AU2012216698B2 - Pneumococcal polysaccharide conjugate vaccine - Google Patents

Abstract

Abstract The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, a multivalent Streptococcus pneumoniae immunogenic 5 composition is provided with various conjugated capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, where the composition comprises serotype 19F capsular saccharide conjugated to diphtheria toxoid (DT) or CRM197, optionally wherein 19F is the only saccharide in the composition conjugated to diphtheria toxoid (DT) or CRM 197.

Description

AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: GlaxoSmithKline Biologicals SA Actual Inventors: Ralph Leon Biemans and Nathalie Marie-Josephe Garcon and Philippe Vincent Hermand and Jan Poolman and Marcelle Paulette Van Mechelen Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: Pneumococcal polysaccharide conjugate vaccine Details of Original Application No. 2006327036 dated 20 Dec 2006 The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 58836AUP01 -la The present application is a divisional application of Australian Application No. 2006327036, which is incorporated in its entirety herein by reference. Field of the Invention The present invention relates to an improved Streptococcus pneumoniae vaccine. 5 Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Children less than 2 years of age do not mount an immune response to most 10 polysaccharide vaccines, so it has been necessary to render the polysaccharides immunogenic by chemical conjugation to a protein carrier. Coupling the polysaccharide, a T-independent antigen, to a protein, a T-dependent antigen, confers upon the polysaccharide the properties of T dependency including isotype switching, affinity maturation, and memory induction. 15 However, there can be issues with repeat administration of polysaccharide protein conjugates, or the combination of polysaccharide-protein conjugates to form multivalent vaccines. For example, it has been reported that a Haemophilus influenzae type b polysaccharide (PRP) vaccine using tetanus toxoid (TT) as the protein carrier was tested in a dosage-range with simultaneous immunization with (free) TT and a 20 pneumococcal polysaccharide-TT conjugate vaccine following a standard infant schedule. As the dosage of the pneumococcal vaccine was increased, the immune response to the PRP polysaccharide portion of the Hib conjugate vaccine was decreased, indicating immune interference of the polysaccharide, possibly via the use of the same carrier protein (Dagan et al., Infect Immun. (1998); 66: 2093-2098) 25 The effect of the carrier-protein dosage on the humoral response to the protein itself has also proven to be multifaceted. In human infants it was reported that increasing the dosage of a tetravalent tetanus toxoid conjugate resulted in a decreased response to the tetanus carrier (Dagan et al, supra). Classical analysis of these effects of combination vaccines have been described as carrier induced epitopic suppression, 30 which is not fully understood, but believed to result from an excess amount of carrier protein (Fattom, Vaccine 17: 126 (1999)). This appears to result in competition for Th cells, by the B-cells to the carrier protein, and B-cells to the polysaccharide. If the B cells to the carrier protein predominate, there are not enough Th-cells available to - lb provide the necessary help for the B-cells specific to the polysaccharide. However, the observed immunological effects have been inconsistent, with the total amount of carrier protein in some instances increasing the immune response, and in other cases diminishing the immune response. 5 Hence there remain technical difficulties in combining multiple polysaccharide conjugates into a single, efficacious, vaccine formulation. Streptococcus pneumoniae is a Gram-positive bacterium responsible for considerable morbidity and mortality (particularly in the young and aged), causing invasive 5 diseases such as pneumonia, bacteraemia and meningitis, and diseases associated with colonisation, such as acute Otitis media. The rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000. In 20% of cases this leads to bacteraemia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment. 10 Pneumococcus is encapsulated with a chemically linked polysaccharide which confers serotype specificity. There are 90 known serotypes of pneumococci, and the capsule is the principle virulence determinant for pneumococci, as the capsule not only protects the inner surface of the bacteria from complement, but is itself poorly immunogenic. Polysaccharides are T-independent antigens, and can not be processed or 15 presented on MHC molecules to interact with T-cells. They can however, stimulate the immune system through an alternate mechanism which involves cross-linking of surface receptors on B cells. It was shown in several experiments that protection against invasive pneumococci disease is correlated most strongly with antibody specific for the capsule, and the 20 protection is serotype specific. Streptococcus pneumoniae is the most common cause of invasive bacterial disease and Otitis media in infants and young children. Likewise, the elderly mount poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol. 42:265-270], hence the increased incidence of bacterial pneumonia in this population [Verghese and 25 Berk, (1983) Medicine (Baltimore) 62:271-285]. The major clinical syndromes caused by S. pneumoniae are widely recognized and discussed in all standard medical textbooks (Fedson DS, Muscher OM. In: Plotkin SA, Orenstein WA, editors. Vaccines. 4rth edition. PhiladelphiaWB Saunders Co, 2004a: 529 588). For instance, Invasive pneumococcal disease (IPD) is defined as any infection in 30 which S. pneumoniae is isolated from the blood or another normally sterile site (Musher DM. Streptococcus pneumoniae. In Mandell GL, Bennett JE, Dolin R (eds). Principles and Practice of Infectious diseases (5th ed). New York, Churchill Livingstone, 2001, p2128 2147). Chronic obstructive pulmonary disease (COPD) is recognised as encompassing several conditions (airflow obstruction, chronic bronchitis, bronchiolitis or small airways 35 disease and emphysema) that often coexist. Patients suffer exacerbations of their 2 condition that are usually associated with increased breathlessness, and often have increased cough that may be productive of mucus or purulent sputum (Wilson, Eur Respir J 2001 17:995-1007). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and/or emphysema 5 (Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society. Am J Respir Crit Care Med. 1995 Nov;152(5 Pt 2):S77 121 ). Exacerbations of COPD are often caused by bacterial (e.g. pneumococcal) infection (Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. Clin Microbiol Rev. 2001 Apr;14(2):336-63). 0 Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. 5 Summary of the Invention The present invention relates to developing an improved formulation of a multiple serotype Streptococcus pneumoniae polysaccharide conjugate vaccine. According to a first aspect, the present invention provides a Streptococcus pneumoniae immunogenic composition comprising 10 or more capsular saccharides from 0 different S. pneumoniae serotypes which are conjugated to a carrier protein, and comprising 3 or more different carrier proteins, wherein the composition comprises serotype 19F capsular saccharide conjugated to diphtheria toxoid (DT), serotype 18C capsular saccharide conjugated to tetanus toxoid (TT) and serotype 1, 4, 5, 6B, 7F, 9V, 14 and 23F capsular saccharides conjugated to protein D from Haemophilus influenzae. 25 According to a second aspect, the present invention provides a method of treating or preventing a disease caused by Streptococcus pneumoniae serotype 19A strain infection said method comprising the step of administering to a subject in need thereof a Streptococcus pneumoniae immunogenic composition comprising 9 or more, 10 or more, 11 of more, 13 or more, or 14 or more capsular saccharides from different S.pneumoniae 30 serotypes conjugated to 2 or more different carrier proteins, wherein the composition comprises serotype 19F capsular saccharide conjugated to CRM197 using CDAP chemistry, but does not comprise capsular saccharide from serotype 19A. 3 According to a third aspect, the present invention provides the use of Streptococcus pneumoniae immunogenic compositions comprising 9 or more, 10 or more, 11 of more, 13 or more, or 14 or more capsular saccharides from different S.pneumoniae serotypes conjugated to 2 or more different carrier proteins, wherein the composition comprises 5 serotype 19F capsular saccharide conjugated to CRM197 using CDAP chemistry, but does not comprise capsular saccharide from serotype 19A in the preparation of a medicament for the treatment or prevention of a disease caused by Streptococcus pneumoniae serotype 19A strain infection. Unless the context clearly requires otherwise, throughout the description and the 0 claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Brief description of Figures Figure 1 Bar chart showing 11 valent conjugate immunogenicity in elderly Rhesus 5 monkeys. The lighter bars represent the GMC after two inoculations with 11 valent conjugate in aluminium phosphate adjuvant. The darker bars represent the GMC after two inoculations with 11 valent conjugate in adjuvant C. Figure 2 Bar chart showing memory B cells for PS3 after inoculation with the 11 valent conjugate in adjuvant C or aluminium phosphate adjuvant. 0 Figure 3 Bar chart showing anti polysaccharide 19F immunogenicity in Balb/C mice for the 4-valent plain polysaccharides and the 4-valent dPly conjugates. Figure 4 Bar chart showing anti polysaccharide 22F immunogenicity in Balb/C mice for the 4-valent plain polysaccharides and the 4-valent PhtD conjugates. Figure 5 Bar chart showing anti-22F IgG response in Balb/c mice. 25 Figure 6 Bar chart showing anti-22F opsono-phagocytosis titres in Balb/c mice. Figure 7 Bar chart comparing IgG responses induced in young C57B1 mice after immunisation with 13 Valent conjugate vaccine formulated in different adjuvants. 3a Figure 8 Bar chart showing the protective efficacy of different vaccine combinations in a monkey pneumonia model. 5 Figure 9 Bar chart showing anti PhtD IgG response in Balb/c mice after immunisation with 22F-PhtD or 22F-AH-PhtD conjugates. Figure 10 Protection against type 4 pneumococcal challenge in mice after immunisation with 22F-PhtD or 22F-AH-PhtD. 10 Description of the Invention The present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more (e.g. 11, 12, 13, 14, or 15 or more) capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more carrier proteins, wherein the 15 vaccine comprises serotype 19F capsular saccharide conjugated to diphtheria toxoid or CRMI197, and wherein the vaccine optionally further comprises protein D from Haemophilus influenzae as free protein or as a further carrier protein or both. For the purposes of this invention, "immunizing a human host against exacerbations of 20 COPD" or "treatment or prevention of exacerbations of COPD" or "reduction in severity of COPD exacerbations" refers to a reduction in incidence or rate of COPD exacerbations (for instance a reduction in rate of 0.1, 0.5, 1, 2, 5, 10, 20% or more) or a reduction in severity of COPD exacerbations as defined above, for instance within a patient group immunized with the compositions or vaccines of the invention. 25 Typically the Streptococcus pneumoniae vaccine of the present invention will comprise capsular saccharide antigens (preferably conjugated), wherein the saccharides are derived from at least ten serotypes of S. pneumoniae. The number of S. pneumonlae capsular saccharides can range from 10 different serotypes (or "V", valences) to 23 30 different serotypes (23V). In one embodiment there are 10, 11, 12, 13, 14 or 15 different serotypes. In another embodiment of the invention, the vaccine may comprise conjugated S. pneumoniae saccharides and unconjugated S. pneumoniae saccharides. Preferably, the total number of saccharide serotypes is less than or equal to 23. For example, the invention may comprise 10 conjugated serotypes and 13 unconjugated saccharides. In a 4 similar manner, the vaccine may comprise 11, 12, 13, 14, 15 or 16 conjugated saccharides and 12, 11, 10, 9, 8 or 7, respectively, unconjugated saccharides. In one embodiment the multivalent pneumococcal vaccine of the invention will be selected 5 from the following serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it is appreciated that one or two other serotypes could be substituted depending on the age of the recipient receiving the vaccine and the geographical location where the vaccine will be administered, e.g. serotype 6A may be included on the list. For example, an 10-valent vaccine may 10 comprise polysaccharides from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent vaccine may also include saccharides from serotype 3. A 12 or 13-valent paediatric (infant) vaccine may also include the 10 or 11 valent formulation supplemented with serotypes 6A and 19A, or 6A and 22F, or 19A and 22F, or 6A and 158, or 19A and 15B, or 22F and 15B, whereas a 13-valent elderly vaccine may include the 11 valent 15 formulation supplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15B, or 8 and 19A, or 8 and 22F, or 12F and 15B, or 12F and 19A, or 12F and 22F, or 15B and 19A, or 15B and 22F. A 14 valent pediatric vaccine may include the 10 valent formulation described above supplemented with serotypes 3, 6A, 19A and 22F; serotypes 6A, 8, 19A and 22F; serotypes 6A, 12F, 19A and 22F; serotypes 6A, 15B, 19A and 22F; serotypes 3, 20 8, 19A and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15B, 19A and 22F; serotypes 3, 6A, 8 and 22F; serotypes 3, 6A, 12F and 22F; or serotypes 3, 6A, 15B and 22F. The composition in one embodiment includes capsular saccharides derived from 25 serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated). In a further embodiment of the invention at least 11 saccharide antigens (preferably conjugated) are included, for example capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In a further embodiment of the invention, at least 12 or 13 saccharide antigens are included, for example a vaccine may comprise capsular 30 saccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F or capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F, although further saccharide antigens, for example 23 valent (such as serotypes 1, 2, 3, 4, 5, 68, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention. 35 5 The vaccine of the present invention may comprise protein D (PD) from Haemophilus influenzae (see e.g. EP 0594610). Haemophilus influenzae is a key causative organism of otitis media, and the present inventors have shown that including this protein in a Streptococcus pneumoniae vaccine will provide a level of protection against Haemophilus 5 influenzae related otitis media (reference POET publication). In one embodiment, the vaccine composition comprises protein D. In one aspect, PD is present as a carrier protein for one or more of the saccharides. In another aspect, protein D could be present in the vaccine composition as a free protein. In a further aspect, protein D is present both as a carrier protein and as free protein. Protein D may be used as a full length protein or 10 as a fragment (WO0056360). In a further aspect, protein D is present as a carrier protein for the majority of the saccharides, for example 6, 7, 8, 9 or more of the saccharides may be conjugated to protein D. In this aspect, protein D may also be present as free protein. The vaccine of the present invention comprises two or more different types of carrier 15 protein. Each type of carrier protein may act as carrier for more than one saccharide, which saccharides may be the same or different. For example, serotypes 3 and 4 may be conjugated to the same carrier protein, either to the same molecule of carrier protein or to different molecules of the same carrier protein. In one embodiment, two or more different saccharides may be conjugated to the same carrier protein, either to the same molecule 20 of carrier protein or to different molecules of the same carrier protein. Each Streptococcus pneumoniae capsular saccharide may be conjugated to a carrier protein independently selected from the group consisting of TT, DT, CRM1 97, fragment C of TT, PhtD, PhtDE fusions (particularly those described in WO 01/98334 and WO 25 03/54007), detoxified pneumolysin and protein D, other than saccharide from serotype 19F which is always conjugated to DT or CRM 197, preferably DT. A more complete list of protein carriers that may be used in the conjugates of the invention is presented below If the protein carrier is the same for 2 or more saccharides in the composition, the 30 saccharides could be conjugated to the same molecule of the protein carrier (carrier molecules having 2 more different saccharides conjugated to it) [see for instance WO 04/083251]. Alternatively the saccharides may each be separately conjugated to different molecules of the protein carrier (each molecule of protein carrier only having one type of saccharide conjugated to it). 35 6 The carrier protein conjugated to one or more of the S. pneumoniae capsular saccharides in the conjugates present in the immunogenic compositions of the invention is optionally a member of the polyhistidine triad family (Pht) proteins, fragments or fusion proteins thereof. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acid sequence 5 sharing 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed in WO 00/37105 or WO 00/39299 (e.g. with amino acid sequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). For example, fusion proteins are composed of full length or fragments of 2, 3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are PhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B, PhtD/E, PhtE/A, 10 PhtE/B and PhtE/D, wherein the proteins are linked with the first mentioned at the N terminus (see for example WO01/98334). Where fragments of Pht proteins are used (separately or as part of a fusion protein), each fragment optionally contains one or more histidine triad motif(s) and/or coiled coil regions 15 of such polypeptides. A histidine triad motif is the portion of polypeptide that has the sequence HxxHxH where H is histidine and x is an amino acid other than histidine. A coiled coil region is a region predicted by "Coils" algorithm Lupus, A et al (1991) Science 252; 1162-1164. In an embodiment the or each fragment includes one or more histidine triad motif as well as at least one coiled coil region. In an embodiment, the or each 20 fragment contains exactly or at least 2, 3, 4 or 5 histidine triad motifs (optionally, with native Pht sequence between the 2 or more triads, or intra-triad sequence that is more than 50, 60, 70, 80, 90 or 100 % identical to a native pneumococcal intra-triad Pht sequence - e.g. the intra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for PhtD). In an embodiment, the or each fragment contains exactly or at least 2, 3 or 4 coiled 25 coil regions. In an embodiment a Pht protein disclosed herein includes the full length protein with the signal sequence attached, the mature full length protein with the signal peptide (for example 20 amino acids at N-terminus) removed, naturally occurring variants of Pht protein and immunogenic fragments of Pht protein (e.g. fragments as described above or polypeptides comprising at least 15 or 20 contiguous amino acids from an amino 30 acid sequence in WOOO/37105 or WOOO/39299 wherein said polypeptide is capable of eliciting an immune response specific for said amino acid sequence in WOOO/37105 or WOOO/39299). In particular, the term "PhtD" as used herein includes the full length protein with the signal 35 sequence attached, the mature full length protein with the signal peptide (for example 20 7 amino acids at N-terminus) removed, naturally occurring variants of PhtD and immunogenic fragments of PhtD (e.g. fragments as described above or polypeptides comprising at least 15 or 20 contiguous amino acids from a PhtD amino acid sequence in WOOO/37105 or WO00/39299 wherein said polypeptide is capable of eliciting an immune 5 response specific for said PhtD amino acid sequence in W000137105 or W00/139299 (e.g. SEQ ID NO: 4 of WO 00/37105 for PhtD). If the protein carrier is the same for 2 or more saccharides in the composition, the saccharides could be conjugated to the same molecule of the protein carrier (carrier 10 molecules having 2 more different saccharides conjugated to it) [see for instance WO 04/083251]. Alternatively the saccharides may each be separately conjugated to different molecules of the protein carrier (each molecule of protein carrier only having one type of saccharide conjugated to it). 15 Examples of carrier proteins which may be used in the present invention are DT (Diphtheria toxoid), TT (tetanus toxid) or fragment C of TT, DT CRM197 (a DT mutant) other DT point mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, 20 Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gin or Ser and/or Ala 158 to Gly and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5917017 or US 6455673; or fragment disclosed in US 5843711, pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13) 25 including ply detoxified in some fashion for example dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for example PhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007), (Pht A-E are described in more detail below) OMPC (meningococcal outer membrane protein - usually extracted from N. meningitidis serogroup B - EP0372501), 30 PorB (from N. meningitidis), PD (Haemophilus influenzae protein D - see, e.g., EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes from various 35 pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as 8 N19 protein (Baraldoi et aI (2004) Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761). 5 Nurkka et al Pediatric Infectious Disease Journal. 23(11):1008-14, 2004 Nov. described an 11 valent pneumococcal vaccine with all serotypes conjugated to PD. However, the present inventors have shown that opsonophagocytic activity was improved for antibodies induced with conjugates having 19F conjugated to DT compared with 19F conjugated to PD. In addition, the present inventors have shown that a greater cross 10 reactivity to 19A is seen with 19F conjugated to DT. It is therefore a feature of the composition of the present invention that serotype 19F is conjugated to DT or CRM 197. In one aspect, serotype 19F is conjugated to DT. The remaining saccharide serotypes of the immunogenic composition may all be conjugated to one or more carrier proteins that are not DT (i.e. only 19F is conjugated to DT), or may be split between one or more 15 carrier proteins that are not DT and DT itself. In one embodiment, 19F is conjugated to DT or CRM 197 and all of the remaining serotypes are conjugated to PD. