneumococcal antigens. Thus the invention provides an immunogenic composition comprising a combination of:

(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 poliovirus antigen; a conjugate of the capsular saccharide antigen from Haemophilus influenzae type B; 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; and a conjugate of the capsular saccharide antigen from serogroup A of Neisseria meningitidis.

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 220.

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 220.

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 220) 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 [221 ]. 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 220, 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.

Immunogenic compositions of particular interest comprise: (i) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, diphtheria toxoid, tetanus toxoid, whole cell pertussis antigens, a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg; (ii) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, diphtheria toxoid, tetanus toxoid, acellular pertussis antigen(s), a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg; (iii) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and conjugate(s) from one or more of meningococcal sero groups A, C, W135 and Y; (iv) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and conjugates from all of meningococcal serogroups A, C, W135 and Y; and (v) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and a meningococcal serogroup B antigen, such as an outer membrane vesicle antigen and/or the combination disclosed in ref. 222. Antibodies

Antibodies against pneumococcal antigens can be used for passive immunisation [223]. Thus the invention provides an antibody that binds to polypeptide comprising one or more of the identified epitopes. Typically, the antibody binds specifically to a polypeptide of the invention. The invention further 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; (c) an antibody which recognises a third amino acid sequence as defined above; (d) an antibody which recognises a fourth amino acid sequence as defined above; (a) an antibody which recognises a fifth amino acid sequence as defined above; and/or (a) an antibody which recognises a sixth amino acid sequence as defined above.

The invention also provides the use of such antibodies and antibody combinations in therapy. The invention also provides the use of such antibodies and 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 an antibody or combination. As described above for immunogenic compositions, these methods and uses allow a mammal to be protected against pneumococcal 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 [224, 225]; F(ab')2 and F(ab) fragments and Fv molecules; non-covalent heterodimers [226, 227]; single-chain Fv molecules (sFv) [228]; dimeric and trimeric antibody fragment constructs; minibodies [229, 230]; humanized antibody molecules [231-233]; 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 234-241, etc.

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

Where the invention concerns an "epitope", this epitope may be a B-cell epitope and/or a T-cell epitope. Such epitopes can be identified empirically {e.g. using PEPS CAN [242,243] or similar methods), or they can be predicted {e.g. using the Jameson-Wolf antigenic index [244], matrix -based approaches [245], MAPITOPE [246], TEPITOPE [247,248], neural networks [249], OptiMer & EpiMer [250, 251], ADEPT [252], Tsites [253], hydrophilicity [254], antigenic index [255] or the methods disclosed in references 256-260, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as "antigenic determinants".

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%. 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.

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. 261. 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. 262.

MODES FOR CARRYING OUT THE INVENTION

Construction of RrgB chimeras

Two different pili have been identified in pneumococcus [2]: PI-1 and PI-2. Knockout studies showed that loss of PI-2 had little effect, but loss of PI-1 reduced a strain's ability to colonise, and thus led to lower bacteremia and lung wash titres. Thus blocking of PI-1 has a better prospect of protecting against pneumococcal disease than blocking PI-2.

PI-1 RrgB protein has three different clades. Fifteen different RrgB amino acid sequences were found in 45 different strains and Figure 9 shows their relationship. The wild-type sequences are >98% conserved within each clade. RrgB protein was found to elicit immune responses which are protective against homologous strains (intra-clade), but which fail to protect against strains having RrgB from a different clade (inter-clade). Thus it was decided to combine multiple RrgB clades into a single composition, thereby increasing the spectrum of strain coverage.

SEQ ID NOs: 1, 2 and 3 are the full-length encoded sequences for RrgB from strains TIGR4, Finland^6B-12 and Taiwan^23F-15. To construct chimeras of these three proteins their N- and C-termini were truncated to give SEQ ID NOs: 4, 5 and 6. Restriction enzymes Nhel, BamHI and Xhol were used in this procedure. To join these fragments to make chimeras linkers SEQ ID NOs: 8 and 10 were used, made of either a Gly-Ser or Leu-Gly dipeptide followed by SEQ ID NO: 7. These linkers provide convenient restriction sites for ligation of fragments. The N-terminus of the chimeras was provided as Met-Ala-Ser, and the C-terminus was a Leu-Gly dipeptide followed by a hexa-His tag (SEQ ID NO: 9) to facilitate purification.

Six chimeras were constructed, referred to hereafter as follows:

RrgB Ι-ΙΙ-ΠΙ = SEQ ID NO: 11

RrgB I-III-II = SEQ ID NO: 13

RrgB III-II-I = SEQ ID NO: 15

RrgB III-I-II = SEQ ID NO: 17

RrgB II-III-I = SEQ ID NO: 19

RrgB ΙΙ-Ι-ΠΙ = SEQ ID NO: 21

Except for the Ι-ΙΠ-ΙΙ chimera (SEQ ID NO: 13) the expressed chimeras had a molecular weight of 205kDa, could be expressed in E.coli in soluble form, and were purified from the soluble proteins. For example, figure 3 shows a gel of the I-II-III chimera at 1.6mg/ml with 90% purity.