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are split between PD, and TT or DT or CRM 197. In a further embodiment, 19F is conjugated to DT or CRM 197 and no more than one saccharide is conjugated to TT. In one aspect of 20 this embodiment, said one saccharide is 18C or 12F. In a further embodiment, 19F is conjugated to DT or CRM 197 and no more than two saccharides are conjugated to TT. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are split between PD, TT and DT or CRM 197. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are split between PD, TT and 25 pneumolysin. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are split between PD, TT and CRM 197. In a further embodiment, 19F is conjugated to DT or CRM197 and the remaining serotypes are split between PD, TT, pneumolysin and optionally PhtD or PhtD/E fusion protein. In a further embodiment, 19F is conjugated to DT or CRM197, 19A is conjugated to pneumolysin or TT, one (two 30 or three) further saccharide(s) is conjugated to TT, one further saccharide is conjugated to PhtD or PhtD/E and all further saccharides are conjugated to PD. In a further embodiment 19F is conjugated to DT or CRMI197, 19A is conjugated to pneumolysin , one (two or three) further saccharide(s) is conjugated to TT, one further saccharide is conjugated to pneumolysin, 2 further saccharides are conjugated to PhtD or PhtD/E and all further 35 saccharides are conjugated to PD. 9 In one embodiment, the immunogenic composition of the invention comprises protein D from Haemophilus influenza. Within this embodiment, If PD is not one of the carrier proteins used to conjugate any saccharides other than 19F, for example 19F is 5 conjugated to DT whilst the other serotypes are conjugated to one or more different carrier proteins which are not PD, then PD will be present in the vaccine composition as free protein. If PD is one of the carrier proteins used to conjugate saccharides other than 19F, then PD may optionally be present in the vaccine composition as free protein. 10 The term "saccharide" throughout this specification may indicate polysaccharide or oligosaccharide and includes both. Polysaccharides are isolated from bacteria and may be sized to some degree by known methods (see for example EP497524 and EP497525) and preferably by microfluidisation. Polysaccharides can be sized in order to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products. 15 Oligosaccharides have a low number of repeat units (typically 5-30 repeat units) and are typically hydrolysed polysaccharides Capsular polysaccharides of Streptococcus pneumoniae comprise repeating oligosaccharide units which may contain up to 8 sugar residues. For a review of the 20 oligosaccharide units for the key Streptococcus pneumoniae serotypes see JONES, Christopher. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Cisnc., June 2005, vol.77, no.2, p.293-324. ISSN 0001-3765. In one embodiment, a capsular saccharide antigen may be a full length polysaccharide, however in others it may be one oligosaccharide unit, or a shorter than native length 25 saccharide chain of repeating oligosaccharide units. In one embodiment, all of the saccharides present in the vaccine are polysaccharides. Full length polysaccharides may be "sized" i.e. their size may be reduced by various methods such as acid hydrolysis treatment, hydrogen peroxide treatment, sizing by emulsiflex@ followed by a hydrogen peroxide treatment to generate oligosaccharide fragments or microfluidization. 30 The inventors have also noted that the focus of the art has been to use oligosaccharides for ease of conjugate production. The inventors have found that by using native or slightly sized polysaccharide conjugates, one or more of the following advantages may be realised: 1) a conjugate having high immunogenicity which is filterable, 2) the ratio of 35 polysaccharide to protein in the conjugate can be altered such that the ratio of 10 polysaccharide to protein (w/w) in the conjugate may be increased (which can have an effect on the carrier suppression effect), 3) immunogenic conjugates prone to hydrolysis may be stabilised by the use of larger saccharides for conjugation. The use of larger polysacchardes can result in more cross-linking with the conjugate carrier and may 5 lessen the liberation of free saccharde from the conjugate. The conjugate vaccines described in the prior art tend to depolymerise the polysaccharides prior to conjugation in order to improve conjugation. The present inventors have found that saccharide conjugate vaccines retaining a larger size of saccharide can provide a good immune response against pneumococcal disease. 10 The immunogenic composition of the invention may thus comprise one or more saccharine conjugates wherein the average size (e.g. weight-average molecular weight; M,) of each saccharide before conjugation is above 80kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa or 10OOkDa. In one embodiment one or more saccharide conjugates of 15 the invention should have an average size of saccharide pre-conjugation of 50-1600, 80 1400, 100-1000, 150-500, or 200-400 kDa (note that where average size is M. , 'kDa' units should be replaced herein with 'x10 3 '). In one embodiment the conjugate post conjugation should be readily filterable through a 0.2 micron filter such that a yield of more than 50, 60, 70, 80, 90 or 95% is obtained post filtration compared with the pre filtration 20 sample. For the purposes of the invention, "native polysaccharide" refers to a saccharde that has not been subjected to a process (e.g. post-purification), the purpose of which is to reduce the size of the saccharide. A polysaccharide can become slightly reduced in size during 25 normal purification procedures. Such a saccharide is still native. Only if the polysaccharide has been subjected to sizing techniques would the polysaccharide not be considered native. For the purposes of the invention, "sized by a factor up to x2" means that the saccharide 30 is subject to a process intended to reduce the size of the saccharide but to retain a size more than half the size of the native polysaccharide. X3, x4 etc. are to be interpreted in the same way i.e. the saccharide is subject to a process intended to reduce the size of the polysaccharide but to retain a size more than a third, a quarter etc. the size of the native polysaccharide. 11 In an aspect of the invention, the immunogenic composition comprises Streptococcus pneumoniae saccharides from at least 10 serotypes conjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each S. pneumoniae saccharide is native polysaccharide. 5 In an aspect of the invention, the immunogenic composition comprises Streptococcus pneumoniae saccharides from at least 10 serotypes conjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each S. pneumoniae saccharide is sized by a factor up to x2, x3, x4, x5, x6, x7, x8, x9 or x1O. In one embodiment of this aspect, the 10 majority of the saccharides, for example 6, 7, 8 or more of the saccharides are sized by a factor up to x2, x3, x4, x5, x6, x7, x8, x9 or x 10. The molecular weight or average molecular weight (or size) of a saccharide herein refers to the weight-average molecular weight (M) of the saccharide measured prior to 15 conjugation and is measured by MALLS. The MALLS technique is well known in the art and is typically carried out as described in example 2. For MALLS analysis of pneumococcal saccharides, two columns (TSKG6000 and 5000PWx) may be used in combination and the saccharides are eluted in water. 20 Saccharides are detected using a light scattering detector (for instance Wyatt Dawn DSP equipped with a 10mW argon laser at 488nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm). In an embodiment the S. pneumoniae saccharides are native polysaccharides or native 25 polysaccharides which have been reduced in size during a normal extraction process. In an embodiment, the S. pneumoniae saccharides are sized by mechanical cleavage, for instance by microfluidisation or sonication. Microfluidisation and sonication have the advantage of decreasing the size of the larger native polysaccharides sufficiently to 30 provide a filterable conjugate. Sizing is by a factor of no more than x20, x1O, x8, x6, x5, x4, x3 or x2. In an embodiment, the immunogenic composition comprises S. pneumoniae conjugates that are made from a mixture of native polysaccharides and saccharides that are sized by 35 a factor of no more than x20. In one aspect of this embodiment, the majority of the 12 saccharides, for example 6, 7, 8 or more of the saccharides are sized by a factor of up to x2, x3, x4, x5 or x6. In an embodiment, the Streptococcus pneumoniae saccharide is conjugated to the carrier 5 protein via a linker, for instance a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, having for example a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The linker has for example between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible linker is ADH. Other linkers include B-propionamido (WO 00/10599), 10 nitrophenyl-ethylamine (Gever et al (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (US4057685), glycosidic linkages (US4673574, US4808700), hexane diamine and 6-aminocaproic acid (US4459286). In an embodiment, ADH is used as a linker for conjugating saccharide from serotype 18C. In an embodiment, ADH is used as a linker for conjugating saccharide from serotype 22F. 15 The saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique. The conjugation method may rely on activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled directly 20 or via a spacer (linker) group to an amino group on the carrier protein. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (for example using iodoacetimide [e.g. ethyl iodoacetimide HCl] or N-succinimidyl 25 bromoacetate or SlAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the protein carrier. Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 30 95/08348 and WO 96/29094 Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98/42721. Conjugation may involve a carbonyl linker which may be formed by reaction of 35 a free hydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 13 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction of with a protein to form a carbamate linkage. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group' reaction of the primary hydroxyl group with CDI to form a CDI carbamate 5 intermediate and coupling the CDI carbamate intermediate with an amino group on a protein. The conjugates can also be prepared by direct reductive amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in 10 EP-0-161-188, EP-208375 and EP-0-477508. A further method involves the coupling of a cyanogen bromide (or CDAP) activated saccharide derivatised with adipic acid dihydrazide (ADH) to the protein carrier by Carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256), for example 15 using EDAC. In an embodiment, a hydroxyl group (preferably an activated hydroxyl group for example a hydroxyl group activated to make a cyanate ester [e.g. with CDAP]) on a saccharide is linked to an amino or carboxylic group on a protein either directly or indirectly (through a 20 linker). Where a linker is present, a hydroxyl group on a saccharide is preferably linked to an amino group on a linker, for example by using CDAP conjugation. A further amino group in the linker for example ADH) may be conjugated to a carboxylic acid group on a protein, for example by using carbodiimide chemistry, for example by using EDAC. In an embodiment, the pneumococcal capsular saccharide(s) is conjugated to the linker first 25 before the linker is conjugated to the carrier protein. Alternatively the linker may be conjugated to the carrier before conjugation to the saccharide. A combination of techniques may also be used, with some saccharide-protein conjugates being prepared by CDAP, and some by reductive amination. 30 In general the following types of chemical groups on a protein carrier can be used for coupling / conjugation: 14 A) Carboxyl (for instance via aspartic acid or glutamic acid). In one embodiment this group is linked to amino groups on saccharides directly or to an amino group on a linker with carbodiimide chemistry e.g. with EDAC. 5 B) Amino group (for instance via lysine). In one embodiment this group is linked to carboxyl groups on saccharides directly or to a carboxyl group on a linker with carbodiimide chemistry e.g. with EDAC, In another embodiment this group is linked to hydroxyl groups activated with CDAP or CNBr on saccharides directly or to such groups on a linker; to saccharides or linkers having an aldehyde group; to saccharides or linkers 10 having a succinimide ester group. C) Sulphydryl (for instance via cysteine). In one embodiment this group is linked to a bromo or chloro acetylated saccharide or linker with maleimide chemistry. In one embodiment this group is activated/modified with bis diazobenzidine. 15 D) Hydroxyl group (for instance via tyrosine). In one embodiment this group is activated/modified with bis diazobenzidine. E) Imidazolyl group (for instance via histidine). In one embodiment this group is 20 activated/modified with bis diazobenzidine. F) Guanidyl group (for instance via arginine). G) Indolyl group (for instance via tryptophan). 25 On a saccharide, in general the following groups can be used for a coupling: OH, COOH or NH2. Aldehyde groups can be generated after different treatments known in the art such as: periodate, acid hydrolysis, hydrogen peroxide, etc. 30 Direct coupling approaches: Saccharide-OH + CNBr or CDAP --- > cyanate ester + NH2-Prot -- > conjugate Saccharide-aldehyde + NH2-Prot -- > Schiff base + NaCNBH3 ----> conjugate Saccharide-COOH + NH2-Prot + EDAC -- > conjugate 35 Saccharide-NH2 + COOH-Prot + EDAC -- > conjugate 15 Indirect coupling via spacer (linker) approaches: Saccharide-OH + CNBr or CDAP -- > cyanate ester + NH2---NH2 -- > saccharide--- 5 NH2 + COOH-Prot + EDAC --- > conjugate Saccharide-OH + CNBr or CDAP ----> cyanate ester + NH2---SH -- > saccharide---SH + SH-Prot (native Protein with an exposed cysteine or obtained after modification of amino groups of the protein by SPDP for instance) -- > saccharide-S-S-Prot 10 Saccharide-OH + CNBr or CDAP --- > cyanate ester + NH2--SH -----> saccharide----SH + maleimide-Prot (modification of amino groups) --- > conjugate Saccharide-OH + CNBr or CDAP -- > cyanate ester + NH2---SH -> Saccharide-SH + 15 haloacetylated-Prot ----> Conjugate Saccharide-COOH + EDAC + NH2----NH2 ---> saccharide----NH2 + EDAC + COOH Prot --- > conjugate 20 Saccharide-COOH + EDAC+ NH2---SH -- > saccharide--SH + SH-Prot (native Protein with an exposed cysteine or obtained after modification of amino groups of the protein by SPDP for instance) --- > saccharide-S-S-Prot Saccharide-COOH + EDAC+ NH2---SH ----- > saccharide----SH + maleimide-Prot 25 (modification of amino groups) ----> conjugate Saccharide-COOH + EDAC + NH2--SH ---> Saccharide-SH + haloacetylated-Prot ----> Conjugate 30 Saccharide-Aldehyde + NH2--NH2 --- > saccharide---NH2 + EDAC + COOH-Prot --- > conjugate Note: instead of EDAC above, any suitable carbodiimide may be used. 16 In summary, the types of protein carrier chemical group that may be generally used for coupling with a saccharide are amino groups (for instance on lysine residues), COOH groups (for instance on aspartic and glutamic acid residues) and SH groups (if accessible) (for instance on cysteine residues. 5 Preferably the ratio of carrier protein to S. pneumoniae saccharide is between 1:5 and 5:1; e.g. between 1:0.5-4:1, 1:1-3.5:1, 1.2:1-3:1, 1.5:1-2.5:1; e.g. between 1:2 and 2.5:1; 1:1 and 2:1 (w/w). In an embodiment, the majority of the conjugates, for example 6, 7, 8, 9 or more of the conjugates have a ratio of carrier protein to saccharide that is greater than 10 1:1, for example 1.1:1. 1.2:1, 1.3:1, 1.4:1, 1.5:1 or 1.6:1. In an embodiment, at least one S. pneumoniae saccharide is conjugated to a carrier protein via a linker using CDAP and EDAC. For example, 18C or 22F may be conjugated to a protein via a linker (for example those with two hydrazino groups at its ends such as 15 ADH) using CDAP and EDAC as described above. When a linker is used, CDAP may be used to conjugate the saccharide to a linker and EDAC may then be used to conjugate the linker to a protein or, alternatively EDAC may be used first to conjugate the linker to the protein, after which CDAP may be used to conjugate the linker to the saccharide. 20 In general, the immunogenic composition of the invention may comprise a dose of each saccharide conjugate between 0.1 and 20g, 1 and 1Opg or 1 and 3pg of saccharide. In an embodiment, the immunogenic composition of the invention contains each S. pneumoniae capsular saccharide at a dose of between 0.1-20pg; 0.5-10pg; 0,5- 5pg or 1 25 3pg of saccharide. In an embodiment, capsular saccharides may be present at different dosages, for example some capsular saccharides may be present at a dose of exactly lpg or some capsular saccharides may be present at a dose of exactly 3pg. In an embodiment, saccharides from serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a higher dose than other saccharides. In one aspect of this embodiment, serotypes 3, 30 18C and 19F (or 4, 18C and 19F) are present at a dose of around or exactly 3 pg whilst other saccharides in the immunogenic composition are present at a dose of around or exactly 1 pg. "Around" or "approximately" are defined as within 10% more or less of the given figure for 35 the purposes of the invention. 17 In an embodiment, at least one of the S. pneumoniae capsular saccharides is directly conjugated to a carrier protein (e.g. using one of the chemistries described above). Preferably the at least one of the S. pneumoniae capsular saccharides is directly 5 conjugated by CDAP. In an embodiment, the majority of the capsular saccharides for example 5, 6, 7, 8, 9 or more are directly linked to the carrier protein by CDAP (see WO 95/08348 and WO 96/29094) The immunogenic composition may comprise Streptococcus pneumoniae proteins, herein 10 termed Streptococcus pneumoniae proteins of the invention. Such proteins may be used as carrier proteins, or may be present as free proteins, or may be present both as carrier proteins and as free proteins. The Streptococcus pneumoniae proteins of the invention are either surface exposed, at least during part of the life cycle of the pneumococcus, or are proteins which are secreted or released by the pneumococcus. Preferably the 15 proteins of the invention are selected from the following categories, such as proteins having a Type 11 Signal sequence motif of LXXC (where X is any amino acid, e.g., the polyhistidine triad family (PhtX)), choline binding proteins (CbpX), proteins having a Type I Signal sequence motif (e.g., Sp101), proteins having a LPXTG motif (where X is any amino acid, e.g., Sp128, Sp130), and toxins (e.g., Ply). Preferred examples within these 20 categories (or motifs) are the following proteins, or immunologically functional equivalents thereof. In one embodiment, the immunogenic composition of the invention comprises at least 1 protein selected from the group consisting of the Poly Histidine Triad family (PhtX), 25 Choline Binding Protein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133. In a further embodiment, the immunogenic composition comprises 2 or more proteins selected from the group consisting of the Poly Histidine Triad family (PhtX), Choline Binding Protein family (CbpX), CbpX truncates, LytX 30 family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA, and Sp128. In one more embodiment, the immunogenic composition comprises 2 or more proteins selected from the group consisting of the Poly Histidine Triad family (PhtX), Choline Binding Protein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins (or fusions), 35 pneumolysin (Ply), and Sp128. 18 The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB, PhtD, and PhtE. The family is characterized by a lipidation sequence, two domains separated by a proline-rich region and several histidine triads, possibly involved in metal or nucleoside binding or 5 enzymatic activity, (3-5) coiled-coil regions, a conserved N-terminus and a heterogeneous C terminus. It is present in all strains of pneumococci tested. Homologous proteins have also been found in other Streptococci and Neisseria. In one embodiment of the invention, the Pht protein of the invention is PhtD. It is understood, however, that the terms Pht A, B, D, and E refer to proteins having sequences disclosed in the citations below as well as 10 naturally-occurring (and man-made) variants thereof that have a sequence homology that is at least 90% identical to the referenced proteins. Preferably it is at least 95% identical and most preferably it is 97% identical. With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, and is also referred 15 to Sp36. As noted above, it is a protein from the polyhistidine triad family and has the type 11 signal motif of LXXC. PhtD is disclosed in WO 00/37105, and is also referred to SpO36D. As noted above, it also is a protein from the polyhistidine triad family and has the type 11 LXXC signal motif. PhtB is disclosed in WO 00/37105, and is also referred to Sp036B. Another member of the PhtB family is the C3-Degrading Polypeptide, as 20 disclosed in WO 00/17370. This protein also is from the polyhistidine triad family and has the type I1 LXXC signal motif. A preferred immunologically functional equivalent is the protein Sp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79kD) is disclosed in W099/15675 which is also considered a member of the PhtX family. PhtE is disclosed in WOOO/30299 and is referred to as BVH-3. Where any Pht protein is referred 25 to herein, it is meant that immunogenic fragments or fusions thereof of the Pht protein can be used. For example, a reference to PhtX includes immunogenic fragments or fusions thereof from any Pht protein. A reference to PhtD or PhtB is also a reference to PhtDE or PhtBE fusions as found, for example, in W00198334. 30 Pneumolysin is a multifunctional toxin with a distinct cytolytic (hemolytic) and complement activation activities (Rubins et al., Am. Respi. Cit Care Med, 153:1339-1346 (1996)). The toxin is not secreted by pneumococci, but it is released upon lysis of pneumococci under the influence of autolysin. Its effects include e.g., the stimulation of the production of inflammatory cytokines by human monocytes, the inhibition of the beating of cilia on 35 human respiratory epithelial, and the decrease of bactericidal activity and migration of 19 neutrophils. The most obvious effect of pneumolysin is in the lysis of red blood cells, which involves binding to cholesterol. Because it is a toxin, it needs to be detoxified (i.e., non-toxic to a human when provided at a dosage suitable for protection) before it can be administered in vivo. Expression and cloning of wild-type or native pneumolysin is known 5 in the art. See, for example, Walker et al. (Infect Immun, 55:1184-1189 (1987)), Mitchell et al. (Biochim Biophys Acta, 1007:67-72 (1989) and Mitchell et al (NAR, 18:4010 (1990)). Detoxification of ply can be conducted by chemical means, e.g., subject to formalin or glutaraldehyde treatment or a combination of both (WO 04081515, PCT/EP2005/010258). Such methods are well known in the art for various toxins. Alternatively, ply can be 10 genetically detoxified. Thus, the invention encompasses derivatives of pneumococcal proteins which may be, for example, mutated proteins. The term "mutated" is used herein to mean a molecule which has undergone deletion, addition or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method. For example, as described above, a mutant ply protein may be 15 altered so that it is biologically inactive whilst still maintaining its immunogenic epitopes, see, for example, W090/06951, Berry et al. (Infect Immun, 67:981-985 (1999)) and W099/03884. As used herein, it is understood that the term "Ply" refers to mutated or detoxified 20 pneumolysin suitable for medical use (i.e., non toxic). Concerning the Choline Binding Protein family (CbpX), members of that family were originally identified as pneumococcal proteins that could be purified by choline-affininty chromatography. All of the choline-binding proteins are non-covalently bound to 25 phosphorylcholine moieties of cell wall teichoic acid and membrane-associated lipoteichoic acid. Structurally, they have several regions in common over the entire family, although the exact nature of the proteins (amino acid sequence, length, etc.) can vary. In general, choline binding proteins comprise an N terminal region (N), conserved repeat regions (RI and/or R2), a proline rich region (P) and a conserved choline binding region 30 (C), made up of multiple repeats, that comprises approximately one half of the protein. As used in this application, the term "Choline Binding Protein family (CbpX)" is selected from the group consisting of Choline Binding Proteins as identified in W097/41151, PbcA, SpsA, PspC, CbpA, CbpD, and CbpG. CbpA is disclosed in W097/41151. CbpD and CbpG are disclosed in WOOO/29434. PspC is disclosed in W097/09994. PbcA is 35 disclosed in W098/21337.SpsA is a Choline binding protein disclosed in WO 98/39450. 20 Preferably the Choline Binding Proteins are selected from the group consisting of CbpA, PbcA, SpsA and PspC. Another preferred embodiment is CbpX truncates wherein "CbpX" is defined above and 5 "truncates" refers to CbpX proteins lacking 50% or more of the Choline binding region (C). Preferably such proteins lack the entire choline binding region. More preferably, the such protein truncates lack (i) the choline binding region and (ii) a portion of the N-terminal half of the protein as well, yet retain at least one repeat region (R1 or R2). More preferably still, the truncate has 2 repeat regions (RI and R2). Examples of such preferred 10 embodiments are NR1xR2 and R1xR2 as illustrated in W099/51266 or W099/51188, however, other choline binding proteins lacking a similar choline binding region are also contemplated within the scope of this invention. The LytX family is membrane associated proteins associated with cell lysis. The N 15 terminal domain comprises choline binding domain(s), however the LytX family does not have all the features found in the CbpA family noted above and thus for the present invention, the LytX family is considered distinct from the CbpX family. In contrast with the CbpX family, the C-terminal domain contains the catalytic domain of the LytX protein family. The family comprises LytA, B and C. With regards to the LytX family, LytA is 20 disclosed in Ronda et al., Eur J Biochem, 164:621-624 (1987). LytB is disclosed in WO 98/18930, and is also referred to as Sp46. LytC is also disclosed in WO 98/18930, and is also referred to as Sp9l. A preferred member of that family is LytC. Another preferred embodiment are LytX truncates wherein "LytX" is defined above and 25 "truncates" refers to LytX proteins lacking 50% or more of the Choline binding region. Preferably such proteins lack the entire choline binding region. Yet another preferred embodiment of this invention are CbpX truncate-LytX truncate chimeric proteins (or fusions). Preferably this comprises NR1xR2 (or R1xR2) of CbpX and the C-terminal portion (Cterm, i.e., lacking the choline binding domains) of LytX (e.g., LytCCterm or 30 Sp9l Cterm). More preferably CbpX is selected from the group consisting of CbpA, PbcA, SpsA and PspC. More preferably still, it is CbpA. Preferably, LytX is LytC (also referred to as Sp9l). Another embodiment of the present invention is a PspA or PsaA truncates lacking the choline binding domain (C) and expressed as a fusion protein with LytX. Preferably, LytX is LytC. 35 21 With regards to PsaA and PspA, both are know in the art. For example, PsaA and transmembrane deletion variants thereof have been described by Berry & Paton, Infect Immun 1996 Dec;64(12):5255-62. PspA and transmembrane deletion variants thereof have been disclosed in, for example, US 5804193, WO 92/14488, and WO 99/53940. 5 Sp128 and Sp130 are disclosed in W0O0076540. Sp125 is an example of a pneumococcal surface protein with the Cell Wall Anchored motif of LPXTG (where X is any amino acid). Any protein within this class of pneumococcal surface protein with this motif has been found to be useful within the context of this invention, and is therefore 10 considered a further protein of the invention. Sp125 itself is disclosed in WO 98/18930, and is also known as ZmpB - a zinc metalloproteinase. Sp101 is disclosed in WO 98/06734 (where it has the reference # y85993). It is characterized by a Type I signal sequence. Sp133 is disclosed in WO 98/06734 (where it has the reference # y85992). It is also characterized by a Type I signal sequence. 15 Examples of preferred Moraxella catarrhalis protein antigens which can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP106 (WO 97/41731 (Antex) & WO 96/34960 (PMC)]; OMP21 or fragments thereof (WO 0018910); LbpA &/or LbpB [WO 98/55606 (PMC)]; TbpA &/or TbpB [WO 97/13785 & WO 97/32980 20 (PMC)]; CopB [Helminen ME, et al. (1993) Infect. Immun. 61:2003-2010]; UspAl and/or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT/EP99/03824); PiIQ (PCT/EP99/03823); OMP85 (PCT/EP0O/01468); lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipol 1 (GB 9918302.2); lipol8 (GB 9918038.2); P6 (PCT/EP99/03038); D15 (PCT/EP99/03822); OmplAl (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and OmpE. 25 Examples of non-typeable Haemophilus influenzae antigens or fragments thereof which can be included in a combination vaccine (especially for the prevention of otitis media) include: Fimbrin protein [(US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides therefrom [eg LB1(f) peptide fusions; US 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of New 30 York)); TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmwl; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); P2; and P5 (WO 94/26304). The proteins of the invention may also be beneficially combined. By combined is meant that the immunogenic composition comprises all of the proteins from within the following 35 combinations, either as carrier proteins or as free proteins or a mixture of the two. For 22 example, in a combination of two proteins as set out hereinafter, both proteins may be used as carrier proteins, or both proteins may be present as free proteins, or both may be present as carrier and as free protein, or one may be present as a carrier protein and a free protein whilst the other is present only as a carrier protein or only as a free protein, or 5 one may be present as a carrier protein and the other as a free protein. Where a combination of three proteins is given, similar possibilities exist. Preferred combinations include, but are not limited to, PhtD + NR1xR2, PhtlD + NR1xR2-Sp9lCterm chimeric or fusion proteins, PhtD + Ply, PhtD + Sp128, PhtD + PsaA, PhtD + PspA, PhtA + NR1xR2, PhtA + NR1xR2-Sp91Cterm chimeric or fusion proteins, PhtA + Ply, PhtA + Sp128, PhtA 10 + PsaA, PhtA + PspA, NR1xR2 + LytC, NR1xR2 + PspA, NR1xR2 + PsaA, NR1xR2 + Sp128, R1xR2 + LytC, R1xR2 + PspA, R1xR2 + PsaA, R1xR2 + Sp128, R1xR2 + PhtD, RlxR2 + PhtA. Preferably, NR1xR2 (or R1xR2) is from CbpA or PspC. More preferably it is from CbpA. Other combinations include 3 protein combinations such as PhtD + NR1xR2 + Ply, and PhtA + NR1xR2 + PhtD. In one embodiment, the vaccine composition 15 comprises detoxified pneumolysin and PhtD or PhtDE as carrier proteins. In a further embodiment, the vaccine composition comprises detoxified pneumolysin and PhtD or PhtDE as free proteins. The present invention further provides a vaccine containing the immunogenic 20 compositions of the invention and a pharmaceutically acceptable excipient. The vaccines of the present invention may be adjuvanted, particularly when intended for use in an elderly population but also for use in infant populations. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or 25 alum, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes. It is preferred that the adjuvant be selected to be a preferential inducer of a THI type of 30 response. Such high levels of Th1-type cytokines tend to favour the induction of cell mediated immune responses to a given antigen, whilst high levels of Th2-type cytokines tend to favour the induction of humoral immune responses to the antigen. The distinction of Th1 and Th2-type immune response is not absolute. In reality an 35 individual will support an immune response which is described as being predominantly 23 Th1 or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) THI and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. (Annual Review of 5 Immunology, 7, p145-173). Traditionally, Thl-type responses are associated with the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by T-cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of 11-4, IL-5, IL-6, IL-10. Suitable adjuvant systems which promote a predominantly Th1 10 response include: Monophosphoryl lipid A or a derivative thereof, particularly 3-de-O acylated monophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with either an aluminum salt (for instance aluminum phosphate or aluminum hydroxide) or an oil-in-water emulsion. In such combinations, antigen and 30-MPL are 15 contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum-adsorbed antigen [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1]. 20 An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 25 95/17210. In one embodiment the immunogenic composition additionally comprises a saponin, which may be QS21. The formulation may also comprise an oil in water emulsion and tocopherol (WO 95/17210). Unmethylated CpG containing oligonucleotides (WO 96/02555) and other immunomodulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of a THI response and are suitable for use in 30 the present invention. Particular adjuvants are those selected from the group of metal Salts, oil in water emulsions, Toll like receptors agonist, (in particular Toll like receptor 2 agonist, Toll like receptor 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like 35 receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof. 24 An adjuvant that can be used with the vaccine compositions of the invention are bleb or outer membrane vesicle preparations from Gram negative bacterial strains such as those taught by W002/09746 - particularly N. meningitidis blebs. Adjuvant properties of blebs 5 can be improved by retaining LOS (lipooligosacccharide) on its surface (e.g. through extraction with low concentrations of detergent [for instanct 0-0.1% deoxycholate]). LOS can be detoxified through the msbB(-) or htrB(-) mutations discussed in W002/09746. Adjuvant properties can also be improved by retaining PorB (and optionally removing PorA) from meningococcal blebs. Adjuvant properties can also be improved by truncating 10 the outer core saccharide structure of LOS on meningococcal blebs - for instance via the lgtB(-) mutation discussed in W02004/014417. Alternatively, the aforementioned LOS (e.g. isolated from a msbB(-) and/or IgtB(-) strain) can be purified and used as an adjuvant in the compositions of the invention. 15 A further adjuvant which may be used with the compositions of the invention may be selected from the group: a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide, an alkyl glucosaminide phosphate, an oil in water emulsion or combinations thereof. A further preferred adjuvant is a metal salt in combination with another adjuvant. It is preferred that the adjuvant is a Toll like receptor agonist in 20 particular an agonist of a Toll like receptor 2, 3, 4, 7, 8 or 9, or a saponin, in particular Qs2l. It is further preferred that the adjuvant system comprises two or more adjuvants from the above list. In particular the combinations preferably contain a saponin (in particular Qs2l) adjuvant and/or a Toll like receptor 9 agonist such as a CpG containing immunostimulatory oligonucleotide. Other preferred combinations comprise a saponin (in 25 particular QS21) and a Toll like receptor 4 agonist such as monophosphoryl lipid A or its 3 deacylated derivative, 3 D - MPL, or a saponin (in particular QS21) and a Toll like receptor 4 ligand such as an alkyl glucosaminide phosphate. Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP 0 671 948 30 81), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP 0 689 454 B1). Other preferred adjuvant systems comprise a combination of 3 D MPL , QS21 and a CpG oligonucleotide as described in US6558670, US6544518. 25 In an embodiment the adjuvant is (or comprises) a Toll like receptor (TLR) 4 ligand, preferably an agonist such as a lipid A derivative particularly monophosphoryl lipid A or more particularly 3 Deacylated monophoshoryl lipid A (3 D - MPL). 5 3 D -MPL is available from GlaxoSmithKline Biologicals North America and primarily promotes CD4+ T cell responses with an IFN-g (Th1) phenotype . It can be produced according to the methods disclosed in GB 2 220 211 A. Chemically it is a mixture of 3 deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably in the compositions of the present invention small particle 3 D- MPL is used. Small particle 3 D 10 -MPL has a particle size such that it may be sterile-filtered through a 0.22pm filter. Such preparations are described in International Patent Application No. WO 94121292. Synthetic derivatives of lipid A are known and thought to be TLR 4 agonists including, but not limited to: 15 OM174 (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o phosphono-3-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-oX-D glucopyranosyldihydrogenphosphate), (WO 95/14026) OM 294 DP (3S, 9 R) -3--[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-(R) 20 3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate) (W099 /64301 and WO 00/0462 ) OM 197 MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9 ((R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1 -dihydrogenophosphate 10-(6 25 aminohexanoate) (WO01/46127) Other TLR4 ligands which may be used are alkyl Glucosaminide phosphates (AGPs) such as those disclosed in W09850399 or US6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in 30 US6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants. Another prefered immunostimulant for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quilaja 35 Saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. 26 in 1974 ("Saponin adjuvants", Archiv. for die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254). Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21). QS-21 is a natural saponin 5 derived from the bark of Quillaja saponaria Molina which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response and is a preferred saponin in the context of the present invention. Particular formulations of QS21 have been described which are particularly preferred, 10 these formulations further comprise a sterol (W096/33739). The saponins forming part of the present invention may be separate in the form of micelles, mixed micelles (preferentially, but not exclusively with bile salts) or may be in the form of ISCOM matrices (EP 0 109 942 B1) , liposomes or related colloidal structures such as worm-like or ring-like multimeric complexes or lipidic/layered structures and lamellae when formulated with 15 cholesterol and lipid, or in the form of an oil in water emulsion (for example as in WO 95/17210). The saponins may preferably be associated with a metallic salt, such as aluminium hydroxide or aluminium phosphate (WO 98/15287). Preferably, the saponin is presented in the form of a liposome, ISCOM or an oil in water emulsion. 20 An enhanced system involves the combination of a monophosphoryl lipid A (or detoxified lipid A) and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disposed in WO 96/33739, A particularly potent adjuvant 25 formulation involving tocopherol with or without QS21 and/or 3D-MPL in an oil in water emulsion is described in WO 95/17210. In one embodiment the immunogenic composition additionally comprises a saponin, which may be QS21. Immunostimulatory oligonucleotides or any other Toll-like receptor (TLR) 9 agonist may 30 also be used.The preferred oligonucleotides for use in adjuvants or vaccines of the present invention are CpG containing oligonucleotides, preferably containing two or more dinucleotide CpG motifs separated by at least three, more preferably at least six or more nucleotides. A CpG motif is a Cytosine nucleotide followed by a Guanine nucleotide. The CpG oligonucleotides of the present invention are typically deoxynucleotides. In a 35 preferred embodiment the intemucleotide in the oligonucleotide is phosphorodithioate, or 27 more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention. Also included within the scope of the invention are oligonucleotides with mixed internucleotide linkages. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in 5 US5,666,153, US5,278,302 and W095/26204. Examples of preferred oligonucleotides have the following sequences. The sequences preferably contain phosphorothioate modified internucleotide linkages. OLIGO 1(SEQ ID NO:1): TCC ATG ACG TTC CTG ACG TT (CpG 1826) 10 OLIGO 2 (SEQ ID NO:2): TCT CCC AGC GTG CGC CAT (CpG 1758) OLIGO 3(SEQ ID NO:3): ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG OLIGO 4 (SEQ ID NO:4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) OLIGO 5 (SEQ ID NO:5): TCC ATG ACG TTC CTG ATG CT (CpG 1668) OLIGO 6 (SEQ ID NO:6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456) 15 Alternative CpG oligonucleotides may comprise the preferred sequences above in that they have inconsequential deletions or additions thereto. The CpG oligonucleotides utilised in the present invention may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such 20 oligonucleotides may be synthesized utilising an automated synthesizer. The adjuvant may be an oil in water emulsion or may comprise an oil in water emulsion in combination with other adjuvants. The oil phase of the emulsion system preferably comprises a metabolisable oil. The meaning of the term metabolisable oil is well known in 25 the art. Metabolisable can be defined as "being capable of being transformed by metabolism" (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 2 5 th edition (1974)). The oil may be any vegetable oil, fish, oil, animal or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this 30 invention and can include commercially available oils such as NEOBEE@ and others. Squalene (2,6,10,15,19, 23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention. Squalene is a metabolisable oil by virtue of the fact that it is an 35 intermediate in the biosynthesis of cholesterol (Merck index, 10"' Edition, entry no.8619). 28 Tocols (e.g. vitamin E) are also often used in oil emulsions adjuvants (EP 0 382 271 31; US5667784; WO 95/17210). Tocols used in the oil emulsions (preferably oil in water emulsions) of the invention may be formulated as described in EP 0 382 271 B1, in that 5 the tocols may be dispersions of tocol droplets, optionally comprising an emulsifier, of preferably less than 1 micron in diameter. Alternatively, the tocols may be used in combination with another oil, to form the oil phase of an oil emulsion. Examples of oil emulsions which may be used in combination with the tocol are described herein, such as the metabolisable oils described above. 10 Oil in water emulsion adjuvants per se have been suggested to be useful as adjuvant compositions (EP 0 399 843B), also combinations of oil in water emulsions and other active agents have been described as adjuvants for vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Other oil emulsion adjuvants have been 15 described, such as water in oil emulsions (US 5,422,109;EP 0 480 982 B2) and water in oil in water emulsions (US 5,424,067;EP 0 480 981 B). All of which form preferred oil emulsion systems (in particular when incorporating tocols) to form adjuvants and compositions of the present invention. 20 Most preferably the oil emulsion (for instance oil in water emulsions) further comprises an emulsifier such as TWEEN 80 and/or a sterol such as cholesterol. A preferred oil emulsion (preferably oil-in-water emulsion) comprises a metabolisible, non toxic oil, such as squalane, squalene or a tocopherol such as alpha tocopherol (and preferably both squalene and alpha tocopherol) and optionally an emulsifier (or surfactant) 25 such as Tween 80. A sterol (preferably cholesterol) may also be included. The method of producing oil in water emulsions is well known to the man skilled in the art. Commonly, the method comprises mixing the tocol-containing oil phase with a surfactant such as a PBS/TWEEN80