Efficacy testing

Various model systems of pneumococcal disease were used for testing efficacy of the chimeras. In a mouse model of intraperitoneal infection, antigens were administered intraperitoneally and the challenge was intraperitoneal. Six-week-old, specific-pathogen -free female BALB/c or CD1 mice were immunized intraperitoneally on days 0, 14, and 28. Immunizations were done using single recombinant proteins (20 μg/mouse) or with a combination of them (10 μg each/mouse), along with aluminium hydroxide or Freund's adjuvant. Controls received identical courses of saline plus adjuvant. Mice were then challenged intraperitoneally with a lethal dose of TIGR4 (typical challenge dose ~lxl0² CFU/mouse), Finland⁶¹³- 12 (~2.xl0⁴ CFU/mouse) or 35B-SME15 (~lxl 0⁴ CFU/mouse). These three strains express RrgB clades I, Π or ΙΠ, respectively, and the TIGR4 strain is very virulent. Efficacy of immunisation is tested by evaluating the effect of vaccination on bacteremia (at 5 and/or 24 hours post infection) and mortality (monitored for at least 10 days following bacterial challenge).

In a model of intravenous infection, antigens were administered intraperitoneally and the challenge was intravenous. Five-week-old CD1 or BALB/c mice were immunized intraperitoneally on days 0, 14, and 28. Immunizations were done using recombinant proteins individually (20 μg/mouse) or with a combination of them (10 μg each/mouse), along with Freund's adjuvant. Controls received identical courses of saline plus adjuvant. Mice were then challenged intravenously with a lethal dose of TIGR4 (typical challenge dose ~5xl0⁶ CFU/mouse), Finland^6B-12 (~2.xl0⁷ CFU/mouse) or 35B- SME15 (~5xl 0⁷ CFU/mouse). Efficacy of vaccine candidates is tested by evaluating the effect of vaccination on bacteremia (at 48 hours post-infection) and mortality (monitored forlO days following bacterial challenge or longer, depending on the infecting strain).

5 For example, CD1 mice were immunised with the chimeras and then challenged with TIGR4. Figure 1 shows bacteremia after the challenge. Geometric mean CFUs were as follows, together with a U-test comparison against the control group:

Figure 2 shows mortality after the challenge. Median survival times in days were as follows:

Figures 30 to 33 show the results of bacteremia and mortality assays for mice immunised intraperitoneally with 20μg of the ΠΙ-ΙΙ-Ι chimera. Figure 30 shows data for i.v. challenge with TIGR4, Figure 31 shows data for i.p. challenge with TIGR4, Figure 32 shows data for i.v. challenge with 35B-SME15 and Figure 33 shows data for i.v. challenge with 6B Finland 12.

5 The following table summarises results obtained in two different models of challenge with three different strains which express, respectively, RrgB in clade I, II or III:

+++ = P<0.01 against control; ++ = P<0.05; + = P<0.1

Therefore the combination of different clades of RrgB allows for broader coverage against pneumococcal strains than single RrgB antigens. In further tests RrgB chimeras were adjuvanted with alum and tested for protection against TIGR4 intraperitoneal challenge. Chimeras I-II-III and ΠΙ-ΙΙ-Ι were highly protective against bacteremia, and the ΙΠ-ΙΙ-Ι chimera was also protective in terms of survival (Figure 7).

Further tests used intranasal challenge after intraperitoneal immunisation with one of four different chimeras (Ι-ΙΙ-ΙΠ, ΠΙ-ΙΙ-Ι, II-III-I, II-I-III). All chimeras showed efficacy or a trend to reduce bacteremia after intranasal TIGR4 challenge. The ΙΙ-ΠΙ-Ι chimera gave good decrease of bacteremia and a non-significant trend of survival increase upon T4 challenge. A PsaA control showed almost no efficacy, measured either by bacteremia or mortality, whereas the Π-ΙΠ-Ι chimera decreased bacteremia and increased survival. Figure 13 shows results for an RrgB ΙΠ-ΙΙ-Ι Chimera in a 24hour bacteremia assay (Figure 13A) and a mortality assay (Figure 13B) in BalB/c mice, immunized intraperitoneally with 2C^g chimera (0-14-28 days) and challenged intranasally with TIGR4.

Antibodies against all five RrgB chimeras were also found to mediate in vitro killing of pneumococci in OPKA. For instance, figure 8 shows results against the TIGR4 strain. Figure 10 shows results against S.pneumoniae serotype 6B in an OPKA assay (rabbits subcutaneously immunized with 100μg of each chimera at days 0, 21 and 35), which shows that no difference in killing percentage is observed between the five chimeras and that the chimeras show killing that is comparable to the conjugate vaccine PCV7. Figure 11 shows that killing is specific and dependent on antibody concentration, showing that by increasing the dilution up to 1/131220, the percentage killing decreases in the tested chimera curves similarly to the positive control.