T

u solution, followed by homogenisation using a homogenizer, it would be clear to a man skilled in the art that a method comprising passing the mixture 30 twice through a syringe needle would be suitable for homogenising small volumes of liquid. Equally, the emulsification process in microfluidiser (M110S Microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted by the man skilled in the art to produce smaller or larger volumes of emulsion. The adaptation could be achieved by 29 routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter. In an oil in water emulsion, the oil and emulsifier should be in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline. 5 The size of the oil droplets found within the stable oil in water emulsion are preferably less than 1 micron, may be in the range of substantially 30-600nm, preferably substantially around 30-500nm in diameter, and most preferably substantially 150-500nm in diameter, and in particular about 150 nm in diameter as measured by photon 10 correlation spectroscopy. In this regard, 80% of the oil droplets by number should be within the preferred ranges, more preferably more than 90% and most preferably more than 95% of the oil droplets by number are within the defined size ranges. The amounts of the components present in the oil emulsions of the present invention are conventionally in the range of from 0.5-20% or 2 to 10% oil (of the total dose volume), such as squalene; 15 and when present, from 2 to 10% alpha tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. Preferably the ratio of oil (preferably squalene): tocol (preferably a-tocopherol) is equal or less than 1 as this provides a more stable emulsion. An emulsifier, such as Tween80 or Span 85 may also be present at a level of about 1 %. In some cases it may be advantageous that the vaccines of the present 20 invention will further contain a stabiliser. Examples of preferred emulsion systems are described in WO 95/17210, WO 99/11241 and WO 99/12565 which disclose emulsion adjuvants based on squalene, c-tocopherol, and TWEEN 80, optionally formulated with the immunostimulants QS21 and/or 3D-MPL. 25 Thus in a particularly, preferred embodiment of the present invention, the adjuvant of the invention may additionally comprise further immunostimulants, such as LPS or derivatives thereof, and/or saponins. Examples of further immunostimulants are described herein and in "Vaccine Design - The Subunit and Adjuvant Approach" 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New 30 York and London, ISBN 0-306-44867-X. In a preferred aspect the adjuvant and immunogenic compositions according to the invention comprise a saponin (preferably QS21) and/or an LPS derivative (preferably 3D-MPL) in an oil emulsion described above, optionally with a sterol (preferably 35 cholesterol). Additionally the oil emulsion (preferably oil in water emulsion) may contain 30 span 85 and/or lecithin and/or tricaprylin. Adjuvants comprising an oil-in-water emulsion, a sterol and a saponin are described in WO 99/12565. Typically for human administration the saponin (preferably QS21) and/or LPS derivative (preferably 3D-MPL) will be present in a human dose of immunogenic 5 composition in the range of 14g - 200pg, such as 10-100gg, preferably 10pg - 504g per dose. Typically the oil emulsion (preferably oil in water emulsion) will comprise from 2 to 10% metabolisible oil. Preferably it will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% (preferably 0.4 - 2%) emulsifier (preferably tween 80 [polyoxyethylene sorbitan monooleate]). Where both squalene and alpha tocopherol are 10 present, preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 (Sorbitan trioleate) may also be present at a level of 0.5 to 1% in the emulsions used in the invention. In some cases it may be advantageous that the immunogenic compositions and vaccines of the present invention will further contain a stabiliser, for example other emulsifiers/surfactants, including caprylic 15 acid (merck index 10* Edition, entry no. 1739), of which Tricaprylin is particularly preferred. Where squalene and a saponin (preferably QS21) are included, it is of benefit to also include a sterol (preferably cholesterol) to the formulation as this allows a reduction in the total level of oil in the emulsion. This leads to a reduced cost of manufacture, 20 improvement of the overall comfort of the vaccination, and also qualitative and quantitative improvements of the resultant immune responses, such as improved IFN-y production. Accordingly, the adjuvant system of the present invention typically comprises a ratio of metabolisable oil:saponin (w/w) in the range of 200:1 to 300:1, also the present invention can be used in a "low oil" form the preferred range of which is 1:1 to 200:1, 25 preferably 20:1 to 100:1, and most preferably substantially 48:1, this vaccine retains the beneficial adjuvant properties of all of the components, with a much reduced reactogenicity profile. Accordingly, the particularly preferred embodiments have a ratio of squalene:QS21 (w/w) in the range of 1:1 to 250:1, also a preferred range is 20:1 to 200:1, preferably 20:1 to 100:1, and most preferably substantially 48:1. Preferably a sterol (most 30 preferably cholesterol) is also included present at a ratio of saponin:sterol as described herein. The emulsion systems of the present invention preferably have a small oil droplet size in the sub-micron range. Most preferably the oil droplet sizes will be in the range 120 to 750 nm, and most preferably from 120-600nm in diameter. 31 A particularly potent adjuvant formulation (for ultimate combination with AIPO4 in the immunogenic compositions of the invention) involves a saponin (preferably QS21), an LPS derivative (preferably 3D-MPL) and an oil emulsion (preferably squalene and alpha tocopherol in an oil in water emulsion) as described in WO 95/17210 or in WO 99/12565 5 (in particular adjuvant formulation 11 in Example 2, Table 1). Examples of a TLR 2 agonist include peptidoglycan or lipoprotein. Imidazoquinolines, such as Imiquimod and Resiquimod are known TLR7 agonists. Single stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), whereas double stranded 10 RNA and poly IC (polyinosinic-polycytidylic acid - a commercial synthetic mimetic of viral RNA). are exemplary of TLR 3 agonists. 3D-MPL is an example of a TLR4 agonist whilst CPG is an example of a TLR9 agonist. The immunogenic composition may comprise an antigen and an immunostimulant 15 adsorbed onto a metal salt. Aluminium based vaccine formulations wherein the antigen and the immunostimulant 3-de-O-acylated monophosphoryl lipid A (3D-MPL), are adsorbed onto the same particle are described in EP 0 576 478 B1, EP 0 689 454 B1, and EP 0 633 784 B1. In these cases then antigen is first adsorbed onto the aluminium salt followed by the adsorption of the immunostimulant 3D-MPL onto the same aluminium salt 20 particles. Such processes first involve the suspension of 3D-MPL by sonication in a water bath until the particles reach a size of between 80 and 500 nm. The antigen is typically adsorbed onto aluminium salt for one hour at room temperature under agitation. The 3D MPL suspension is then added to the adsorbed antigen and the formulation is incubated at room temperature for 1 hour, and then kept at 4oC until use. 25 In another process, the immunostimulant and the antigen are on separate metal particles, as described in EP 1126876. The improved process comprises the adsorption of immunostimulant, onto a metallic salt particle, followed by the adsorption of the antigen onto another metallic salt particle, followed by the mixing of the discrete metallic particles 30 to form a vaccine. The adjuvant for use in the present invention may be an adjuvant composition comprising an immunostimulant, adsorbed onto a metallic salt particle, characterised in that the metallic salt particle is substantially free of other antigen. Furthermore, vaccines are provided by the present invention and are characterised in that the immunostimulant is adsorbed onto particles of metallic salt which are substantially free 32 from other antigen, and in that the particles of metallic salt which are adsorbed to the antigen are substantially free of other immunostimulant. Accordingly, the present invention provides an adjuvant formulation comprising immunostimulant which has been adsorbed onto a particle of a metallic salt, characterised 5 in the composition is substantially free of other antigen. Moreover, this adjuvant formulation can be an intermediate which, if such an adjuvant is used, is required for the manufacture of a vaccine. Accordingly there is provided a process for the manufacture of a vaccine comprising admixing an adjuvant composition which is one or more immunostimulants adsorbed onto a metal particle with an antigen. Preferably, the antigen 10. has been pre-adsorbed onto a metallic salt. Said metallic salt may be identical or similar to the metallic salt which is adsorbed onto the immunostimulant. Preferably the metal salt is an aluminium salt, for example Aluminium phosphate or Aluminium hydroxide. The present invention further provides for a vaccine composition comprising immunostimulant adsorbed onto a first particle of a metallic salt, and antigen adsorbed 15 onto a metallic salt, characterised in that first and second particles of metallic salt are separate particles. LPS or LOS derivatives or mutations or lipid A derivatives described herein are designed to be less toxic (e.g. 3D-MPL) than native lipopolysaccharides and are interchangeable 20 equivalents with respect to any uses of these moieties described herein. They may be TLR4 ligands as described above. Other such derivatives are described in W0020786737, W09850399, WO0134617, WO0212258, WO03065806. In one embodiment the adjuvant used for the compositions of the invention comprises a 25 liposome carrier (made by known techniques from a phospholipids (such as dioleoyl phosphatidyl choline [DOPC]) and optionally a sterol [such as cholesterol]). Such liposome carriers may carry lipid A derivatives [such as 3D-MPL - see above] and/or saponins (such as QS21 - see above). In one embodiment the adjuvant comprises (per 0.5 mL dose) 0.1-10mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (e.g. 0.4-0.6, 0.9-1.1, 0.5 or 1 mg) 30 phospholipid (for instance DOPC), 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) sterol (for instance cholesterol), 5-60, 10-50, or 20-30 pg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 pg) lipid A derivative (for instance 3D MPL), and 5-60, 10-50, or 20-30 pg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 sg) saponin (for instance QS21). 35 33 This adjuvant is particularly suitable for elderly vaccine formulations. In one embodiment the vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more from serotypes 3, 6A, 19A, and 22F), wherein the 5 GMC antibody titre induced against one or more (or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferior to that induced by the Prevnar@ vaccine in human vaccinees. In one embodiment the adjuvant used for the compositions of the invention comprises an 10 oil in water emulsion made from a metabolisable oil (such as squalene), an emulsifier (such as Tween 80) and optionally a tocol (such as alpha tocopherol). In one embodiment the adjuvant comprises (per 0.5 mL dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6mg (e.g. 2-3, 5-6, or 10-11 mg) metabolisable oil (such as squalene), 0.1-10, 0.3-8, 0.6-6, 0.9-5, 1-4, or 2-3 mg (e.g. 0.9-1.1, 2-3 or 4-5 mg) emulsifier (such as Tween 80) and optionally 0.5-20, 1 15 15, 2-12, 4-10, 5-7 mg (e.g. 11-13, 5-6, or 2-3 mg) tocol (such as alpha tocopherol). This adjuvant may optionally further comprise 5-60, 10-50, or 20-30 pg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 pg) lipid A derivative (for instance 3D-MPL). 20 These adjuvants are particularly suitable for infant or elderly vaccine formulations. In one embodiment the vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all the following serotypes: 4, 68, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more from serotypes 3, 6A, 19A, and 22F), wherein the GMC antibody titre induced against one or more (or all) the vaccine 25 components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferior to that induced by the Prevnar@ vaccine in human vaccinees. This adjuvant may optionally contain 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125 0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) sterol (for instance cholesterol), 5-60, 30 10-50, or 20-30 pg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 sg) lipid A derivative (for instance 3D-MPL), and 5-60, 10-50, or 20-30 jig (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 jig) saponin (for instance QS21). This adjuvant is particularly suitable for elderly vaccine formulations. In one embodiment 35 the vaccine composition comprising this adjuvant comprises saccharide conjugates 34 derived from at least all the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more from serotypes 3, 6A, 19A, and 22F), wherein the GMC antibody titre induced against one or more (or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferior to that induced by the Prevnar@ 5 vaccine in human vaccinees. In one embodiment the adjuvant used for the compositions of the invention comprises aluminium phosphate and a lipid A derivative (such as 3D-MPL). This adjuvant may comprise (per 0.5 mL dose) 100-750, 200-500, or 300-400 pg Al as aluminium phosphate, 10 and 5-60, 10-50, or 20-30 gg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 g) lipid A derivative (for instance 3D-MPL). This adjuvant is particularly suitable for elderly or infant vaccine formulations. In one embodiment the vaccine composition comprising this adjuvant comprises saccharide 15 conjugates derived from at least all the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more from serotypes 3, 6A, 19A, and 22F), wherein the GMC antibody titre induced against one or more (or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferior to that induced by the Prevnar@ vaccine in human vaccinees. 20 The vaccine preparations containing immunogenic compositions of the present invention may be used to protect or treat a mammal susceptible to infection, by means of administering said vaccine via systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous 25 routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage). Although the vaccine of the invention may be administered as a single dose, components thereof may also be co 30 administered together at the same time or at different times (for instance pneumococcal saccharide conjugates could be administered separately, at the same time or 1-2 weeks after the administration of the any bacterial protein component of the vaccine for optimal coordination of the immune responses with respect to each other). For co-administration, the optional Th1 adjuvant may be present in any or all of the different administrations. In 35 addition to a single route of administration, 2 different routes of administration may be 35 used. For example, saccharides or saccharide conjugates may be administered IM (or ID) and bacterial proteins may be administered IN (or ID). In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses. 5 The content of protein antigens in the vaccine will typically be in the range 1-100pg, preferably 5-50pg, most typically in the range 5 - 25pg. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced. Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant 10 approach" (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. The vaccines of the present invention may be stored in solution or lyophilized. Preferably the solution is lyophilized in the presence of a sugar such as sucrose or lactose. It is still 15 further preferable that they are lyophilized and extemporaneously reconstituted prior to use. Lyophilizing may result in a more stable composition (vaccine) and may possibly lead to higher antibody titers in the presence of 3D-MPL and in the absence of an aluminum based adjuvant. 20 in one aspect of the invention is provided a vaccine kit, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilised form, and further comprising a vial containing an adjuvant as described herein. It is envisioned that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilised immunogenic composition. 25 Although the vaccines of the present invention may be administered by any route, administration of the described vaccines into the skin (ID) forms one embodiment of the present invention. Human skin comprises an outer "horny" cuticle, called the stratum corneum, which overlays the epidermis. Underneath this epidermis is a layer called the 30 dermis, which in turn overlays the subcutaneous tissue. Researchers have shown that injection of a vaccine into the skin, and in particular the dermis, stimulates an immune response, which may also be associated with a number of additional advantages. Intradermal vaccination with the vaccines described herein forms a preferred feature of the present invention. 35 36 The conventional technique of intradermal injection, the "mantoux procedure", comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26-31 gauge) facing upwards the needle is inserted at an angle of between 10-15*. Once the bevel of the needle is inserted, the barrel of the needle is 5 lowered and further advanced whilst providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby fonning a bleb or bump on the skin surface, followed by slow withdrawal of the needle. More recently, devices that are specifically designed to administer liquid agents into or 10 across the skin have been described, for example the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described for example in WO 01/13977; US 5,480,381, US 5,599,302, US 5,334,144, US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US 5,383,851, US 5,893,397, US 5,466,220, US 5,339,163, US 5,312,335, US 5,503,627, US 5,064,413, US 5,520, 639, US 4,596,556, US 4,790,824, US 15 4,941,880, US 4,940,460, WO 97/37705 and WO 97/13537. Alternative methods of intradermal administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or applied to the surface of the skin (transdermal or transcutaneous delivery WO 98/20734 ; WO 98/28037). 20 When the vaccines of the present invention are to be administered to the skin, or more specifically into the dermis, the vaccine is in a low liquid volume, particularly a volume of between about 0.05 mi and 0.2 ml. 25 The content of antigens in the skin or intradermal vaccines of the present invention may be similar to conventional doses as found in intramuscular vaccines (see above). However, it is a feature of skin or intradermal vaccines that the formulations may be "low dose". Accordingly the protein antigens in "low dose" vaccines are preferably present in as little as 0.1 to 10pg, preferably 0.1 to 5 p.g per dose; and the saccharide (preferably 30 conjugated) antigens may be present in the range of 0.01-1pg, and preferably between 0.01 to 0.5 pg of saccharide per dose. As used herein, the term "intradermal delivery" means delivery of the vaccine to the region of the dermis in the skin. However, the vaccine will not necessarily be located exclusively 35 in the dermis. The dermis is the layer in the skin located between about 1.0 and about 2.0 37 mm from the surface in human skin, but there is a certain amount of variation between individuals and in different parts of the body. In general, it can be expected to reach the dermis by going 1.5 mm below the surface of the skin. The dermis is located between the stratum corneum and the epidermis at the surface and the subcutaneous layer below. 5 Depending on the mode of delivery, the vaccine may ultimately be located solely or primarily within the dermis, or it may ultimately be distributed within the epidermis and the dermis. The present invention further provides an improved vaccine for the prevention or 10 amelioration of Otitis media caused by Haemophilus influenzae by the addition of Haemophilus influenzae proteins, for example protein D in free or conjugated form. In addition, the present invention further provides an improved vaccine for the prevention or amelioration of pneumococcal infection in infants (e.g., Otitis media), by relying on the addition of one or two pneumococcal proteins as free or conjugated protein to the S. 15 pneumoniae conjugate compositions of the invention. Said pneumococcal free proteins may be the same or different to any S. pneumoniae proteins used as carrier proteins. One or more Moraxella catarrhalis protein antigens can also be included in the combination vaccine in a free or conjugated form. Thus, the present invention is an improved method to elicit a (protective) immune response against Otitis media in infants. 20 In another embodiment, the present invention is an improved method to elicit a (protective) immune response in infants (defined as 0-2 years old in the context of the present invention) by administering a safe and effective amount of the vaccine of the invention [a paediatric vaccine]. Further embodiments of the present invention include the 25 provision of the antigenic S. pneumoniae conjugate compositions of the invention for use in medicine and the use of the S. pneumoniae conIjugates of the invention in the manufacture of a medicament for the prevention (or treatment) of pneumococcal disease. In yet another embodiment, the present invention is an improved method to elicit a 30 (protective) immune response in the elderly population (in the context of the present invention a patient is considered elderly if they are 50 years or over in age, typically over 55 years and more generally over 60 years) by administering a safe and effective amount of the vaccine of the invention, preferably in conjunction with one or two S. pneumoniae proteins present as free or conjugated protein, which free S. pneumoniae proteins may be 35 the same or different as any S. pneumoniae proteins used as carrier proteins. 38 A further aspect of the invention is a method of immunising a human host against disease caused by S. pneumoniae and optionally Haemophilus influenzae infection comprising administering to the host an immunoprotective dose of the immunogenic composition or 5 vaccine or kit of the invention. A further aspect of the invention is an immunogenic composition of the invention for use in the treatment or prevention of disease caused by S.pneumoniae and optionally Haemophilus influenzae infection. 10 A further aspect of the invention is use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of diseases caused by S. pneumoniae and optionally Haemophilus influenzae infection. 15 The terms "comprising", "comprise" and "comprises" herein are intended by the inventors to be optionally substitutable with the terms "consisting of", "consist of" and "consists of, respectively, in every instance. Embodiments herein relating to "vaccine compositions" of the invention are also 20 applicable to embodiments relating to "immunogenic compositions" of the invention, and vice versa. All references or patent applications cited within this patent specification are incorporated by reference herein. 25 In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner. 30 Examples Example 1: EXPRESSION OF PROTEIN D Haemophilus Influenzae protein D Genetic construction for protein D expression 39 Starting materials The Protein D encoding DNA Protein D is highly conserved among H. influenzae of all serotypes and non typeable strains. The vector pHIC348 containing the DNA sequence encoding the entire 5 protein D gene has been obtained from Dr. A. Forsgren, Department of Medical Microbiology, University of Lund, Malmo General Hospital, Malmo, Sweden. The DNA sequence of protein D has been published by Janson et al. (1991) Infect. Immun. 59: 119 125. 10 The expression vector pMG1 The expression vector pMG1 is a derivative of pBR322 (Gross et al., 1985) in which bacteriophage A derived control elements for transcription and translation of foreign inserted genes were introduced (Shatzman et al., 1983). In addition, the Ampicillin resistance gene was exchanged with the Kanamycin resistance gene. 15 The E. coli strain AR58 The E. coli strain AR58 was generated by transduction of N99 with a P1 phage stock previously grown on an SA500 derivative (galE::TN10, lambdaKir c1857 AHI). N99 and SA500 are E. coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the 20 National Institute of Health. The expression vector pMG I For the production of protein D, the DNA encoding the protein has been cloned into the expression vector pMG 1. This plasmid utilises signals from lambdaphage DNA 25 to drive the transcription and translation of inserted foreign genes. The vector contains the promoter PL, operator OL and two utilisation sites (NutL and NutR) to relieve transcriptional polarity effects when N protein is provided (Gross et al., 1985). Vectors containing the PL promoter, are introduced into an E. coli lysogenic host to stabilise the plasmid DNA. Lysogenic host strains contain replication-defective lambdaphage DNA 30 integrated into the genome (Shatzman et al., 1983). The chromosomal lambdaphage DNA directs the synthesis of the cI repressor protein which binds to the OL repressor of the vector and prevents binding of RNA polymerase to the PL promoter and thereby transcription of the inserted gene. The ci gene of the expression strain AR58 contains a temperature sensitive mutant so that PL directed transcription can be regulated by 35 temperature shift, i.e. an increase in culture temperature inactivates the repressor and 40 synthesis of the foreign protein is initiated. This expression system allows controlled synthesis of foreign proteins especially of those that may be toxic to the cell (Shimataka & Rosenberg, 1981). 5 The E. coli strain AR58 The AR58 lysogenic E. coli strain used for the production of the protein D carrier is a derivative of the standard NIH E. coli K12 strain N99 (F- su- galK2, lacZ- thr ). It contains a defective lysogenic lambdaphage (galE::TN10, lambdaKi[ c1857 AH1). The Kil phenotype prevents the shut off of host macromolecular synthesis. The c1857 mutation 10 confers a temperature sensitive lesion to the cl repressor. The AH1 deletion removes the lambdaphage right operon and the hosts bio, uvr3, and chIA loci. The AR58 strain was generated by transduction of N99 with a P1 phage stock previously grown on an SA500 derivative (galE::TN10, lambdaKi[ c1857 AH1). The introduction of the defective lysogen into N99 was selected with tetracycline by virtue of the presence of a TN10 transposon 15 coding for tetracyclin resistance in the adjacent galE gene. Construction of vector pMGMDPPrD The pMG 1 vector which contains the gene encoding the non-structural S1 protein of Influenzae virus (pMGNSI) was used to construct pMGMDPPrD. The protein D gene 20 was amplified by PCR from the pHIC348 vector (Janson et al. 1991 Infect. Immun. 59:119-125) with PCR primers containing Ncol and Xbal restriction sites at the 5' and 3' ends, respectively. The Ncol/Xbal fragment was then introduced into pMGNS1 between Ncol and Xbal thus creating a fusion protein containing the N-terminal 81 amino acids of the NS1 protein followed by the PD protein. This vector was labelled pMGNS1 PrD. 25 Based on the construct described above the final construct for protein D expression was generated. A BamHI/BamHl fragment was removed from pMGNS1PrD. This DNA hydrolysis removes the NS1 coding region, except for the first three N-terminal residues. Upon religation of the vector a gene encoding a fusion protein with the following N-terminal amino acid sequence has been generated: 30 --- MDP SSHSSNMANT--- NS1 Protein D 41 The protein D does not contain a leader peptide or the N-terminal cysteine to which lipid chains are normally attached. The protein is therefore neither excreted into the periplasm nor lipidated and remains in the cytoplasm in a soluble form. The final construct pMG-MDPPrD was introduced into the AR58 host strain by 5 heat shock at 37 0C. Plasmid containing bacteria were selected in the presence of Kanamycin. Presence of the protein D encoding DNA insert was demonstrated by digestion of isolated plasmid DNA with selected endonucleases. The recombinant E. coli strain is referred to as ECD4. Expression of protein D is under the control of the lambda PL promoter/ OL 10 Operator. The host strain AR58 contains a temperature-sensitive cl gene in the genome which blocks expression from lambda PL at low temperature by binding to OL- Once the temperature is elevated cl is released from OL and protein D is expressed. Small-scale preparation 15 At the end of the fermentation the cells are concentrated and frozen. The extraction from harvested cells and the purification of protein D was performed as follows. The frozen cell culture pellet is thawed and resuspended in a cell disruption solution (Citrate buffer pH 6.0) to a final OD6so = 60. The suspension is passed twice through a high pressure homogenizer at P = 1000 bar. The cell culture homogenate 20 is clarified by centrifugation and cell debris is removed by filtration. In the first purification step the filtered lysate is applied to a cation exchange chromatography column (SP Sepharose Fast Flow). PD binds to the gel matrix by ionic interaction and is eluted by a step increase of the ionic strength of the elution buffer. In a second purification step impurities are retained on an anionic exchange matrix 25 (Q Sepharose Fast Flow). PD does not bind onto the gel and can be collected in the flow through. In both column chromatography steps fraction collection is monitored by OD. The flow through of the anionic exchange column chromatography containing the purified protein D is concentrated by ultrafiltration. 30 The protein D containing ultrafiltration retentate is finally passed through a 0.2 pm membrane. Large Scale Preparation 42 The extraction from harvested cells and the purification of protein D was performed as follows. The harvested broth is cooled and directly passed twice through a high pressure homogenizer at a Pressure of around 800 bars. In the first purification step the cell culture homogenate is diluted and applied to a 5 cation exchange chromatography column (SP Sepharose Big beads). PD binds to the gel matrix by ionic interaction and is eluted by a step increase of the ionic strength of the elution buffer and filtrated. In a second purification step impurities are retained on an anionic exchange matrix (Q Sepharose Fast Flow). PD does not bind onto the gel and can be collected in the flow 10 through. In both column chromatography steps fraction collection is monitored by OD. The flow through of the anionic exchange column chromatography containing the purified protein D is concentrated and diafiltrated by ultrafiltration. The protein D containing ultrafiltration retentate is finally passed through a 0.2 pm 15 membrane. Example 1b: EXPRESSION OF PhtD The PhtD protein is a member of the pneumococcal histidine-triad (Pht) protein family 20 characterized by the presence of histidine-triads (HXXHXH motif). PhtD is a 838 aa molecule and carries 5 histidine triads (see MedImmune WOOO/37105 SEQ ID NO: 4 for amino acid sequence and SEQ ID NO: 5 for DNA sequence). PhtD also contains a proline rich region in the middle (amino acid position 348-380). PhtD has a 20 aa-N-terminal signal sequence with a LXXC motif. 25 Genetic construct The gene sequence of the mature MedImmune PhtD protein (from aa 21 to aa 838) was transferred recombinantly to E. coli using the in-house pTCMP14 vector carrying the pX promoter. The E. coli host strain is AR58, which carries the c857 thermosensitive repressor, allowing heat-induction of the promotor. 30 Polymerase chain reaction was realized to amplify the phtD gene from a MedImmune plasmid (carrying the phtD gene from Streptococcus pneumoniae strain Norway 4 (serotype 4)-- SEQ ID NO: 5 as described in WO 00/37105). Primers, specific for the phID gene only, were used to amplify the phtD gene in two fragments. Primers carry either the Ndel and KpnI or the KpnI and Xbal restriction sites. These primers do not hybridize with 35 any nucleotide from the vector but only with phtD specific gene sequences. An artificial ATG start codon was inserted using the first primer carrying the NdeI restriction site. The generated PCR products were then inserted into the pGEM-T cloning vector (Promega), 43 and the DNA sequence was confirmed. Subcloning of the fragments in the TCMP14 expression vector was then realized using standard techniques and the vector was transformed into A R58 E. coli. PhtD Purification 5 PhID purification is achieved as follows: " Growth of E.coli cells in the presence of Kanamycin: growth 30 hours at 30 *C then induction for 18 hours at 39.5 "C " Breakage of the E.coli cells from whole culture at OD 2115 in presence of EDTA 5 mM 10 and PMSF 2 mM as protease inhibitors: Rannie, 2 passages, 1000 bars. " Antigen capture and cells debris removal on expanded bed mode Streamline Q XL chromatography at room temperature (20 0 C); the column is washed with NaCl 150 mM + Emplgen 0.25% pH 6.5 and eluded with NaCI 400 mM + Empigen 0.25% in 25 mM 15 potassium phosphate buffer pH 7.4. = Filtration on Sartobran 150 cartridge (0.45 + 0.2 pm) " Antigen binding on Zn** Chelating Sepharose FF IMAC chromatography at pH 7.4 in 20 presence of 5 mM imidazole at 4*C: the column is washed with Imidazole 5 mM and Empigen 1% and eluted with 50 mM imidazole, both in 25 mM potassium phosphate buffer pH 8.0. o Weak anion exchange chromatography in positive mode on Fractogel EMD DEAE at 25 pH 8.0 (25 mM potassium phosphate) at 4 0 C; the column is washed with 140 mM NaC and eluted at 200 mM NaCl while contaminants (proteins and DNA) remain adsorbed on the exchanger. " Concentration and ultrafiltration with 2 mM Na/K phosphate pH 7.15 on 50 kDa 30 membrane. " Sterilising filtration of the purified bulk on a Millipak-20 0.2 pm filter cartridge. Example 1c: EXPRESSION OF PNEUMOLYSIN 35 Pneumococcal pneumolysin was prepared and detoxified as described in W02004/081515 and W02006/032499. Example 2: 40 Preparation of conjugates It is well known in the art how to make purified pneumococcal polysaccharides. For the purposes of these examples the polysaccharides were made essentially as 44 described in EP072513 or by closesly-related methods. Before conjugation the polysaccharides may be sized by microfluidisation as described below. The activation and coupling conditions are specific for each polysaccharide. These are given in Table 1. Sized polysaccharide (except for PS5, 6B and 23F) was dissolved in 5 NaCl 2M, NaCl 0.2M or in water for injection (WFI). The optimal polysaccharide concentration was evaluated for all the serotypes. All serotypes except serotype 18C were conjugated directly to the carrier protein as detailed below. Two alternative serotype 22F conjugates were made; one conjugated directly, one through an ADH linker. From a 100 mg/ml stock solution in acetonitrile or acetonitrile/water 50%/50% 10 solution, CDAP (CDAP/PS ratio 0.5-1.5 mg/mg PS) was added to the polysaccharide solution. 1.5 minute later, 0.2M-0.3M NaOH was added to obtain the specific activation pH. The activation of the polysaccharide was performed at this pH during 3 minutes at 25 *C. Purified protein (protein D, PhtD, pneumolysin or DT) (the quantity depends on the initial PS/carrier protein ratio) was added to the activated polysaccharide and the coupling 15 reaction was performed at the specific pH for up to 2 hour (depending upon serotype) under pH regulation. In order to quench un-reacted cyanate ester groups, a 2M glycine solution was then added to the mixture. The pH was adjusted to the quenching pH (pH 9.0). The solution was stirred for 30 minutes at 25 *C and then ovemight at 2-8 *C with continuous slow stirring. 20 Preparation of 18C: 18C was linked to the carrier protein via a linker - Adipic acid dihydrazide (ADH) Polysaccharide serotype 18C was microfluidized before conjugation. Derivatization of tetanus toxoId with EDAC For derivatization of the tetanus toxoid, purified TT was diluted at 25 mg/ml in 0.2M NaCl 25 and the ADH spacer was added in order to reach a final concentration of 0.2M. When the dissolution of the spacer was complete, the pH was adjusted to 6.2. EDAC (1-ethyl-3-(3 dimethyl-aminopropyl) carbodiimide) was then added to reach a final concentration of 0.02M and the mixture was stirred for 1 hour under pH regulation. The reaction of condensation was stopped by increasing pH up to 9.0 for at least 30 minutes at 25 0 C. 30 Derivatized TT was then diafiltrated (10 kDa CO membrane) in order to remove residual ADH and EDAC reagent. TTA bulk was finally sterile filtered until coupling step and stored at -70 0 C. Chemical coupling of T1 to PS 18C Details of the conjugation parameters can be found in Table 1. 45 2 grams of microfluidized PS were diluted at the defined concentration in water and adjusted to 2M NaCl by NaCl powder addition. CDAP solution (100 mg/ml freshly prepared in 50/50 v/v acetonitrile/WFI) was added to reach the appropriate CDAP/PS ratio. 5 The pH was raised up to the activation pH 9.0 by the addition of 0.3M NaOH and was stabilised at this pH until addition of TTAH. After 3 minutes, derivatized TTAH (20 mg/mI in 0.2 M NaCl) was added to reach a ratio TTAH /PS of 2; the pH was regulated to the coupling pH 9.0. The solution was left one hour under pH regulation. 10 For quenching, a 2M glycine solution, was added to the mixture PS/TTAH/CDAP. The pH was adjusted to the quenching pH (pH 9.0). The solution was stirred for 30 min at 25 *C, and then left overnight at 2-8"C with continuous slow stirring. 15 PS22FH-PhtD conjugate In a second conjugation method for this saccharide (the first being the direct PS22-PhtD conjugation method shown in Table 1), 22F was linked to the carrier protein via a linker - Adipic acid dihydrazide (ADH). Polysaccharide serotype 22F was microfluidized before conjugation. 20 PS 22F derivatization Activation and coupling are performed at 25*C under continuous stirring in a temperature controlled waterbath. 25 Microfluidized PS22F was diluted to obtain a final PS concentration of 6 mg/ml in 0.2M NaCl and the solution was adjusted at pH 6.05 ± 0.2 with 0.1N HCL. CDAP solution (100 mg/ml freshly prepared in acetonitrile/WFI, 50/50) was added to reach the appropriate CDAPIPS ratio (1.5/1 ww). The pH was raised up to the activation pH 9.00 ±0.05 by the addition of 0.5M NaOH and 30 was stabilised at this pH until addition of ADH. After 3 minutes, ADH was added to reach the appropriate ADH/PS ratio (8.9/1 w/w); the pH was regulated to coupling pH 9.0. The solution was left for 1 hour under pH regulation. The PSAH derivative was concentrated and diafiltrated. 35 Coupling 46 PhtD at 10 mg/mI in 0.2M NaCl was added to the PS22FAH derivative in order to reach a PhtD/PS22FAH ratio of 4/1 (w/w). The pH was adjusted to 5.0 1 0.05 with HCl. The EDAC solution (20 mg/ml in 0.1M Tris-HCI pH 7.5) was added manually in 10 min (250 0 / min) to reach 1 mg EDAC/mg PS22FAH. The resulting solution was incubated for 150 min 5 (though 60 mins was also used) at 25*C under stirring and pH regulation. The solution was neutralized by addition of 1M Tris-HCI pH 7.5 (1/10 of the final volume) and let 30 min at 25*C. Prior to the elution on Sephacryl S400HR, the conjugate was clarified using a 5pm 10 Minisart filter. The resulting conjugate has a final PhtD/PS ratio of 4.1 (w/w), a free PS content below 1% and an antigenicity (a-PS/a-PS) of 36.3% and anti-PhtD antigenicity of 7.4%. 15 Purification of the conjugates: The conjugates were purified by gel filtration using a Sephacryl S400HR gel filtration column equilibrated with 0.15M NaCl (S500HR for 18C) to remove small molecules (including DMAP) and unconjugated PS and protein. Based on the different molecular sizes of the reaction components, PS-PD, PS-TT, PS-PhtD, PS-pneumolysin or PS-DT 20 conjugates are eluted first, followed by free PS, then by free PD or free DT and finally DMAP and other salts (NaCl, glycine). Fractions containing conjugates are detected by UV 28 o0m. Fractions are pooled according to their Kd, sterile filtered (0.22Mm) and stored at +2-8'C. The PS/Protein ratios in the conjugate preparations were determined. 25 Specific actlvatlon/couplinnlquenchinq conditions of PS S. pneumoniae-Protein D/TT/DT/PhtD/Plyconlugates Where "pfluid" appears in a row header, it indicates that the saccharide was sized by 30 microfluidisation before conjugation. Sizes of saccharides following microfluidisation are given in table 2. Table I Specific activation/couplina/guenchinq conditions of PS S. neumoniae-Protein DITT/DT/PhtD/Plyconluqates 35 Serotype 1 4 5 6A 6B 7F Spfluld fluid fluid PS 2.5 2.5 7.1 5.0 5.0 5.0 conc.(migIml) 47 PS WFI WFI WFI NaCl 2M NaCl 2M NaCl 2M dissolution PD 10.0 10.0 5.0 5.0 5.0 10.0 conc.(mg/ml) Initial PD/PS 1.5/1 1.5/1 1/1 1/1 1.1/1 1. 2/1 Ratio (w/w) CDAP conc. 0.50 0.50 0.79 0.83 0.83 0.75 (mg/mg PS) pH.=pH.=pHq 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0 9.5/9.5/9.0 Serotype 9V 14 18C 19A 19F 22F 23F fluid pfluid fluid pfluid pfluid fluid PS 5.0 5.0 4.5 15.0 9.0 6.0 2.38 conc.(mg/ml) PS NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 0.2M NaCl 2M dissolution Carrier 10.0 10.0 20.0 (TT) 10.0 ' 20.0 10.0 5.0 protein (Ply) (DT) (PhtD) conc.(mg/ml) initial carrier 1.2/1 1.2/1 2/1 2.5/1 1.5/1 3/1 1/1 protein/PS Ratio (w/w) CDAP conc. 0.50 0.75 0.75 1.5 1.5 1.5 0.79 (mg/mg PS) pH.