Figure 12 shows a 48 hour bacteremia (Figure 12A) and mortality (Figure 12B) assay using a ΠΙ-ΙΙ-Ι chimera (immunised i.p. and challenged i.p. with 35B-SME15) is comparable when using different chimera doses (2 μg and 20 μg).

Figure 14 shows that RrgB ΙΠ-ΙΙ-Ι chimera is protective using MF59 adjuvant in BalB/c mice, intraperitoneal immunisation with 20 μg chimera (0-14-28 days) and challenged intransally.

Figure 15 shows that RrgB ΙΠ-ΙΙ-Ι chimera is protective upon subcutaneous immunization in BalB/c mice, immunized subcutaneously and challenged intraperitoneally with TIGR4 (130CFU/mouse). Figure 15A shows a 24hour bacteremia assay and Figure 15B shows a mortality assay.

Figure 16 shows that RrgB ΠΙ-ΙΙ-Ι chimera elicits production of functional antibodies in a passive protection study, compared to a Normal Rabbit Serum (NRS) control, in a 24hour bacteremia assay. Figure 17 shows that antibodies are functional in OPA against strains of the three clades and Figure 18 shows that the OPA activity is specifically due to the antibodies against RrgB ΠΙ-ΙΙ-Ι chimera. Figure 19A shows that single RrgB domains confer protection in vivo. Specifically, the data show % survival of BalB/c mice immunised with the RrgB Dl (TIGR4) domain or the RrgB D4 (TIGR4) domain (i.p. immunization 2C^g, 0-14-28 days; i.p. challenge with TIGR4 l OOCFU). The possible relevance of antibodies in the protection elicited by RrgB Dl and RrgB D4 domains was investigated by a passive serum transfer experiment, with groups of 8 mice. The results are shown in Figure 19B. The results were consistent with those obtained by active immunization with the corresponding antigens, although in this experiment the level of infection and mortality achieved in the controls was lower. Both anti-Dl and anti-D4 sera elicited significant protection against bacteremia (P < 0.05), with a reduction of the CFU geomean by 2.6 and 1.6 Logs as compared with that of the control group, respectively. In terms of mortality course, only anti-Dl serum afforded significant protection (P < 0.05), and gave 100% survival rate (P < 0.05), while anti-D4 afforded a non-significant protective trend, although giving 60% survival rate. When comparing the two immunized groups, no significant differences in the protective results were observed, both in terms of bacteremia and survival. The results obtained by passive serum transfer indicate that immunization with RrgB Dl and RrgB D4 domains elicits functional antibodies that may play a role in the protection.

Figure 23 shows a 48 hour bacteremia (Figure 23 A) and mortality (Figure 23B) assay using a ΠΙ-ΙΙ-Ι chimera when combined with different combinations of further polypeptide antigens (20μg antigens with alum; immunised i.p. and challenged i.v. with 6B-Finland 1.2E+08 CFU/mouse). In both (A) and (B): column 1 shows a combination of spr0057, spr0096 and spr2021 ; column 2 shows a combination of SP2216-1 , SP1732-3 and PsaA; column 3 shows RrgB ΠΙ-ΙΙ-Ι chimera; column 4 shows RrgB ΙΠ-ΙΙ-Ι chimera combined with spr0057, spr0096 and spr2021 ; column 5 shows RrgB III-II-I chimera combined with SP2216-1 , SPl 732-3 and PsaA; and column 6 shows an alum control. These data show that the efficacy of a combination of SP2216-1 , SPl 732-3 and PsaA is significantly increased when combined with the RrgB chimera.

Figure 24 shows a 48 hour bacteremia (Figure 24A) and mortality (Figure 24B) assay using a ΠΙ-ΙΙ-Ι chimera when combined with different combinations of further polypeptide antigens (20μg antigens with alum; immunised i.p. and challenged i.v. with 35B-SME15 5.2E+07 CFU/mouse). In both (A) and (B): column 1 shows a combination of spr0057, spr0096 and spr2021 ; column 2 shows a combination of SP2216-1 , SP1732-3 and PsaA; column 3 shows RrgB ΠΙ-ΙΙ-Ι chimera; column 4 shows RrgB ΙΠ-ΙΙ-Ι chimera combined with spr0057, spr0096 and spr2021 ; column 5 shows RrgB III-II-I chimera combined with SP2216-1 , SPl 732-3 and PsaA; and column 6 shows an alum control. These data show that the RrgB ΠΙ-ΙΙ-Ι chimera, and the combinations of the RrgB ΠΙ-ΙΙ-Ι chimera with other antigens, are all protective.

Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data in BALB/c mice using a ΙΠ- II-I chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control (i.p. immunisation, i.p. challenge with TIGR4 2.1E+02 CFU/mouse). These data show that both the his-tagged and tag-less chimeras significantly protect against TIGR4 both in terms of bacteremia and survival, with the tag-less chimera showing the most significant protection. Figure 26 shows similar data i.e. a 24 hour bacteremia assay in BALB/c mice using a ΙΠ-ΙΙ-Ι chimera that contains a polyhistidine tag compared to a tag-less ΙΠ-ΙΙ-Ι chimera and an alum control, further compared to a combination of spr0057, spr0096 and spr2021 antigens, and a combination of the spr0057, spr0096 and spr2021 antigens with the tag-less ΙΠ-ΙΙ-Ι chimera, (i.p. immunisation, i.p. challenge with TIGR4 1.6E+02 CFU/mouse). Figures 27 and 28 show data for i.v. challenge with 35B-SME15 (Figure 27) and 6BFinlandl2 (Figure 28), showing that tag-less ΠΙ-ΙΙ-Ι chimera showed the same protective efficacy as his-tagged ΙΠ-ΙΙ-Ι chimera against 35B-SME15 and 6BFinlandl2 i.v. challenge. Similarly, Figure 29 shows that both tag-less and his-tagged ΙΠ-ΙΙ-Ι chimeras are protective against i.v. TIGR4 challenge.

Figure 34 shows the results of 48 hour bacteremia and mortality assays for ΠΙ-ΙΙ-Ι chimera comparing a TIGR4 challenging strain over-expressing pilus to a challenging strain that only expresses very low amounts of pilus. These data show that protection is very good when the pilus is overexpressed and also when the pilus is only present at very low levels. Figure 35 shows similar bacteremia data for both ΠΙ-ΙΙ-Ι and Π-Ι-ΙΠ chimeras comparing a 6BFinll2 challenging strain over- expressing pilus (Figure 35A) to a 6BFinll2 challenging strain under-expressing pilus (Figure 35B). The chimeras show significant protection against both the strain overexpressing and the strain underexpressing pilus. Antimicrobial Resistance

Figure 36 shows that pilus-1 is more prevalent in pneumococcal strains that are resistant to antiobiotics (erythromycin-resistance, penicillin-resistance and multiple-drug-resistance) compared to strains that are susceptible to antibiotics. There is a significant association between pilus-1 presence and antibiotic resistance. An increase in the presence of pilus-1 in antibiotic-resistant strains has also been observed in the multi-resistant PMEN strain collection (data not shown). These data suggest that immunising against pilus-1 using an immunogenic composition including multiple RrgB clades will have the additional advantage of protecting against pneumococci that are resistant to antibiotic treatment, for example erythromycin-resistant strains, penicillin-resistant strains and multiply-resistant strains.

Monoclonal antibodies

Monoclonal antibodies were raised against the RrgB from TIGR4. Four mAbs were studied in more detail (named 23B8 B6, 23F8/10, 23E1/A9 and 30A8/A8). 23B8/B6 and 23F8/10 bound to the full- length RrgB from TIGR4, to the Dl domain fragment, and also to a D1 -D2-D3 fragment, but not to a D4 fragment. Conversely, 23E1/A9 bound to the full-length protein and so the D4 domain fragment but not to a D1-D2-D3 fragment, or a D4 fragment. 30A8/A8 bound to the full-length RrgB protein but not to any of the domain fragments. The mAbs did not bind to RrgB protein from Finland^6B-12 or 23F strains, but they did bind to all five chimeras which were expressed. The binding results are shown in figure 5 and confirm that the RrgB retains epitopes in its hybrid form.

As shown in figure 4A, each of the four tested anti-TIGR4 mAbs was able to reduce bacteremia in a passive protection test, with the best results coming from 23F8/10. Each of the four tested anti- TIGR4 mAbs also guaranteed a significant (p<0.01 for all MAbs except 23B8 B6, P=0.021) survival increase in a mortality assay (Figure 4B).

To determine the epitope recognized by each of the four protective MAbs, the different RrgB domains were cloned, as single domains (Dl, D2, D3, D4) or as multi-domain fragments (Dl-3, D2- 4, D3-4), expressed in E.Coli as His-tagged polypeptides and successfully purified in a soluble form by affinity chromatography on His-trap high performance columns (GE Healthcare). The recombinant proteins were then probed in western blot analysis against the MAbs by using FL RrgB clade I and BSA as positive and negative controls respectively.

The results, as shown in Figure 20, showed that monoclonal antibodies have a different and specific reactivity on the recombinant proteins. Both mAb 23F8/10 and mAb 23 B8/B6 were able to specifically recognize the N-terminal domain Dl, the mAb 23 E1/A9 recognized the C-terminal D4, while 30A8/A8, was able to detect only D2-4, suggesting the recognition of a conformational epitope between D2 and D4. These data were then subsequently confirmed by ELISA (data not shown).