=pHc=PHq 9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Note: pHa,c,q corresponds to the pH for activation, coupling and quenching, respectively 5 Characterlsation: 48 Each conjugate was characterised and met the specifications described in Table 2. The polysaccharide content (pg/ml) was measured by the Resorcinol test and the protein content (pg/ml) by the Lowry test. The final PS/PD ratio (w/w) is determined by the ratio of the concentrations. 5 Free polysaccharide content (%): The free polysaccharide content of conjugates kept at 4 0 C or stored 7 days at 37*C was determined on the supernatant obtained after incubation with a-carrier protein antibodies and saturated ammonium sulfate, followed by a centrifugation. 10 An a-PS/a-PS ELISA was used for the quantification of free polysaccharide in the supernatant. The absence of conjugate was also controlled by an a-carrier protein/a-PS ELISA. Antigenicity: 15 The antigenicity on the same conjugates was analyzed in a sandwich-type ELISA wherein the capture and the detection of antibodies were a-PS and a-Protein respectively. Free protein content (%): 20 Unconjugated carrier protein can be separated from the conjugate during the purification step. The content of free residual protein was determined using size exclusion chromatography (TSK 5000-PWXL) followed by UV detection (214 nm). The elution conditions allowed separating the free carrier protein and the conjugate. Free protein content in conjugate bulks was then determined versus a calibration curve (from 0 to 50 25 4g/ml of carrier protein). Free carrier protein in % was obtained as follows: % free carrier (free carrier (Ag/ml)/ (Total concentration of corresponding carrier protein measured by Lowry (pg/ml) * 100%). Stability: 30 Molecular weight distribution (K,,) and stability was measured on a HPLC-SEC gel filtration (TSK 5000-PWXL) for conjugates kept at 4*C and stored for 7 days at 37 0 C. The 10/11/13/14-valent characterization is given in Table 2 (see comment thereunder). 49 The protein conjugates can be adsorbed onto aluminium phosphate and pooled to form the final vaccine. Conclusion: 5 Immunogenic conjugates have been produced, that have since been shown to be components of a promising vaccine. TABLE 2 - characteristics of the conjugates PS PS size Carrier/PS Free PS Antigenicity Conj. Size Conjugates (Dax10 3 Ratio (Elisa) Free Carrier (Elisa) (kDa) 349- 1.0%- 1499 PS1-PD 382* 1.5-1.6 1.2% 3.9%-4.8% 87%-95% 1715 4.7- 1303 PS4-PD 93-100* 1.5-1.6 6.5% 3.2%-4.0% 90%-96% 1606 8.7- 93%- 1998 PS5-PD*** 367-443 0.80 11.2% 2.2%-3.8% 108% 2352 1100 PS6A-PD 1540 0.61 4.5% Not done 45.9% Not done PS6B- 1069- 1.3- 4778 PD** 1391 0.7-0.8 1.6% <2.0% 68%-75% 5235 255- 3907 PS7F-PD 264* 1 1.1-1.2 <1% <1.4% 58% 4452 258- 9073 PS9V-PD 280* 1.3-1.5 <1% <1.3% 67%-69% 9572 232- 3430 PS14-PD 241* 1.4 <1% <1.5% 70% 3779 1.5- 5464 PS18C-TT~ 89-97* 2.2-2.4 2.2% <4% 46%-56% 6133 PS19A-Ply* 151 3.2 <1% 29% 133- 4.1%- <1.2%- 2059 PS19F-DT 143* 1.4-1.5 5.9% <1.3% 82%-88% 2335 PS22F PhtD* 159-167 2.17 5.8 Not done 37% Not done PS22F-AHPhtD*159-167 3.66-4.34 <1% Not done 28-31% Not done PS23F- 1.4- 137%- 2933 PD*** 914-980 0.5 1.9% 3.7%-4.9% 154% 3152 * PS size following microfluidizatlon of the native PS A 10 valent vaccine was made by mixing serotype 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 10 23F conjugates (e.g. at a dose of 1, 3, 1, 1, 1, 1, 1, 3, 3, 1 pg of saccharide, respectively per human dose). An 11 valent vaccine was made by further adding the serotype 3 conjugate from Table 5 (e.g. at 1 pig of saccharide per human dose). A 13 valent vaccine was made by further adding the serotypes 19A and 22F conjugates above (with 22F either directly linked to PhtD, or alternatively through an ADH linker) [e.g. at a dose of 3 15 pg each of saccharide per human dose]. A 14 valent vaccine may be made by further 50 adding the serotype 6A conjugate above [e.g. at a dose of 1 ig of saccharide per human dose. Example 3: Evidence that inclusion of Haemphllus influenzae protein D in an 5 immunogenic composition of the invention can provide improved protection against acute otitis media (AOM). Study design. The study used an 11Pn-PD vaccine - comprising serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 10 18C, 19F and 23F each conjugated to protein D from H. influenzae (refer to Table 5 in Example 4). Subjects were randomized into two groups to receive four doses of either the 11 Pn-PD vaccine or Havrix at approximately 3, 4, 5 and 12-15 months of age. All subjects received GSK Biologicals' Infanrix- hexa (DTPa-HBV-IPV/Hib) vaccine concomitantly at 3, 4 and 5 months of age. Infanrix- hexa is a combination of Pediarix and Hib mixed before 15 administration. Efficacy follow-up for the "According-to-Protocol" analysis started 2 weeks after administration of the third vaccine dose and continued until 24-27 months of age. Nasopharyngeal carriage of S. pneumoniae and H. influenzae was evaluated in a selected subset of subjects. Parents were advised to consult the investigator if their child was sick, had ear pain, 20 spontaneous perforation of the tympanic membrane or spontaneous ear discharge. If the investigator suspected an episode of AOM, the child was immediately referred to an Ear, Nose and Throat (ENT) specialist for confirmation of the diagnosis. A clinical diagnosis of AOM was based on either the visual appearance of the tympanic membrane (i.e. redness, bulging, loss of light reflex) or the presence of middle ear fluid 25 effusion (as demonstrated by simple or pneumatic otoscopy or by microscopy). In addition, at least two of the following signs or symptoms had to be present: ear pain, ear discharge, hearing loss, fever, lethargy, irritability, anorexia, vomiting, or diarrhea. If the ENT specialist confirmed the clinical diagnosis, a specimen of middle ear fluid was collected by tympanocentesis for bacteriological testing. 30 For subjects with repeated sick visits, a new AOM episode was considered to have started if more than 30 days had elapsed since the beginning of the previous episode. In addition, an AOM episode was considered to be a new bacterial episode if the isolated bacterium/serotype differed from the previous isolate whatever the interval between the two consecutive episodes. 35 51 Trial results A total of 4968 infants were enrolled, 2489 in the 11 Pn-PD group and 2479 in the control group. There were no major differences in the demographic characteristics or risk factors between the two groups. 5 Clinical episodes and A OM case definition During the per protocol follow-up period, a total of 333 episodes of clinical AOM were recorded in the 11 Pn-PD group and 499 in the control group. Table 3 presents the protective efficacy of the 11Pn-PD vaccine and both 7-valent vaccines previously tested in Finland (Eskola et al N Engl J Med 2001; 344: 403 - 409 10 and Kilpi et alClin Infect Dis 2003 37:1155-64) against any episode of AOM and AOM caused by different pneumococcal serotypes, H. influenzee, NTHi and M. catarrhalis. Statistically significant and clinically relevant reduction by 33.6% of the overall AOM disease burden was achieved with 11 Pn-PD, irrespective of the etiology (table 3). The overall efficacy against AOM episodes due to any of the 11 pneumococcal serotypes 15 contained in the 11Pn-PD vaccine was 57.6% (table 3). Another important finding in the current study is the 35.6% protection provided by the 11Pn-PD vaccine against AOM caused by H. influenzae (and specifically 35.3% protection provided by NTHi). This finding is of major clinical significance, given the 20 increased importance of H. influenzae as a major cause of AOM in the pneumococcal conjugate vaccine era. In line with the protection provided against AOM, the 11 Pn-PD vaccine also reduced nasopharyngeal carriage of H. influenzae following the booster dose in the second year of life. These findings are in contrast with previous observations in Finland where, for both 7-valent pneumococcal conjugate vaccines, an increase in AOM 25 episodes due to H. influenzae was observed, (Eskola et al and Kilpi et al) as evidence of etiological replacement. A clear correlation between protection against AOM episodes due to Hi and antibody levels against the carrier Protein D could not be established, as post-primary anti-PD igG 30 antibody concentrations in 11Pn-PD vaccinees, that remained Hi AOM episode-free, were essentially the same as post-primary anti-PD IgG antibody levels measured in 11Pn-PD vaccinees that developed at least one Hi AOM episode during the efficacy follow-up period. However, although no correlation could be established between the biological impact of the vaccine and the post-primary IgG anti-PD immunogenicity, it is reasonable 52 to assume that the PD carrier protein, which is highly conserved among H. influenzae strains, has contributed to a large extent in the induction of the protection against Hi. The effect on AOM disease was accompanied by an effect on nasopharyngeal carriage 5 that was of similar magnitude for vaccine serotype pneumococci and H. influenzae (Figure 1). This reduction of the nasopharyngeal carriage of H. influenzae in the PD-conjugate vaccinees supports the hypothesis of a direct protective effect of the PD-conjugate vaccine against H. influenzae, even if the protective efficacy could not be correlated to the anti-PD IgG immune responses as measured by ELISA. 10 In a following experiment a chinchilla otitis media model was used with serum pools from infants immunised with the 11 valent formulation of this example or with the 10 valent vaccine of Example 2 (see also Table 1 and 2 and comments thereunder). Both pools induce a significant reduction of the percentage of animals with otitis media versus the 15 pre-immune serum pool. There is no significant difference beteen the 10 and 11 valent immune pools. This demonstrates that both vaccines have a similar potential to induce protection against otitis media caused by non typeable H. influenzae in this model. 20 53 C1. CD. C -z oo r-U Sb .2 ~ ~ ~ - Z. ~ e ~~ C-6 ~ (Lo 't C1 -C - C " a ,.C1 M I. II I-0 -i % ag ~ o r- tnW1 Lo C knne en3 C-4 C"fj *.i ecm )S n ~ LA UoeU 0 I-- Example 4: Selection of carrier protein for serotype 19F ELISA assay used 5 The 22F inhibition ELISA method was essentially based on an assay proposed in 2001 by Concepcion and Frasch and was reported by Henckaerts et al., 2006, Clinical and Vaccine Immunology 13:356-360. Briefly, purified pneumococcal polysaccharides were mixed with methylated human serum albumin and adsorbed onto Nunc MaxisorpTM (Roskilde, DK) high binding microtitre plates overnight at 4 0 C. The plates were blocked 10 with 10% fetal bovine serum (FBS) in PBS for 1 hour at room temperature with agitation. Serum samples were diluted in PBS containing 10% FBS, 10 pg/mL cell-wall polysaccharide (SS) and 2 pg/mL of pneumococcal polysaccharide of serotype 22F (ATCC), and further diluted on the microtitre plates with the same buffer. An internal reference calibrated against the standard serum 89-SF using the serotype-specific IgG 15 concentrations in 89-SF was treated in the same way and included on every plate. After washing, the bound antibodies were detected using peroxidase-conjugated anti-human IgG monoclonal antibody (Stratech Scientific Ltd., Soham, UK) diluted in 10% FBS (in PBS), and incubated for 1 hour at room temperature with agitation. The color was developed using ready-to-use single component tetramethylbenzidine peroxidase enzyme 20 immunoassay substrate kit (BioRad, Hercules, CA, US) in the dark at room temperature. The reaction was stopped with H2SO4 0.18 M, and the optical density was read at 450 nm. Serotype-specific IgG concentrations (in pg/mL) in the samples were calculated by referencing optical density points within defined limits to the internal reference serum curve, which was modelized by a 4-parameter logistic log equation calculated with 25 SoftMax Pro T M (Molecular Devices, Sunnyvale, CA) software. The cut-off for the ELISA was 0.05 pg/mL IgG for all serotypes taking into account the limit of detection and the limit of quantification. Opsonophagocytosis assay 30 At the WHO consultation meeting in June 2003, it was recommended to use an OPA assay as set out in Romero-Steiner et al Clin Diagn Lab Immunol 2003 10 (6): pp101 9 1024. This protocol was used to test the OPA activity of the serotypes in the following tests. 35 Preparation of conjugates 55 In studies llPn-PD&Di-001 and 1lPn-PD&Di-007, three 11-valent vaccine formulations (Table 4) were included in which 3pg of the 19F polysaccharide was conjugated to diphtheria toxoid (19F-DT) instead of lpg polysaccharide conjugated to protein D (19F PD). Conjugation parameters for the studies 11Pn-PD, 11 Pn-PD&Di-001 and 11 Pn 5 PD&Di-007 are disclosed in Tables 5, 6 and 7 respectively. Anti-pneumococcal antibody responses and OPA activity against serotype 19F one month following primary vaccination with these 19F-DT formulations are shown in Table 8 and 9 respectively. 10 Table 10 shows 22F-ELISA antibody concentrations and percentages of subjects reaching the 0.2 pg/mL threshold before and after 23-valent plain polysaccharde booster vaccination. The opsonophagocytic activity was shown to be clearly improved for antibodies induced 15 with these 19F-DT formulations as demonstrated by higher seropositivity rates (opsonophagocytic titers > 1:8) and OPA GMTs one month following primary vaccination (Table 9). One month after 23-valent plain polysaccharide booster vaccination, opsonophagocytic activity of 19F antibodies remained significantly better for children primed with 19F-DT formulations (Table 11). 20 Table 12 presents immunogenicity data following a 11Pn-PD booster dose in toddlers previously primed with 19F-DT or 19F-PD conjugates compared to a 4t consecutive dose 25 of Prevnai@. Given the breakthrough cases reported after the introduction of Prevnar@ in the US, the improved opsonophagocytic activity against serotype 19F when conjugated to the DT carrier protein may be an advantage for the candidate vaccine. Table 13 provides ELISA and OPA data for the 19F-DT conjugate with respect to the cross-reactive serotype 19A. It was found that 19F-DT induces low but significant OPA 30 activity against 19A. 56 Table4 Pneumococcal conjugate vaccine formulations used in clinical studies. Formulation Pneumococcal serotype pg/carrier protein AP* 1 3 4 5 6B 7F 9V 14 18C 19F 23F mg 11Pn-PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD < 0.8 19F-DT Form 1 3/PD 3/PD 3/PD 3/PD 10/DT 3/PD 3/PD 3/PD 3/PD 3/DT 5/DT s0.35 19F-DT Form 2 3/PD 2/PD 2/PD 3/PD 5/DT 3/PD 2/PD 2/PD 2/PD 3/DT 5/DT S 0.35 19F-DT Form 3 3/PD 3/PD 3/PD 3/PD 3/PD 3/PD 3/PD 3/PD 3/PD 3/DT 3/PD 0.5 Table 5 Specific activation/coupllg/quenchinq conditions of PS 5 S.pneumoniae-ProtelnD/TT/DTconjugates Serotype 1 3 4 5 6B 7F Native pfluid Native Native Native Native PS 1.5 2 2.0 7.5 5.5 3.0 conc.(mg/ml) PS dissolution NaCl NaCl 2M WFI WFI NaCl 2M NaCl 2M 150mM PD 5.0 5.0 5.0 5.0 5.0 5.0 conc.(mg/ml) Initial PS/PD 1/0.7 1/1 1/1 1/1 1/1 1/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) _ I pHa=pH.=pHg 9.0/9.0/9.0 9.5/9.5/9.0 8.8/8.8/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 Coupling time 60 mins 60 mins 45 mins 40 mins 60 mins 60 mins Serotype 9V 14 18C 19F 23F Native Native Native Native Native PS 1.75 2.5 1.75 4.0 2.5 conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M WFI NaCl 2M NaCl 2M PD 5.0 5.0 5.0 5.0 5.0 conc.(mg/ml) Initial PS/PD 1/0.75 1/0.75 1/1.2 1/1 1/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH,=pHc=pHp 8.5/8.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0 Coupling time 60 mins 60 mins 45 mins 30 mins 60 mins 57 Table 6 Specific activation/couplinq/quenchinq conditions of PS S.pneumoniae-Protein D/DTconlugates for the 11 Pn-PD&Di-001 study 5 Serotype 1 3 4 5 6B 7F pfluid fluid fluid fluid fluid Native PS 4 2.0 2.5 7.5 10 3.0 conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M NaCl 2M NaCI 2M NaCl 2M NaCI 2M PD 10.0 5.0 5.0 5.0 20 (DT) 5.0 conc.(mg/ml) NaCI 2M NaCl 2M Initial PD/PS 1.2/1 1/1 1/1 1/1 1.5/1 1/1 Ratio (w/w) I CDAP conc. 1.50 0.75 . 1.5 2 1.5 0.75 (mg/rng PS) pHa=pH,=pHg 9.0/9.019.0 9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9/9/9 Coupling time 60 mins 60 mins 60 mins 60 mins 60 mins 60 mins Serotype 9V 14 18C 19F 23F Native Native fluid pfluid pfluid PS 1.75 2.5 5.0 9.0 10 conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M Carrier protein 5.0 5.0 5.0 20 (DT) 10 (DT) conc.(mg/ml) Initial carrier 0.75/1 0.75/1 1.2/1 1.5/1 1.5/1 protein/PS Ratio (w/w) CDAP conc. 0.75 0.75 1.5 1.5 0.75 (mg/mg PS) pH,=pH,=pH, 8.5/8.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Coupling time 60 mins 60 mins 30 mins 60 mins 60 mins Table 7 Specific activation/couplingqguenchinq conditions of PS 10 S.pneumoniae-Protein D/DTconiugates for the 11 Pn-PD&Di-007 study Serotype 1 3 4 5 6B 7F Native fluid Native Native Native fluid PS 1.5 2.0 2 7.5 5.5 5.0 58 conc.(mg/ml) PS dissolution NaCl NaCl 2M WFI WFI NaC1 2M NaCI 2M 150mM PD 5.0 5.0 5.0 5.0 5 10 conc.(mg/mi) Initial PD/PS 0.7/1 1/1 1 1/1 1/1 1.2/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH,=pHe=pH, 9.0/9.0/9.0 9.5/9.5/9.0 8.8/8.8/9.0 9.0/9.0/9.0 9.5/9.519.0 9.5./9.5/9 Coupling time 60 mins 60 mins 45 mins 40 mins 60 mins 60 mins Serotype 9V 14 18C 19F 19F 23F fluid fluid Native fluid pfluid fluid PS 5.0 5.0 1.75 9.0 10.0 9.5 conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M WFI NaCi 2M NaCI 2M NaCI 2M Carrier protein 10 10.0 5.0 20 (DT) 5.0 (PD) 10 conc.(mg/ml) Initial carrier 1.2/1 1.2/1 1.2/1 1.5/1 1.2/1 1/1 protein/PS Ratio (w/w) CDAP conc. 0.5 0.75 0.75 1.5 0.75 0.75 (mg/mg PS) pHa=pHc=pHq 9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Coupling time 60 mins 60 mins 45 mins 120 mins 120 mins 60 mins Table 8 Percentage of subjects with 19F antibody concentration 0.20 pigImL and 19F antibody Geometric mean antibody concentrations (GMCs 5 with 95% Cl; pg/mL) one month following 1pg 19F-PD, 3pg 19F-DT or Prevnar (2pg 19F-CRM) primary vaccination (Total cohort) 11Pn.PD&Di-001 (22F.ELISA) 11 Pn.PD&Di-007 (22F-ELISA) Group N [ % 2 0.20 gg/mL GMC (pg/mL) N % 20.20 pglmL GMC (pg/mL) (95% Cl) (95% CI) (95% CI) (95% Cl) 59 11Pn-PD 152 98.7 1.93 50 100 2.78 (95.3-99.8) (1.67.222) (92.9-100) (2.31-3.36) 19F-DT Form 1 r 146 99.3 2.88 - (96.2-100) (2.45-3.38) 19F-DT Form 2 " 150 96.0 2.43 - (91.5-98.5) (2.01-2.94) 19F-DT Form 3 r - - - 50 96.0 3.70 1 1 (86.3-99.5) (2.58-5.30) Prevnar 148 98.6 2.98 41 97.6 2.91 1 _ 1 (95.2-99.8) (2.60-3.41) (87.1-99.9) | (2.15-3.94) The composition of the different formulations is provided in table 4. Table 9 Percentage of subjects with 19F OPA titer 2 1:8 and 19F OPA GMTs one month following primary vaccination with 1pg 19F-PD, 3pg 19F 5 DT or Prevnar (2pg 19F-CRM) (Total cohort) 11Pn-PD&DI-001 11PnPD&Di-007 Group N 21:8 GMT N 21:8 GMT (95% CI) (95% Cl) (95% CI) (95% Cl) 11Pn-PD 136 84.6 77.8 46 95.7 167.8 (77.4-90.2) (58.1-104.4) (85.2-99.5) (118.1-238.6) 19F-OT Form 1 137 95.6 263.2 - - (90.7-98.4) (209.4-330.7) 19F-DT Form 2 F 139 92.1 218.9 - - (86.3-96.0) (166.5-287.9) 19F-DT Form 3 - - - 49 91.8 403.1 (80.4-97.7) (225.7-719.9) Prevnar 131 86.3 82.6 38 81.6 65.0 (79.2-91.6) (61.1-111.6) (65.7-92.3) (37.7-112.2) The composition of the different formulations is provided In Table4. Table 10 Percentage of subjects with 19F antibody concentration : 0.20 pg/mL and 19F antibody GMCs (pg/mL) prior to and one month following 23 10 valent plain polysaccharide booster in children primed with 1pg 19F PD, 3pg 19F-DT or Prevnar(2pg 19F-CRM) (Total cohort) I 1Pn-PD&Di-002 (221F ELISA) Prior to booster vaccinaion One month post 23-valent PS booster Primary group N % t 0.20 pglmL GMC (pg/mi) N % 2t 0.20 pl.&n GMC (pigiml) (95% CQ_) (95% C1) (95% CI) (95% CI) 11 IPn-PD 70 77.1 0.67 67 94.0 11.50 (65.6-86.3) (0.45-0.98) (85.4-98.3) (7.76-17.03) 19F-DT Form 1" 68 91.2 0.71 69 98.6 14.50 (81.8-96.7 (0.54-0.94) (92.2-100) (1.72 . ) 1 9F-DT Form 2F 74 81.1 0.59 72 95.8 9.90 (70.3-89.3) (0.43-0.80) (88.3-99.1) (6.74-14.54) Prevnar 65 64.6 0.40 67 100 9.40 (51.8-76.1) (0.27-0.60) (94.6-100) (6.95-1 2 .71) rThe composition of the different formulations is provided in Table4. Table 11 Percentage of subjects with 19F OPA titer 21:8 and 19F OPA GMTs prior to and one month following 23-valent plain polysaccharide 60 booster in children primed with 1pg 19F-PD, 3ig 19F-DT or Prevnar (2pg 19F-CRM) (Total cohort) 11Pn-PD&DI-002 Prior to booster vaccination f One month post 23-valent PS booster Primary group N % 1:8 GMT N % 2 1:8 GMT (95% CI) (95% CI) (95% CI) (95% Cl) 11Pn-PO 29 27.6 10.9 28 82.1 400.0 (12.7-47.2) (5.0.23.7) (63.1-93.9) (157.3-1058.3) 19F-OT Form 1 r 19 47.4 18.1 18 94.4 1063.8 (24.4-71.1) (7.2-45.7) (72.7-99.9) (386.6-2927.5) 19F-DT Form 2 r 27 33.3 8.5 28 100 957.6 1 (16.5-54.0) (4.7-15.3) (87.7-100) (552.8-1659.0) Pevnar 24 12.5 9.1 23 82.6 380.9 (2.7-32.4) (3.4-19.6) (61.2-95.0) (133.2-1089.5) r The composition of the different formulations is provided in Table4. 