Monoclonal antibodies were also raised against the RrgB from Finland^6B-12. Two particular mAbs (2A5/29, 3A5/19) bound to the full-length RrgB from Finland⁶¹³- 12, but not to the RrgB protein from TIGR4 or 23F strains. The mAbs also bound to all five chimeras which were expressed. The binding results are shown in figure 6. Epitope Mapping of Protective mAb 23F8/10

To map the region on the Dl domain containing the protective epitope recognized by mAb 23 F8/10 mass spectrometry analysis, Western Blot detection and limited proteolysis of the recombinant proteins were used in combination. This approach can be summarized in four main steps: (i) enzymatic or chemical partial cleavage of the protein, (ii) definition of sequence coverage of the generated fragments by MS analysis after their separation by SDS-PAGE, (iii) western blot analysis of the generated fragments, (iv) comparison of positive and negative bands in westem blot in order to localize the epitope.

The first step was to obtain from the full length RrgB a significant number of polypeptides showing a well resolved pattern after separation on SDS-PAGE. The protease selected for these experiments was trypsin, which cleaves proteins at the C-terminal side of arginine (R) and lysine (K) residues. 20 μg of full length RrgB was digested and the products of the digestion were separated with SDS- PAGE (5 μg of the full length protein, and 12 μg of the product of digestion). As noted above, and as shown in Figure 20, the monoclonal antibody 23F8/10 recognized both the full length recombinant RrgB and the RrgB Dl, as well as a high number of polypeptides derived from the cleavage of the full length protein with trypsin. The identification of both the positive and negative bands in the western blot analysis (with respect to the same Coomassie stained sample) was important for the epitope identification. The western blot with monoclonal antibody 23F8/C10 is shown as Figure 21. About 20 Coomassie-stained proteolitic fragments, comprising both western blot (immunoblotted with MAb 23F8/C10) positive (green arrows) and negative (red arrows) bands, were excised from the gel and in situ digested with trypsin O/N and analyzed by MALDI-TOF/TOF mass spectrometry, in order to define the sequence coverage for each of them. The sequence coverage obtained for each analyzed fragment was defined between the most "N-terminal" and the most "C-terminal" tryptic peptides identified in the PMF spectra {peptide mass fingerprints). A schematic sequence coverage of the electrophoretic pattern of the trypsin products derived from full length RrgB, in association with western blot results, was prepared. This analysis suggested that the 23F8/10 epitope is between amino acid 32 and amino acid 141 of full length RrgB.

The same strategy was then used on the N-terminal domain Dl, in order to narrow the region containing the epitope recognized by MAb 23F8/C10. 20 μg of Dl was digested and the products of the digestion were separated with SDS-PAGE (5 μg of the full length protein, and 12 μg of the product of digestion). Unlike full length digested RrgB, in this experiment the monoclonal antibody 23F8/10 recognized only full length Dl and some of the polypeptides derived from trypsin Dl digestion. Afterwards, both positive and negative bands were taken into consideration for further analysis. About 10 Coomassie-stained peptide fragments, comprising both positive and negative bands, were excised from the gel and in situ digested with trypsin O/N and analyzed by MALDI- TOF/TOF mass spectrometry, in order to define the sequence coverage for each of them. The sequence coverage obtained for each analyzed fragment was defined between the most "N-terminal" and the most "C-terminal" tryptic peptides identified in the PMF spectra ( peptide mass fingerprints ). The sequence coverage of the electrophoretic band of the trypsin products derived from RrgB Dl domain, as previously established in association with the results of the western blot, suggested that the region recognised by MAb 23F8/10 containing the protective epitope is from amino acid residue 55 to amino acid residue 89 of RrgB. The Dl amino acid sequence (for which no structural data are yet available) was modeled onto the domain 1 crystal structure of the S. pyogenes pilus backbone Spy0128 (overall homology about 27%). The residues that the data suggest are the epitope (aa 55- 89) were mapped onto the model (Figure 22A). In a 3D reconstruction of the electron density map of the pilus, obtained performing a rigid body fitting of the RrgB Dl-4 structure, this epitope is shown to be surface exposed (Figure 22 B & 22C). Epitope mapping on Dl and D4 domains of RrgB.

Further experiments were undertaken to identify linear epitopes within the RrgB Dl and RrgB D4 domains. A PepScan approach covering residues 25 to 190 (Dl) and 444 to 628 (D4) of TIGR4 RrgB was applied. Overlapping 15-mer peptides with an offset of 5 residues were synthesized in situ on a glass fiber membrane. Membranes were conditioned by wetting with ethanol and washing 3 times for 5 min in TTBS (50 mM Tris-HCl, pH 7.0; 137 mM NaCl; 2.7 mM KCl; 0.05 % Tween 20). After overnight blocking at 4°C in MBS (2 % dry milk in TTBS), membranes were incubated for 1.5 h at 37°C with the mouse polyclonal anti-Dl (TIGR4) and anti-D4(TIGR4) antisera (1 :3000 in MBS) previously used for passive immunization experiments (in Figure 19B) followed by secondary goat anti-mouse IgG alkaline phosphatase conjugated antibodies (Promega, 1 : 5000 in MBS) and signals were developed by using Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega). For image processing, membranes were scanned using an Epson V750 Pro scanner at 800 dpi, 48 bit color depth and with gamma 1.0 full linear response. Incubation with the anti-Dl serum revealed a single linear epitope spanning two adjacent peptides and covering residues 40 to 59 (Dl-1) (Figure 37A). Incubation of the membrane with anti-D4 serum resulted in detection of a single epitope, D4-1 (RrgB residues 494-508) (Figure 37B).

Interestingly, this analysis with polyclonal antibodies against RrgB Dl and D4 revealed in both domains the presence of linear epitopes, which could contribute to the protective activity exerted by the Dl and D4 domains. The two identified epitopes, when mapped onto the computer model of the entire RrgB molecule, appear to be surface exposed (Figure 38B). Taken together, these results suggest that RrgB contains multiple protective epitopes, thus explaining the potential of this vaccine candidate. Noteworthy, the protective efficacy exerted by the combination of the four RrgB domains D1+D2+D3+D4 is comparable to that afforded by the full-length RrgB. This observation suggests that, although the existence of possible conformational epitopes involving residues from different domains cannot be excluded, their contribution to the protective efficacy might not be essential.

To determine whether the linear epitopes identified at residues 40 to 59 (in Dl) and 494 to 508 (in D4) of TIGR4 RrgB retain the clade specificity of full length RrgB, Fluorescence Activated Cell Sorting ("FACS") and Western Blot experiments were performed, as follows.

A Fluorescence Activated Cell Sorting ("FACS") experiment was performed on TIGR4 ("clade I"), 6B Finlandl2 ("clade Π"), and 35BSME15 ("clade III") S.pneumoniae using mouse anti-Dl (TIGR4), anti-D4 (TIGR4), anti-Dl+D2+D3+D4 (TIGR4) and anti-full length RrgB (TIGR4) antibodies. The data (not shown) indicate that all of these TIGR4 antibodies are clade I (SEQ ID NO.1) specific and do not bind to RrgB from other clades (SEQ ID NO.2 and SEQ K) No.3).

The results of a Western Blot using the Anti-D4 (TIGR4) polyclonal antibodies (that were used in the epitope mapping experiments above) to probe various bacterial mutanolysates and recombinant proteins are shown in Figure 40. This Figure shows that antibodies that bind to the linear epitope at residues numbers 494 to 508 (in D4) of RrgB are TIGR4 (clade I) specific. This is supported by an alignment of the epitope identified in the TIGR 4 sequence (SEQ ID No. l) with the same residue numbers from the other two RrgB clades (from SEQ ID Nos. 2 and 3), which shows that the residues at these positions are dissimilar:

D4 494 to 508

Cladel VTTKDALDRAVAAYN— 15

Clade3 — KQALDAAIAAYTNAA 15

Clade2 -YTNAADKQAAQALVD- 15

* * *

The results of a Western Blot using the anti-Dl (TIGR4) polyclonal antibodies (that were used in the epitope mapping experiments above) to probe various bacterial mutanolysates and recombinant proteins are shown in Figure 41. This anti-Dl antibody shows a weak cross-reaction with clade ΠΙ (SEQ ID NO.3) when probed on bacterial lysates, but does not show cross reactivity against clade Π. Again, this is confirmed by an alignment of the epitope identified in the TIGR 4 sequence (SEQ ID No.l) with the same residue numbers from the other two RrgB clades (from SEQ ID Nos. 2 and 3). Dl 40 to 59

Cladel HKLLATDGDMDKIANELETG 20

Clade3 HKLLMTDQELDAWNSDAITT 20

Clade2 VTKTLTIHKLLLSEDDLKTW 20

The result obtained probing the anti-Dl antibody on the recombinant proteins is not conclusive as the antibody recognizes RrgB clade I, but also clade II and ΙΠ and a His tagged unrelated protein. This could mean that a fraction of the IgGs is directed against the His tag (in common among the 4 proteins and absent in the BSA preparation loaded in lane 10).

Figure 42 shows the results of a control for each of the Western Blots described above, using anti- RrgB ΠΙ-ΙΙ-Ι chimera (lanes 1 to 4) and anti-RrgB TIGR4 (lanes 6 to 10) antibodies probed against the same bacterial lysates and recombinant proteins that were tested in the Examples above.

In summary, these data show TIGR4 (SEQ ID NO:l) contains two linear epitopes, at residue numbers 40 to 59 (Dl) and 494-508 (D4). These epitopes appear to be surface exposed and are thought to contribute to the protective activity exerted by the Dl and D4 domains (which is demonstrated by Figure 19). The TIGR4 D4 epitope is clade specific and the TIGR4 Dl epitope has a small cross-reaction with the clade III sequence (SEQ ID No.3). Modelling SEQ ID NO.2 and SEQ ID NO.3 onto the structure obtained for SEQ K) NO. l shows that the epitopes identified in SEQ ID NO:l occupy a similar position is SEQ ID NOs. 2 and 3. It is reasonable to consider that the residues at the same positions in the other clades, i.e. at residue numbers 40 to 59 and 494 to 508 in each of SEQ ID NO.2 and SEQ ID NO.3, will also be useful epitopes.