5 Table 12 Percentage of subjects with antibody concentrations 0.2 pg/mL, OPA 2 1:8 and GMCs/GMTs against 19F pneumococci one month following 11 Pn-PD or Prevnar booster in children primed with I pg 19F-PD, 3pg 19F-DT or Prevnar (2pg 19F-CRM) (Total cohort) I1Pn-PD&Di.002 22F.ELISA assay OPA assay Primary group N % > 0.20IpgmL GMC (pg/ml) N % : 1:8 GMT (95% CI) (95% Cl) (95% Cl) (95% CI) 11Pn-PD 70 100 4.52 21 100 255.6 - (94.9-100) (3.7-5.5) | (83.9-100) (135.5-481.9) 19F-DT Form 1 66 98.5 3.45 23 95.7 374.0 (91.8-100 (2.8-4.3 (78.1-99.9) (192.6-726.2) 19F-DT Form 2 r 70 98.6 3.80 29 96.6 249.1 1 1 (92.3-100) (2.9-4.9) (82.2-99.9) [144.7-428.7) Prevnar 69 97.1 2.56 31 96.8 528.7 (89.9-99.6) (2.0-3.3) (83.3-99.9) (319.4-875.2) rThe composition of the different formulations is provided in Table4. 10 Table 13 Percentage of subjects with antibody concentrations 2 0.2 pg/mL, OPA 2 1:8 and GMCs/GMTs against 19A pneumococci one month following primary vaccination with 1pg 19F-PD, 3pg 19F-DT or Prevnar(2pg 19F-CRM) (Total cohort) 11Pn-PD&DI-001 22F-ELISA assay OPA assay Group N % 0.20 pg/mL GMC (pg/mL) N %. 1:8 GMT (95% CI) (95% CI) (95% CI) (95% Cl) 1lPn-PD 45 28.9 0.09 52 7.7 5.2 (16.4-44.3) (0.07-0.11) (2.1-18.5) (4.0-6.8) 19F-DT Form 2r 51 29.4 0.11 59 27.1 12.4 (17.5-43.8) (0.08-0.16) _ (16.4-40.3) (7.6-20.3) Prevnar 55 18.2 0.10 61 3.3 4.6 1 (9.1-30.9) (0.08-0.12) (0.4-11.3) (3.8-5.6) r The composition of the different formulations Is provided in TabIe4 61 Example 5: Adjuvant experiments in preclinical models: impact on the immunogenicty of pneumococcal I1-valent polysaccharide conjugates in elderly Rhesus monkeys 5 To optimize the response elicited to conjugate pneumococcal vaccines in the elderly population, GSK formulated an 1 1-valent polysaccharide (PS) conjugate vaccine with a novel adjuvant Adjuvant C - see below. 10 Groups of 5 elderly Rhesus monkeys (14 to 28 years-old) were immunized intramuscularly (IM) at days 0 and 28 with 500 pl of either 11-valent PS conjugates adsorbed onto 315 pg of AIPO4 or 11-valent PS conjugates admixed with Adjuvant C. In both vaccine formulations, the 11-valent PS conjugates were each composed of the 15 following conjugates PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS7F-PD, PS9V-PD, PS14 PD, PS18C-PD, PS19F-PD, PS23F-DT and PS6B-DT. The vaccine used was 1/5 dose of of the human dose of the vaccine (5 Vg of each saccharide per human dose except for 6B [10 pg]) conjugated according to Table 6 conditions (Example 4), except 19F was made according to the following CDAP process conditions: sized saccharide at 9 mg/ml, PD at 5 20 mg/mI, an initial PD/PS ratio of 1.211, a CDAP concentration of 0.75 mg/mg PS, pHa=pHc=pHq 9.0/9.0/9.0 and a coupling time of 60 min. Anti-PS ELISA IgG levels and opsono-phagocytosis titres were dosed in sera collected at day 42. Anti-PS3 memory B cell frequencies were measured by Elispot from peripheral 25 blood cells collected at day 42. According to the results shown here below, Adjuvant C significantly improved the immunogenicity of 11-valent PS conjugates versus conjugates with AIPO4 in elderly monkeys. The novel adjuvant enhanced the IgG responses to PS (Figure 1) and the 30 opsono-phagocytosis antibody titres (Table 14). There was also supportive evidence that the frequency of PS3-specific memory B cells is increased by the use of Adjuvant C (Figure 2). Table 14, Conjugate Immunogenicity In elderly Rhesus monkeys (post-il opsono 35 phagocytosis titres) PSI PS3 PS4 PS5 PS6B PS7F PSOV PS14 PS1SC PS19F PS23F 11-a ent Pre-imn ei <8 5 <U $ <8 16 .8 <8 0 <8 .8 AIP04 day i4 post 11 S 181 64 40 64 4096 42 37 169 64 <84 11 talent Pre-immune 5 a <8 5 a 37 <6 <C .8 <8 48 AdJ.C day 14 post II 776 1351 891 676 6208 16384 111 161 7132 2048 <4 62 B Cell Elispot The principle of the assay relies on the fact that memory B cells mature into plasma cells 5 in vitro following cultivation with CpG for 5 days. In vitro generated antigen-specific plasma cells can be easily detected and therefore be enumerated using the B-cell elispot assay. The number of specific plasma cells mirrors the frequency of memory B cells at the onset of the culture. 10 Briefly, in vitro generated plasma cells are incubated in culture plates coated with antigen. Antigen-specific plasma cells form antibody/antigen spots, which are detected by conventional immuno-enzymatic procedure and enumerated as memory B cells. In the present study, Polysaccharides have been used to coat culture plates in order to enumerate respective memory B cells. Results are expressed as a frequency of PS specific 15 memory B cells within a million of memory B cells. The study shows that Adjuvant C may be able to alleviate the known problem of PS3 boostability (see 5th International Symposium on Pneumococci and Pneumococcal Diseases, April 2-6 2006, Alice Springs, Central Australia. 20 Specificities of immune responses against a serotype 3 pneumococcal conjugate. Schuerman L, Prymula R, Poolman J. Abstract book p 245, P010.06). Example 6, Effectiveness of detoxified Pneumolysin (dPly) as a protein carrier to enhance the immunogenicity of PS 19F in young Balb/c mice 25 Groups of 40 female Balb/c mice (4-weeks old) were immunized IM at days 0, 14 and 28 with 50 pl of either 4-valent plain PS or 4-valent dPly-conjugated PS, both admixed with Adjuvant C. Both vaccine formulations were composed of 0.1 pg (quantity of saccharide) of each of 30 the following PS: PS8, PS12F, PS19F and PS22F. Anti-PS ELISA IgG levels were dosed in sera collected at day 42. The anti-PS19F response, shown as an example in Figure 3, was strongly enhanced in mice given 4-valent dPly conjugates compared to mice immunized with the plain PS. The same improvement was observed for the anti-PS8, 12F and 22F IgG responses (data not 35 shown). 40 63 Example 7, Effectiveness of Pneumococcal Histidine Triad Protein D (PhtD) as a 5 protein carrier to enhance the immunogenicity of PS 22F in young Balb/c mice Groups of 40 female Balb/c mice (4-weeks old) were immunized IM at days 0, 14 and 28 with 50 pl of either 4-valent plain PS or 4-valent PhtD-conjugated PS, both admixed with Adjuvant C. 10 Both vaccine formulations were composed of 0.1 pg (quantity of saccharide) of each of the following PS: PS8, PS12F, PS19F and PS22F. Anti-PS ELISA IgG levels were dosed in sera collected at day 42. The anti-PS22F response, shown as an example in Figure 4, was strongly enhanced in mice given 4-valent PhtD conjugates compared to mice immunized with the plain PS. The 15 same improvement was observed for the anti-PS8, 12F and 1 9F IgG responses (data not shown). Example 8, Immunogenicity in elderly C57B1 mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD 20 Groups of 30 old C57BI mice (>69-weeks old) were immunized IM at days 0, 14 and 28 with 50 pl of either 11 -valent PS conjugates or 13-valent PS conjugates, both admixed with Adjuvant C (see below). The 11-valent vaccine formulation was composed of 0.1 pg saccharide of each of the 25 following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine discussed under Table 2). The 13-valent vaccine formulation contained in addition 0.1 pg of PSi 9A-dPly and PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccine discussed under Table 2 [using directly-conjugated 22F]). In group 2 30 and 4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147 from WO 03/054007) was used. In group 6 19A was conjugated to diphtheria toxoid and 22F to protein D. Anti-PSI9A and 22F ELISA IgG levels were dosed in individual sera collected at day 42. 35 The ELISA IgG response generated to the other PS was measured in pooled sera. 19A-dPly and 22F-PhtD administered within the 13-valent conjugate vaccine formulation were shown immunogenic in old C57B1 mice (Table 15). The immune response induced against the other PS was not negatively impacted in mice given the 13-valent formulation compared to those immunized with the 11-valent formulation. 40 Table 15, PS Immunogenicity In old C57BI mice (post-IlIl IgG levels) 64 Old C57 Black mice ELISA GROUP 1 GROUP 2 GROUP3 GROUP4 GROUP 5 GROUP6 11V 11V 11V 11V 11V 11V 19A-dPly 19A-dPly 19A-dPly 19A-dPly gmbs formol gmbs formol 19A-DT 22F-PhtD 22F-PhtD 22F-PhtD-E 22F-PhtD-E 22F-PD 0.1pg/50pl 0.1pg/50pI 0.1pg/50pl 0.ipg/50pl 0.1pg/5Opl 0.1pg/50pl Adj C Adj C Adj C Adj C Adj C Ad[ C 1 average 19.30 20.20 24.40 12.80 12.10 13.60 Pool 3 average 6.32 4.84 5.21 6.74 2.38 2.54 Pool 4 average 60.9 67.1 51.4 47.4 45.5 41.1 Pool 5 average 1.34 3.81 3.06 2.75 1.26 1.23 Pool 6B average 4.41 4.12 5.88 1.58 2.31 5.64 Pool 7F average 0.83 0.81 1.65 1.98 0.89 0.99 Pool 9V average 13.8 23.7 20.0 13.1 15.5 9.6 Pool 14 average 25.73 42.96 34.12 32.53 23.97 15.60 Pool 18C average 13.4 20.1 11.9 9.1 8.3 8.4 Pool 19F average 57.5 90.0 63.8 36.5 47.0 69.1 Pool 23F average NR NR NR NR NR NR Pool 19A GMC 0.06 0.09 0.25 0.08 0.23 0.19 65 Ic 0.04-0.1 0.05-0.14 0.15-0.41 0.06-0.12 0.14-0.38 0.09-0.3 %sero 33% 47% 83% 53% 80% 73% 22F GMC NR 5.81 3.76 0.54 0.85 2.02 IC 3.2-10.6 1.8-7.9 0.3-1.1 0.4-1.7 1.2-3.4 /sero 0% 97% 90% 77% 87% 97% Example 9, immunogenicity in young Balb/c mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD 5 Groups of 30 young Balb/c mice (4-weeks old) were immunized IM at days 0, 14 and 28 with 50 pl of either 11-valent PS conjugates or 13-valent PS conjugates, both admixed with Adjuvant C (see below). The 11-valent vaccine formulation was composed of 0.1 pg saccharide of each of the 10 following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine discussed under Table 2). The 13-valent vaccine formulation contained in addition 0.1 pg of PS19A-dPly and PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccine discussed under Table 2 [using directly-conjugated 22F]). In group 2 15 and 4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147 from WO 03/054007) was used. In group 6 19A was conjugated to diphtheria toxoid and 22F to protein D. Anti-PS19A and 22F ELISA IgG levels were dosed in individual sera collected at day 42. 20 The ELISA IgG response generated to the other PS was measured in pooled sera. 19A-dPly and 22F-PhtD administered within the 13-valent conjugate vaccine formulation were shown immunogenic in young Balb/c mice (Table 16). The immune response induced against the other PS was not negatively impacted in mice given the 13-valent formulation compared to those immunized with the 1 1-valent formulation. 25 Table 16, PS immunogenicity in young Balb/c mice (post-il IgG levels) BalbC mice EUSA GROUP GROUP2 GROUP3 GROUP4 GROUP5 GROUP 11V 11V 11V 11V 11V 11V 19A-dPly 19A-dPly 19A-dPly 19A-dPly gmbs formol gmbs formal 19A-DT 22F-PhtD 22F-PhtD 22F-PhtD-E 22F-PhtD-E 22F-PD 0.1pg/50pl 0.1pg/50pl 0.1pg/50pl 0.1 pg/50pl 0.1pg/50p1 0.1pg/50pl Adj C Adj C Adi C Adj C Adj C Adl C 1 average 131.70 101.20 83.00 82.40 67.90 85.50 66 Pool 3 average 21.85 10.38 12.53 8.83 8.73 14.98 Pool 4 average 147.4 127.0 104.4 95.0 113.6 114.2 Pool 5 average 21.38 20.29 18.26 18.95 18.02 23.04 Pool 6B average 1.97 4.76 3.72 2.35 1.43 1.05 Pool 7F average 7.69 4.58 4.77 4.24 3.92 3.94 Pool 9V average 30.1 30.7 26.5 21.4 23.4 28.3 Pool 14 average 28.78 27.67 26.23 21.54 24.34 13.73 Pool 18C average 53.4 52.37 48.5 57.8 47.8 75.8 Pool 19F average 186.6 157.7 169.3 178.9 181.9 223.2 Pool 23F average 4.98 3.9 5.11 0.57 3.13 4.57 Pool 19A GMC 0.4 32.8 25.1 21.6 18.9 23.5 IC 0.2-0.6 26.4-40.7 20.6-30.6 17.5-26.7 15.1-23.5 19.5-28.5 %sero 93% 100% 100% 100% 100% 100% 22F GMC NR 3.99 3.76 6.27 8.70 18.76 IC 1.9-8.42 1.8-8 3.8-10.4 5.4-13.9 15.2-23.1 %sero 0% 93% 100% 100% 100% 100% Example 10, Immunogenicity in Guinea Pigs of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD 5 Groups of 20 young Guinea Pigs (Hartley Strain; 5.weeks old) were immunized IM at days 0, 14 and 28 with 125 pl of either 11-valent PS conjugates or 13-valent PS conjugates, both admixed with Adjuvant C (see below). 67 The 11 -valent vaccine formulation was composed of 0.25 pg saccharide of each of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine discussed under Table 2). The 13-valent vaccine formulation contained in 5 addition 0.1 pg of PS1 9A-dPly and PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccine discussed under Table 2 [using directly-conjugated 22F]). In group 2 and 4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147 from WO 03/054007) was used. In 10 group 6 19A was conjugated to diphtheria toxoid and 22F to protein D. Anti-PS19A and 22F ELISA IgG levels were dosed in individual sera collected at day 42. The ELISA IgG response generated to the other PS was measured in pooled sera. Table 17, PS Immunogenicity in young Balb/c mice (post-Ill IgG levels) 15 Guinea pigs EUSA GROUP 1 GROUP2 GROUP3 GROUP4 GROUP GROUP6 11V 11V 11V 11V 11V 11V 19A-dPly 19A-dPly 19A-dPly 19A-dPly gmbs formol gmbs formol 19A-DT 22F-PhtD 22F-PhtD 22F-PhtD E 22F-PhtD-E 22F-PD 0.1pg/50pl 0.1pg/50pl 0.1 pg/50pI 0.1pg/50p 0.1pg/50pl 0.1pg/50pl Adj C Adi C Adj C Adj C Adi C Adj C I average 78.00 77.21 76.15 68.77 68.59 81.04 Pool 3 average 7.75 9.31 12.73 7.94 4.75 9.59 Pool 4 average 130.7 94.4 132.6 166.8 85.0 101.3 Pool 5 average 109.10 117.10 110.70 158.40 74.10 100.40 Pool 6B average 3.14 4.26 14.4 7.63 6.3 7.52 Pool 7F average 154.2 216.0 240.0 181.0 142.0 179.1 Pool 9V average 90.69 105.45 98.20 93.45 54.12 73.05 68 Pool 14 average 71.19 77.18 46.53 59.67 38.47 53.69 Pool 18C average 109.4 122.3 137.1 79.9 73.7 83.1 Pool 19F average 73.9 102.5 112.2 75.5 62.3 72.1 Pool 23F average 19.19 30.74 29.44 31.52 19.13 24.94 Pool 19A GMC 0.4 25.58 41.49 14.25 27.49 6.74 IC 0.24-0.68 12-54.5 24.4-70.5 5.9-34.6 16.6-45.4 4-11.3 %sero 75% 100% 100% 100% 100% 100% 22F GMC 0.12 2.51 3.67 45.74 30.68 96.38 IC 0.09-0.16 0.94-6.73 1.59-8.42 29.3-71.4 17-53.3 73.5-126.4 %sero 10% 95% 95% 100% 100% 100% Example 11: Formulations being made and tested 5 a) The following formulations are made (using the 13 valent vaccine from table 1 and serotype 3 from table 5 - see comment on 14 valent vaccine discussed under Table 2 [using directly-conjugated 22F or through an ADH linker]). The saccharides are formulated with aluminium phosphate and 3D-MPL as shown below. 10 14V 25wp MPL 14V Iu, I MPL Stim of BAC Aluainium content -> FF Sum or BAC Alumininm content -> FF _ po rer Do PS carrier pg PS L ralin PS/Al pg Al PS carrier pg PS MPL ratio PS/Al pgA I PD I 10 10 1 PD I 10 10 3 PD 1 10 10 3 PD I t0 10 4 PD 3 10 30 4 PD 3 10 30 5 PD I 10 10 5 P1D 1 10 10 0 1 0 6A PD 1 0 10 6B PD 1 10 10 6B PD I 10 10 7F PD 1 1n 10 7F PD 1 10 10 9V PD 1 I10 10 9V PD 1 10 10 14 PD 1 10 10 14 PD 1 10 10) 18C TT,_ 3 i5 45 l8C T. 3 15 45 19A diPly _3 10 30 19A dPy 3 10 30 19F DT 3 10 30 1 DT 3 1 0 30 22F Pht_310 30 22F PhtD 3 10 30 23F PD 1 10 10 23F PD 1 10 10 BACMPL5D/200 25 4 100 BACMPL 50200 10 4 40 FF Aluminium Sum - 355 FF Aluminium conmi Sum - 295 cotlent 69 b) The same saccharide formulation is adjuvanted with each of the following adjuvants: - In the table herebelow the concentration of the emulsion components per 500pl dose is shown. 5 Adjuvant Al Adjuvant A2 Adjuvant A3 Ingredients 250.l o/w 125pd o/w 50p1 o/w emulsion emulsion emulsion alpha 11.88mg 5.94mg 2.38mg Tocopherol Squalene 10.7mg 5.35mg 2.14mg Tween 80 4.85mg 2.43mg 0.97mg Adjuvant A4 Adjuvant A5 Adjuvant A6 Adjuvant A7 Ingredients 2501p o/w 250l 1 i o/w 125V 1 o/w 50[d o/w emulsion emulsion emulsion emulsion alpha 11.88mg 11.88mg 5.94mg 2.38mg Tocopherol Squalene 10.7mg 10.7mg 5.35mg 2.14mg Tween 80 4.85mg 4.85mg 2.43mg 0.97mg 3D-MPL 50kg 25pg 25pg 10pg 10 c) The saccharides are also formulated with two liposome based adjuvants: Composition of Adjuvant B1 Qualitative Quantitative (per 0.5 ml dose) 15 Liposornes: - DOPC 1 mg - cholesterol 0.25 mg 3DMPL 50 pg QS21 50 pg 20 KH2PO4 13.124 mg Buffer Na2HP0 10.290 mg Buffer NaCl 2.922 mg (100 mM) WF! q.s. ad 0.5 ml Solvent 25 pH 6.1 1. Total P04concentration = 50 mM Composition of Adjuvant B2 Qualitative Quantitative (per 0.5 mL dose) 30 Liposomes: -0OPC 0.5 mg -cholesterol 0.125 mg 3DMPL 25 pg QS21 25 pg 70 KH2PO4 13.124 mg Buffer Na2HPO4 10.290 mg Buffer NaCl 2.922 mg (100 mM) 5 WFI q.s. ad 0.5 ml Solvent pH 6.1 d) The saccharides are also formulated with Adjuvant C (see above for other compositions where this adjuvant has been used): 10 Qualitative Quantitative (per 0.5 mL dose) Oil in water emulsion: 50 pl -squalene 2.136 mg -w-tocopherol 2.372 mg 15 - Tween 80 0.97 mg -cholesterol 0.1 mg 3DMPL 50 pg QS21 50 pg KH2PO4 0.470 mg Buffer 20 Na2HP041 0.219 mg Buffer NaCl 4.003 mg (137 mM) KCI0.101 mg (2.7 mM) 25 WFI q.s. ad 0.5 ml Solvent pH 6.8 Example 12, Impact of conjugation chemistry on 22F-PhtD conjugate Immunogenicity In Balb/c mice 30 Groups of 30 female Balb/c mice were immunised by the intramuscular (IM) route at days 0, 14 and 28 with 13-valent PS formulations containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F (dose: 0.3 pg saccharide / PS for PS 4, 18C, 19A, 19F and 22F and 0.1 pg saccharide I PS for the other PS). PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid, 19A to formol 35 detoxified Ply, 22F to PhtD and the other PS to PD. Two formulations, constituted of either 22F-PhtD prepared by direct CDAP chemistry or 22F-AH-PhtD (ADH-derivitized PS), were compared. See Example 2, Table 1 and comment under Table 2 for characteristics of 13 valent vaccine made either with 22F directly conjugated or via an ADH spacer. The vaccine formulations were supplemented 40 with adjuvant C. 71 Anti-PS22F ELISA IgG levels and opsono-phagocytosis titres were measured in sera collected at day 42. 22F-AH-PhtD was shown much more immunogenic than 22F-PhtD in terms of both IgG levels (figure 5) and opsono-phagocytic titres (figure 6). 5 Example 13, Impact of new adiuvants on immuno-genicity of Streptoccoccus pneumoniae capsule PS conjugates Groups of 40 female Balb/c mice were immunised by the IM route at days 0, 14 and 28 10 with 13-valent PS formulations containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F (dose: 0.3 pg / PS for PS 4, 18C, 19A, 19F and 22F and 0.1 pg / PS for the other PS). PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid, 19A to formol detoxified Ply, 22F to PhtD and the other PS to PD. See Example 2, Table 1 and 15 comment under Table 2 for characteristics of 13 valent vaccine made with 22F directly conjugated. Four formulations, supplemented with either AIPO 4 , adjuvant A1, adjuvant A4 or adjuvant A5, were compared. Anti-PS, Ply, PhtD and PD ELISA IgG levels were measured in sera collected at day 42 20 and pooled per group. The following ratio was calculated for each antigen: IgG level induced with the new adjuvant tested / IgG level induced with AIPO 4 . All the new adjuvants tested improved at least 2-fold the immune responses to 13-valent conjugates compared to the classical AIPO 4 formulation (figure 7). Example 14, protective efficacy of a PhtDidetoxified Ply combo In a pneumococcal 25 monkey pneumonia model Groups of 6 Rhesus monkeys (3 to 8 years-old), selected as those having the lowest pre existing anti-19F antibody levels, were immunized intramuscularly at days 0 and 28 with either 11-valent PS conjugates (i.e. 1 pg of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and 3 pg 30 of PS 4, 18C and 19F) or PhtD (10 pg) + formol-detoxified Ply (10 pg) or the adjuvant alone. 72 PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid and the other PS to PD. See Example 2, Table 1 and comment under Table 2 for characteristics of 11 valent vaccine. All formulations were supplemented with adjuvant C. Type 19F pneumococci (5.108 cfu) were inoculated in the right lung at day 42. Colonies 5 were counted in broncho-alveolar lavages collected at days 1, 3 and 7 post-challenge. The results were expressed as the number of animals per group either dead, lung colonized or cleared at day 7 after challenge. As shown in figure 8, a good protection close to statistical significance (despite the low number of animals used) was obtained with 11-valent conjugates and the PhtD+dPly 10 combo (p < 0.12. Fisher Exact test) compared to the adjuvant alone group. Example 15, impact of conjugation chemistry on the anti-PhtD antibody response and the protective efficacy against a type 4 challenge induced by 22F-PhtD conjugates 15 Groups of 20 female OF1 mice were immunised by the intramuscular route at days 0 and 14 with 3 pg of either 22F-PhtD (prepared by direct CDAP chemistry) or 22F-AH-PhtD (ADH-derivitized PS), or the adjuvant alone. Both monovalent 22F conjugates were made by the processes of Example 2 (see also Table 1 and Table 2). Each formulation was 20 supplemented with adjuvant C. Anti-PhtD ELISA IgG levels were measured in sera collected at day 27. Mice were challenged intranasally with 5.106 cfu of type 4 pneumococci at day 28 (i.e. a 25 pneumococcal serotype not potentially covered by the PS present in the vaccine formulation tested). The mortality induced was monitored until day 8 post-challenge. 22F-AH-PhtD induced a significantly higher anti-PhtD IgG response and better protection against type 4 challenge than 22F-PhtD. 30 73

Claims (18)

1. A Streptococcus pneumoniae immunogenic composition comprising 10 or more capsular saccharides from different S. pneumoniae serotypes which are conjugated to a carrier protein, and comprising 3 or more different carrier proteins, wherein the composition comprises: serotype 19F capsular saccharide conjugated to diphtheria toxoid (DT); serotype 18C capsular saccharide conjugated to tetanus toxoid (TT); serotype 1, 4, 5, 6B, 7F, 9V, 14 and 23F capsular saccharides conjugated to protein D from Haemophilus influenzae.

2. An immunogenic composition according to claim 1 wherein 19F capsular saccharide is directly conjugated to the carrier protein.

3. An immunogenic composition according to claim 1 wherein 19F capsular saccharide is conjugated to the carrier protein via a linker.

4. An immunogenic composition according to claim 3 wherein the linker is bifunctional.

5. The immunogenic composition of claim 3 or claim 4 wherein the linker is ADH.

6. The immunogenic composition of any one of claims 3 to 5, wherein the linker is attached to the carrier protein by carbodiimide chemistry, or using EDAC chemistry.

7. The immunogenic composition of any preceding claim wherein the 19F saccharide is conjugated to the carrier protein or to the linker using CDAP chemistry.

8. The immunogenic composition of any preceding claim wherein the ratio of carrier protein to 19F saccharide is between 5:1 and 1:5, 4:1 and 1:1 or 2:1 and 1:1, or 1.5:1 and 1.4:1 (w/w).

9. The immunogenic composition of any preceding claim wherein the average size (e.g. M,) of the 19F saccharide is above 100 kDa.

10. The immunogenic composition of any preceding claim wherein the dose of the 19F saccharide conjugate is between 1 and 10 pg, 1 and 5 pg, or 1 and 3 pg of saccharide.

11. An immunogenic composition according to any preceding claim further comprising serotype 6A and/or 15B.

12. An immunogenic composition according to any preceding claim further comprising serotype 19A. 74

13. An immunogenic composition according to any preceding claim further comprising serotype 22F.

14. An immunogenic composition according to any preceding claim which comprises pneumolysin as free or carrier protein.

15. An immunogenic composition according to any preceding claim which comprises a PhtX protein, PhtD protein, a PhtBD fusion protein or a PhtDE fusion protein as free or carrier protein.

16. A method of treating or preventing a disease caused by Streptococcus pneumoniae serotype 19A strain infection said method comprising the step of administering to a subject in need thereof a Streptococcus pneumoniae immunogenic composition comprising 9 or more, 10 or more, 11 of more, 13 or more, or 14 or more capsular saccharides from different S.pneumoniae serotypes conjugated to 2 or more different carrier proteins, wherein the composition comprises serotype 19F capsular saccharide conjugated to CRM197 using CDAP chemistry, but does not comprise capsular saccharide from serotype 19A.

17. Use of Streptococcus pneumoniae immunogenic compositions comprising 9 or more, 10 or more, 11 of more, 13 or more, or 14 or more capsular saccharides from different S.pneumoniae serotypes conjugated to 2 or more different carrier proteins, wherein the composition comprises serotype 19F capsular saccharide conjugated to CRM197 using CDAP chemistry, but does not comprise capsular saccharide from serotype 19A in the preparation of a medicament for the treatment or prevention of a disease caused by Streptococcus pneumoniae serotype 19A strain infection.

18. A Streptococcus pneumoniae immunogenic composition according to claim 1; a method of treatment according to claim 16; or use according to claim 17, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 75