Solution structure of the Dl domain.

In order to get further insights over the three-dimensional structure of the most protective domain RrgB Dl, the polypeptide including amino acids 20-193 of the RrgB protein was expressed as a C- terminal His-tag fusion protein in E. coli, purified and subjected to NMR spectroscopy. The Dl fragment consists of 181 amino acids, including a His6-tag tail and lacks residues 1-19 corresponding to the predicted signal sequence. The 1H-15N HSQC spectra shows well dispersed resonances indicative of an overall well folded protein.

The 3D structure of RrgB Dl domain shows a common IgG- ke β sandwich fold (41 A ^χ 48 A ^χ 30 A) and a topology of secondary structure elements as shown in Figure 38 A and B. The core of the structure is characterized by seven parallel and antiparallel β strands: β1(36-39), β4(80-85), β7(119- 121), β8(127-130), β9(138-143), β10(166-169), βΐ 1(178-180). These β strands are arranged in two sheets (comprising βΐ, β8, βΐ ΐ and β4, β7, β9, βΐθ, respectively) packed against each other and flanked by two long segments (40-78,87-117) located between strands βΐ and β4 and strands β4 and β7 respectively. An a-helix (49-57), stabilized by two short β strands β2 (42-44) and β3 (73-75), is inserted within the first segment, and two additional β strands β5 (89-91) and β6 (97-101) forming a β-hairpin structure are inserted within the second one. In addition, in 50% of the 20 conformers of the RrgB Dl family a β-sheet is formed by two short hydrogen-bonded β-strands (stretches 161 -163 and 184-187).

Overall, the structure is well defined with the exception of three long loops located between the a- helix and strands β3 (stretch 56-69), between strands β9 and βΐθ (148-162), and between strands βΐθ and βΐ ΐ (residues 173-177). From heteronuclear relaxation measurements, it appears that the residues in the first two mentioned loops have lower and higher than average heteronuclear NOE and longitudinal Rl values, respectively. This behavior is a consequence of local internal motions in the ns-ps timescale occurring on a faster timescale than the overall rotational correlation time and accordingly, a correlation time can be fitted for the previous mentioned loops. In addition to these fast internal motions, slow conformational exchange processes on the ms^s timescale affect some residues located in the region 155-164 of the second loop, since transversal R2 relaxation rate values higher than the average are observed. Overall the present data indicate that this loop regions experience higher flexibility and in agreement with this behavior they are characterized by a very low number of long ranges 1H-1H NOEs and, for a few residues, the assignment of the backbone atoms is missing (Glul43, Hisl45, Serl26, Serl48, Thrl49, Tyrl 50, Vall52, Glyl60), likely as a consequence of an increased local mobility. The loop between strands β9 and βΐθ is indeed the most disordered region with an RMSD value of about 2 A . Moreover, conformational exchange processes on the ms^s timescale have been observed for the loop between strands βΐθ and βΐ ΐ comprising residues 174-178.

The core of the protein is characterized by hydrophobic interactions between aliphatic as well as aromatic residues located on the first sheet (strands βΐ, β8, βΐ ΐ) and residues located on the other sheet (strands β4, β7, β9, βΐθ). Contacts between two complementarily charged side chains of residues Lys41 and Glul43 are also observed. These residues are also conserved within the various S. pneumoniae strains and the detected salt bridge contributes to stabilize the protein core.

In addition, the aliphatic side chains of residues Met48, Ile52, Ala53 and Leu56, all located on the same side on the a-helix, form hydrophobic interactions with aliphatic residues belonging to the two β2 and β3 strands; the latter interactions determine the position of the helix with respect to the rest of the protein.

A search for structurally related proteins in Protein Data Bank (PDB) through the DALI Server identified as the closest structurally homologues to RrgB Dl domain the following regions: the C- terminal β sandwich domain CNA3 of BcpA (PDB ID code 3KPT; rmsd: 2.4 A), which is the major pilin subunit of Bacillus cereus, the N-terminal domain of the Corynebacterium diphtheriae SpaA pilus backbone protein (PDB ID code 3HTL; rmsd: 2.1 A) and the Nl domain of the Streptococcus agalactiae minor pilin GBS52 (PDB ID code 2PZ4; rmsd: 3.4 A). Likewise RrgB Dl, none of these domains contain intra-molecular isopeptide bonds. However, only in the case of SpaA the Dl domain is homologue to the N-terminus of the protein (residues 54-192; see Figure 39A for the superimposition).

In an attempt to determine the orientation of Dl with respect to the x-ray structure of RrgB D2-D4, a rigid body fitting of RrgB Dl NMR structure was attempted within the electron density map of the native pilus obtained at 22A resolution from cryo-electron microscopy and single particle approach. A previously performed fitting of the RrgB D2-D4 crystal structure into the EM density map of the native pilus left an empty space, likely occupied by the missing Dl domain. However, when the rigid body fitting approach was applied for Dl, we found that simultaneous positioning of the long loops and the a-helix was not possible, indicating that the surface taken up by the isolated Dl domain significantly differs with respect to the full polymer.

Therefore, in order to obtain a model of the full-length RrgB protein architecture we positioned the Dl NMR on top of the x-ray D2-D4 crystal structure in a way that the four C-terminal amino acids of Dl can be superimposed to the corresponding residues present within the N-terminal portion of the D2-D4 crystal (Figure 39B). This model represents only one of the possible orientations that Dl can assume with respect to the rest of the RrgB protein, as it potentially has 360° of flexibility.

RrgB chimeras as carrier proteins

In addition to acting as vaccine components, the RrgB chimeras are suitable for use as carrier proteins in saccharide-carrier conjugates. The Ι-ΙΙ-ΙΠ and ΠΙ-ΙΙ-Ι chimeras were conjugated to a saccharide immunogen and IgG responses (GMT) against the saccharide were then measured by ELISA. Results were compared to a number of other pneumococcal proteins, and also to N19 and CRM197 as positive controls. Results from study VLVII were as follows: CRM197 N19 I-II-III ΙΠ-ΙΙ-Ι 1287 LRP 1875

2688 1004 638 133 25 114 114

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

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Claims

1. An immunogenic composition comprising:

(a) a first polypeptide comprising a first amino acid sequence, where the first amino acid sequence comprises or consists of: SEQ ID NO.100, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 100, or an amino acid sequence that competes with SEQ ID NO. 100 for binding to an antibody raised against SEQ ID NO.100, or a fragment of at least 7 amino acids of SEQ ID NO.100; and/or

(b) a second polypeptide, comprising a second amino acid sequence, where the second amino acid sequence comprises or consists of: SEQ ID NO.101, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 101, or an amino acid sequence that competes with SEQ ID NO.101 for binding to an antibody raised against SEQ ID NO. 101 , or a fragment of at least 7 amino acids of SEQ ID NO.101 ; and/or

(c) a third polypeptide, comprising a third amino acid sequence, where the third amino acid sequence comprises or consists of: SEQ ID NO.102, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 102, or an amino acid sequence that competes with SEQ ID NO.102 for binding to an antibody raised against SEQ ID NO.102, or a fragment of at least 7 amino acids of SEQ ID NO.102; and/or

(d) a fourth polypeptide, comprising a fourth amino acid sequence, where the fourth amino acid sequence comprises or consists of: SEQ ID NO.103, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 103, or an amino acid sequence that competes with SEQ ID NO.103 for binding to an antibody raised against SEQ ID NO.103, or a fragment of at least 7 amino acids of SEQ ID NO.103; and/or

(e) a fifth polypeptide, comprising a fifth amino acid sequence, where the fifth amino acid sequence comprises or consists of: SEQ ID NO.104, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 104, or an amino acid sequence that competes with SEQ ID NO.104 for binding to an antibody raised against SEQ ID NO.104, or a fragment of at least 7 amino acids of SEQ ID NO.104; and/or

(f) a sixth polypeptide, comprising a sixth amino acid sequence, where the sixth amino acid sequence comprises or consists of: SEQ ID NO.105, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 105, or an amino acid sequence that competes with SEQ ID NO.105 for binding to an antibody raised against SEQ ID NO.105, or a fragment of at least 7 amino acids of SEQ ID NO.105.

2. An immunogenic composition according to claim 1, wherein the first, second, third, fourth, fifth and/or sixth polypeptide contains 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 30 or fewer, or 25 or fewer amino acid residues.

3. A polypeptide comprising a first, second, third, fourth, fifth and/or sixth amino acid sequence as defined in claim 1.

4. A polypeptide comprising amino acid sequence:

A-{-X-L-} _n-B

wherein: each X is an amino acid sequence of first amino acid sequence, second amino acid sequence, third amino acid sequence, fourth amino acid sequence, fifth amino acid sequence or sixth amino acid sequence as defined in claim 1 ; L is an optional linker amino acid sequence; A is an optional N terminal amino acid sequence; B is an optional C terminal amino acid sequence; n is an integer of 2 or more.

5. A polypeptide according to claim 3 or claim 4, consisting of 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 34, 33 or fewer amino acid residues.

6. An immunogenic composition according to claim 1 or claim 2, or a polypeptide according to any of claims 3 to 5, comprising two, three, four, five or six different amino acid sequences.

7. An immunogenic composition or polypeptide according to claim 6, comprising amino acid sequences from two or three different clades.

8. An immunogenic composition or polypeptide according to claim 7, comprising at least one amino acid sequence selected from two or more of the following groups:

(d) the first and second amino acid sequence;

(e) the third and fourth amino acid sequence; and

(f) the fifth and sixth amino acid sequence.

9. A bacterium which expresses a polypeptide according to any of claims 3 to 8.

10. An antibody that binds to a polypeptide according to any of claims 3 to 8.